Fat Injection From Filling to Regeneration

50
Part I • Principles and Basic Concepts
Fig. 2-7
Finally, I create blueprints or the procedures using copies o the pencil tracings o the patient’s ace
rom di erent angles. T ese blueprints allow the patient to participate in the planning and to better
understand the procedure. Areas to be augmented can be marked on the blueprints with annotations
about the layer o planned inf ltration (against bone, in muscle, or under mucosa or skin). Green marks
indicate placement o structural at to change the shape o an area. Yellow highlighting designates areas
that will be enlarged or f lled without changing their shape. Purple marks show areas where atty tissue

51
Chapter 2 • Analysis o the Patient
will be removed. Red or brown marks indicate access incisions or placing or removing at. o complete
this blueprint, I list the general complications as I have described them to the patient (see Chapter ).
T is blueprint becomes a visual record o my discussion with the patient. I consult this plan during and
a er the procedure. Given the three-dimensional nature o sculptural at gra ing, this stage o the plan-
ning process is crucial.
I n f ormed C on sent
I believe that the most important part o the in ormed consent process is not the printed operative
consent document itsel but rather the time spent with the patient in preparing or the procedure. In ad-
dition to a verbal explanation o the procedure and the perioperative course by the surgeon and nurses,
the patient should be given printed material or re erence. I provide patients with an individualized
computer-generated explanation o what they need to do be ore the surgery, what they can expect on the
day o surgery, and the expected postoperative course.
T e patient must be prepared or postoperative swelling and bruising and the potential or complications
with structural at gra ing. Although there is a high rate o long-term patient satis action, the patient
who is not prepared or any expected or unexpected postoperative sequelae will be less likely to express
satis action with the results.
T e changes created by Coleman at gra ing o the ace can be measured in millimeters or ractions o
millimeters. No matter how per ect the result may be and how closely the result resembles the preop-
erative plan, the patient can still desire urther small alterations. Some patients who request this type o
procedure are obsessively preoccupied with each millimeter o their aces.
T e possibility o revision and additional procedures should be discussed with all patients be ore surgery
and the charge or any such procedures should be documented, pre erably in writing.
T h e D a y o f S ur g ery
REVIEW OF THE PLAN
On the day o surgery, the blueprints are reviewed and the plan discussed again with the patient. Patients
almost always have questions that have not been answered at previous consultations or by the support
staf . T e patient reads and signs the operative consent orm that delineates the planned procedure in
words he or she can understand.
I also obtain a photographic consent be ore the procedure. I help prepare prospective patients by show-
ing them photographs o patients on whom I have per ormed structural at gra ing in the past. o do
this, I need the patients’ permission to show their photographs to other patients and to physicians as well
as to publish them in medical publications or popular consumer magazines.

52
Part I • Principles and Basic Concepts
MARKINGS
Fig. 2-8
On the day o the procedure, colored marks (Penmark laundry markers work well) are drawn on the
patient’s ace using the blueprints created at the second consultation as guides. T e markings outline
both the areas to be augmented and the amounts o tissue to be inf ltrated. T e markings can be used to
remind the surgeon o the specif c levels o placement against the skin, against bone, or in intermediate
levels.
I use colors similar to those used in the tracings, with the exception o yellow, because it is di cult to see
on the ace during the procedure. Green marks the locations or structural changes and orange delin-
eates where no at will be placed. From the green marks to the orange marks will be a transition or eath-

53
Chapter 2 • Analysis o the Patient
ering zone. I use purple to indicate special considerations, such as the purple diamonds on this patient’s
cheeks, which designate the proposed height o the cheek. I also use purple to show areas o removal o
atty tissue. Finally, I use the color red to denote incision sites.
Patients requently ask i the markers wash o . I assure them that the markings usually wipe o during
the procedure. Any residual marks can be removed with oil-based creams or emulsions. Water-based
solutions such as alcohol or acetone should not be used. I the ink enters the 1 to mm incision site, the
dermis o the resultant incisional scar can become tattooed. For this reason, I use a red laundry marker
to plan the incision sites, because it will usually not leave a tattoo.
Next, while the patient looks in a mirror, I explain that the markings designate where at will be added or
removed. T is is the last chance or the patient to participate in the procedure be ore a sedative is given.
T e patient is able to conf rm or reject the f nal plan or atty tissue placement. Finally, the markings are
photographed.
I the patient later says, “Well, I didn’t really understand you were putting at there. I thought you were
going to put at into my lips also!” I can point to the documented markings on the photographs in that
area to gently remind her o the original plan she agreed to. People orget. I a patient says, “I didn’t recall
that you were going to put at in my orehead,” the photographs that show green markings all over the
orehead will limit the discussion. I insist that all patients care ully examine the markings on their aces
in a hand mirror so that they will understand where atty tissue will be placed or removed.
Markings are placed on the skin as the ultimate three-dimensional guide to placement o atty tissue.
T ey not only indicate the areas o tissue placement, but also are a topographic map to delineate the
volumes o placement within a certain area to create specif c contour changes.
Fig. 2-9
For example, a youth ul chin usually has a central atness and lateral protuberances. I plan the size and
orm guided by old photographs o the patient’s chin or photographs o a person whose chin the patient
likes. I delineate the contour objective with concentric circles placed like a topographic map o two
mounds separated by a valley. T e arther apart the circles are placed, the squarer the chin will appear.
T e closer together the circles are placed, the more pointed the chin will appear. I use this type o topo-
graphic manipulation o the plan on most areas o the ace.
Markings play a vital role in the last part o the planning phase, or the in ormed consent, and as a guide
or the placement o at gra s.

54
Part I • Principles and Basic Concepts
C oncl usi on
T e surgeon must have a clear understanding o the patient’s desires, expectations, and physical condi-
tion be ore a plan or Coleman at gra ing can be developed. T e changes produced by this technique
are usually quite subtle and the surgeon must be able to clearly visualize the proposed alterations. Preop-
erative planning is the key to a success ul result and a satisf ed patient.
REFERENCES
1. Coleman SR. Structural at gra ing. Aesthet Surg J 18:386, 1998.
2. Gorney M. Reconstruction o your sel image. Plast Reconstr Surg 1: 36, 1973.
3. Hurwitz D, Coleman SR, Katz A. Structural at gra ing o the ace: lessons rom a teacher and his student.
Key Issues Plast Surg 17:1 , 2000.

55
C h a pt e r 3
T b g m n sms sp ns b s g d s n mp
nd s d, d sp d d s s d . On b s s m s n s d s p m d n
g n mb m n sp m ns d p s ss , s p ss b p d dd n n -
m n b p ss s n d n s g d nd m m n n n
g ng.1 T n s d p b m s s m n n ng m g d d p s ss ,
n p b s ns n nd d n, w p p ns
m s m s .
T d s p p ns w s m p p s w n d p s ss p mp d -
ns d n p n n pp ns .2 In f d p s nd ns -
s g , d n n p g ng s ng w d n w n p p -
g n n.3 Ad p s ss s d b ns d d n s f , b s s g n
n s. Un d s s m p p s d p s -d d s m s (ADSCs),
b m nsp n s w s s mp b d p s s
nd p s m p g s p nsp n n. H w , d
s ng s p d n s , d ng d b s b mp n nd g n s
nd x g n s d p s g m .4 T b nsp n d s s s
b n d b d, g ng s q s n w s ss g s m s
s ng g d d p s ( s ) m n s d p s
( s p m n ).5
d s d b s d n n nq b ADSCs, s , nd
x n nd m d n m n n g n p ss s. nsw s q s ns,
w m s s ns d mp x ns ps ADSCs s b s w x n -
s, p s g (b nsp n ) p ( nsp n ), n b d m n ng
d n nd — s, n .
In s p w w d s ss n p xp n ng g s n b s s niche
theory. W w p d g n d f n n niche nd xp n w n n s pp
d n s n s m b g nd p p m z n w, x d n p
p n g d .
he Niche heor y or Fat
Gr a t Sur vival
Andrea Sbarbati, Giamaica Conti

56
Part I • Principles and Basic Concepts
T h e S tem C el
N i ch e o f F a t
Niche
Cell
niche
Stem
cell
niche
Fat:
ADSC
niche
Fig. 3-1
T rough a comprehensive discussion o the def nition o niche and stem cell niche, we will introduce the
concept o at as the niche of adipose-derived stromal cells.
HE NICHE
T e Word Web Online Dictionary provides the ollowing our def nitions or niche: (1) a position partic-
ularly well suited to the person who occupies it; (2) a small concavity; (3) an enclosure that is set back or
indented; and (4) (ecology) the status o an organism within its environment and community (a ecting
its survival as a species). A f h meaning can be derived rom economics, where the term “niche market”
(also known as “target market”) re ers to a specif c targetable portion (subset) o the market.
In a physical space, a niche is a recess, a restricted portion o that space, that is usually associated with
an idea o suitability and that allows whoever or whatever occupies it to satis y his needs and express
properties inherent in his identity. Within complex systems such as an ecosystem or a market, a niche
includes the range o elements and interactions that allow the long-term survival o a given species or
business, respectively.
T e Cell Niche
T e physiology o any cell is strictly regulated by the interactions that it establishes with its microen-
vironment (niche). T ese interactions involve physical communications with neighboring cells or the
surrounding extracellular matrix and the binding o circulating or paracrine actors released by distant
or local cells, respectively. In other words, the niche concept, which originally re erred to interactions
between species and their environment in the context o the whole ecosystem, can be extended to de-
scribe the complex relationships inside a single organism that allow any cell to survive, grow, di erenti-
ate, and reproduce.

57
Chapter 3 • he Niche heory or Fat Gra t Survival
Blood circulating factors
Cell-matrix interactions
Cell-cell interactions
Paracrine factors
Fig. 3-2
T e cell niche concept applies to interactions with neighboring cells or the surrounding extracellular ma-
trix, but also to long-distance interactions through the blood circulation, lymphatic system, and nerve
pathways. T us the phenotypic identity o any single cell and its reaction to a given stimulus are not de-
termined only by the genetic or epigenetic equipment o the cell, but are also strongly dependent on the
specif c niche context in which the cell resides. A change in niche parameters, or transplantation o cells
into niches other than their original ones, can drastically a ect cell physiology and alter its properties.
T e cell niche theory can obviously be applied to any kind o cell, but the impact o this concept is partic-
ularly signif cant in the context o stem cell biology. T e characteristic that primarily distinguishes stem
cells rom di erentiated cells is their plasticity, or capacity to change their identity in relation to their
external environment, or niche. Changes in the niche o a stem cell might plausibly have a pro ound
impact on the plasticity o the stem cell, resulting in its arrest, through cell commitment; its activation
toward a regenerative or reparative unction; or its aberrant evolution toward neoplastic behavior. Con-
sidering the same concept in terms o clinical applications, the outcome o therapeutically aimed cell
gra ing into an exogenous environment could be understood, predicted, and controlled only on the
basis o knowledge, or prediction, o the local and distant actors that govern the physiology o gra ed
cells and their responsiveness to incoming stimuli.
T e cell niche theory is only in its in ancy, and our preliminary considerations are obviously simplistic.
In this chapter we will discuss recent insights into di erent examples o stem cell niches, with the ambi-
tious aim o def ning common biologic paradigms that could help us to better understand and predict
the outcome o therapeutic gra ing o the cells we are interested in—ADSCs—along with their niche—
at.
Stem Cell Niches
T e concept o a niche as a specialized microenvironment housing stem cells was f rst proposed by Scho-
f eld6 almost 40 years ago with re erence to mammalian hematology. Although the f eld o stem cell bi-
ology is still young and very con using, several aspects have been clarif ed or certain stem populations,
providing re erence concepts with the potential to def ne a common paradigm o the stem cell niche.
T e f rst well-known stem cell niche is that o hematopoietic stem cells (HSCs). HSCs are mainly ound
in bone marrow and, to a lesser extent, in extramedullary tissues, such as the spleen and liver. In bone
marrow, HSCs predominantly line the endosteum. Both osteoblasts and osteoclasts have been shown
to be involved in the determination o HSC ate. Endosteal osteoblasts are thought to provide a variety
o actors that regulate HSC proli eration and contribute to the homeostatic maintenance o the HSC
reservoir,7 such as osteopontin, KI ligand, and Notch ligand (Jagged-1). It is possible to observe how
di erent environmental actors can in uence the signaling o the HSC niche.

58
Part I • Principles and Basic Concepts
Table 3-1 Environmental Factors Inf uencing Signaling o the Hematopoietic
Stem Cell Niche
Factor
Role in Niche Stimulation
NO CH

Notch signaling seems to repress di erentiation programs, thus avoring
stem cell maintenance and proli eration.
Wnt

T e Wnt pathway is implicated in HSC homeostasis and ate
determination. It uses beta-catenin as the critical signal transducer and
is well known to be necessary or the ormation o mesoderm during
embryogenesis.
Serotonin
Serotonin encourages hematopoiesis and prevents apoptosis o
hematopoietic progenitors in vitro.
RANKL and osteoclast activation

T e association o production o RANKL and osteoclast activation
seems to mobilize HSCs in blood circulation and their di erentiation.
(SDF-1)/CXCL12

T is plays an important role in HSC tra cking inside and outside bone
marrow toward destination sites.
Granulocyte colony–stimulating
actor (G-CSF)–induced osteoblast
suppression and bone marrow
CXCL12 downregulation
Leads to a trans er o HSCs into the circulation.
Ca concentration

In uences homing o HSCs into the endosteal niche. A scarcity o HSCs
in close proximity to the endosteal sur ace correlates with de ective
adhesion to extracellular matrix protein collagen type I.
In bone marrow, as well as in the spleen and liver, HSCs can also be ound adjacent to sinusoids, which
points to the perivascular area o sinusoids as another HSC niche. T e connection o HSCs with sinu-
soids could ensure homeostatic blood cell production and prompt responses to hematologic stresses. It
has been suggested that endosteal niches maintain HSC quiescence over the long term, whereas perivas-
cular niches maintain HSCs over shorter periods, supporting ate determination and circulation.
Neural stem cells (NSCs) are cells with the molecular and morphologic eatures o astrocytes that re-
side in the subventricular zone (SVZ) o the lateral ventricle wall and the subgranular zone (SGZ) o
the dentate gyrus o the hippocampus. In addition to the NSCs (type B cells), the neural niche contains
rapidly dividing (transit ampli ying) cell populations, called type C cells in the SVZ and type D cells in
the SGZ, lineage-committed (postmitotic) migratory neuroblasts (type A cells), glial cells, blood vessels,
and vessel-associated cytotypes (pericytes, f broblasts, smooth muscle cells, and macrophages). A large
number o molecules are known to modulate the balance between NSC homeostasis and cell ate de-
termination, such as growth actors (noggin, bone morphogenetic proteins [BMP], epidermal growth
actor, f broblast growth actor, vascular endothelial growth actor, pigment-epithelium–derived actor,
platelet-derived growth actor), and neurotransmitters (catecholamine, serotonin, gamma-aminobutyric
acid [GABA], glutamate, and nitric oxide).8

59
Chapter 3 • he Niche heory or Fat Gra t Survival
Table 3-2 Modulating Factors for Neural Stem Cells
Factor
Role in Niche Stimulation
NO CH and Wnt

It is demonstrated that they are involved in NSC homeostasis.
(SDF-1)/CXCL12

T is has been reported to be involved in NSC tra cking.
Serotonin
T is actor encourages NSC proli eration.
Recently an important role in NSC niche homeostasis has been recognized or a highly organized extra-
vascular basal lamina, which is regularly arranged along the subependymal layer and consists o exten-
sively branched stems terminating in bulbs immediately beneath the ependyma. All the cytotypes com-
posing the NSC niche are in direct contact with one or more branches o this basal lamina, suggesting
direct or indirect involvement o cell-matrix interactions in NSC ate regulation.9
T e precursors o enterocytes and goblet cells are located in apposite niches within the intestinal crypts.
T e niches consist o intestinal stem cells (ISCs), transit-ampli ying ( A) cells, and pericryptal f bro-
blasts, so they are made up o an endodermal derived epithelium surrounded by cells o mesodermal
origin. Cell signaling between these two tissue layers plays a critical role in coordinating patterning and
organogenesis o the gut and its derivatives. T e table below summarizes or this stem cell niche the sig-
naling occurring between stem cells and the environment.
Table 3-3 Factors Involved in the Signaling Between Stem Cells and the Extracellular
Environment
Factor
Role in Niche Stimulation
Wnt

It is the predominant orce controlling cell proli eration,
di erentiation, and apoptosis along the crypt-villus axis.
noggin and BMP

T ese play antagonistic roles in ISC ate determination.
Other organ- or tissue-specif c stem cell niches (hair ollicle, rodent incisor, dental pulp, kidney, liver,
and lung) have been described in recent years. Although the picture is still somewhat con used and
several aspects are controversial, some substantial analogies can be ound between all the various stem
cell niches, despite evident di erences in cytoarchitecture and histologic eatures. In particular, the ol-
lowing elements seem to recur as universal regulators o stem cell homeostasis and ate determination:
(1) canonical Wnt pathway, (2) cell-matrix interaction, ( ) neurotransmitters, (4) interaction with blood
vessels, and ( ) interaction with cells o mesodermal origin.

60
Part I • Principles and Basic Concepts
T e Fat Niche
*
*
*
*
*
*
A
B
C

D
Fig. 3-3
wo distinct types o adipose tissue, white and brown, can be ound in mammals. Each o these exam-
ples (A) contains blood vessels (B) and stromal cells (C) within the stromal septa that surround white
(B and C, asterisks; D, lef panel) or brown (D, right panel) adipocytes.10 T ese tissues have a distinct
anatomic structure and unction; however, both o them contain stromal cells with molecular eatures
and multilineage plasticity analogous to those o bone marrow mesenchymal stem cells. T ese putative
mesenchymal stem cells are thought to be direct precursors o preadipocytes. T e variety o terms used
to describe them re ects the controversy about their histogenetic kinship with bone marrow mesenchy-
mal stem cells.
T e number o viable ADSCs in the stromal vascular raction (SVF) obtained rom lipoaspirates was
estimated on the basis o the colony– orming unit f broblasts (CFU-F) assay. T is procedure consists o
seeding × 4 SVF cells in mm cell culture dishes and counting f broblast-like colonies macroscop-
ically visible a er days o culture. It assumes that any colony has been ormed by single cells that have
been capable o adhering to the plate and proli erating. T e CFU-F is considered a reliable quantitative
estimation o viable ADSCs. Using this assay, it was estimated that cc o human lipoaspirate contained
approximately 4 cells capable o orming f broblast-like colonies, corresponding to .5% to 5% o the
total SVF population.11,12
A large body o literature has demonstrated that the signals that control the embryonic origin o ADSCs
and their di erentiation ate in adult adipose tissue include the same pathways involved in homeostasis

61
Chapter 3 • he Niche heory or Fat Gra t Survival
o other adult and embryonic stem populations, such as Nodal, Wnt, BMP, and f broblast growth actor
(FGF).12 T e complex orchestration o these signals can have di erent e ects, depending on the con-
centration, stage o di erentiation, and extrinsic actors o the niche, such as cell-matrix and cell-cell
interactions, the presence o vasculature, and the level and type o innervation.
Despite extensive research into the intracellular signaling involved in ADSC homeostasis and di eren-
tiation, little is known about the role o cell-cell and cell-matrix interaction in this respect. In a recent
study by Zannettino et al,13 immuno uorescent staining o human adipose tissue sections revealed that
markers o mesenchymal and perivascular cells colocalized to the region adjacent to blood vessels. T is
f nding suggests that ADSCs reside in perivascular niches and prompts the speculation that, as or HSCs,
perivascular structures (cells and extracellular matrix) could provide signals that balance the mainte-
nance o ADSCs in an undi erentiated state with their commitment toward a di erentiative ate. ran
et al1 demonstrated the perivascular origin o adipocytes, conf rming the hypothesis described earlier.
Based on our hypothesis o the vascular origin o adipocytes, clinical applications o ADSCs, particularly
in the context o plastic and reconstructive surgery, can be practiced without eradicating cells rom their
at niche by means o at transplantation.
T he F a t N i ch e i n P l a sti c S ur g ery
Some recent evidence suggests that in ADSC-based therapies, the direct transplantation o ADSCs along
with their niche ( at niche), without their isolation in culture, can allow a more avorable outcome and
can prevent eventual collateral e ects, such as their neoplastic trans ormation.11
FAT NICHE TRANSPLANTATION
T e use o at gra ing or aesthetic purposes was documented or the f rst time in 89 in Germany,
when Neuber3 f lled out depressed scars in a patient’s ace by transplanting autologous at harvested rom
the man’s arm. Since then, ree at gra ing has ound various applications in cranio acial, orthopedic,
breast, cosmetic, and even brain surgery, so that historically, at was the f rst and remains the ideal f ller
or surgical interventions.14-16
Recently, evidence o the dynamic and regenerative nature o at gra s has emerged rom di erent ar-
eas o plastic surgery as well as rom molecular and cell biology. Clinical practice has shown that the
long-term outcome o at gra ing can include rejuvenation o skin texture.15,16 Rigotti et al15 recently re-
ported clinical evidence o a regenerative e ect exerted by ree at transplantation on radiation-induced
mammary ischemia. T eir 0-month-long ollow-up study proved that autologous at transplantation
determined signif cant clinical improvement in patients with progressive secondary lesions ollowing
radiation treatment.
Fat Niche Transplantation: Preinjection Adipose-Derived Stromal Cell Survival
In the “regenerative protocols” used in the pioneering work o Coleman, at was centri uged at 000 rpm
be ore transplantation. T is practice was empirically determined with the aim o reducing the volume o
at and removing as much debris, oil, blood, and water as possible without causing signif cant damage to
the tissues to be transplanted.17
Ultrastructural studies o at a er centri ugation demonstrated damage to mature adipocytes, which
showed interruptions o the cytoplasmic membrane and various degrees o degeneration, ranging up to
cellular necrosis. However, the SVF appeared to be well preserved, with minimal loss.15

62
Part I • Principles and Basic Concepts
Centrifugation
(rpm)
3000
6000
10,000
Collagenas e
diges tion
Volume
reduction
Yield of
SVF cells
CFU-F as say
ADSCs per 10
SVF cells
4
Fig. 3-4
In our study, we compared the e ects o di erent protocols o centri ugation in terms o at volume
reduction, SVF cell number, and the yield o viable and sel -renewing ADSCs rom lipoaspirates or
autologous transplantation.11 Centri ugation at 3000, 000, and 0,000 rpm or 3 minutes reduced the
volume o at harvested to an average 0% or less o the initial volume, with little di erence between the
protocols. However, a er collagenase digestion o lipoaspirate samples, 0,000 rpm–centri uged samples
provided, on average, three ourths as many SVF cells as at samples centri uged at 3000 rpm. Noncentri-
uged samples also provided ewer cells on average than those obtained rom 3000 rpm–centri uged at
samples. From the 000 rpm–centri uged samples we obtained variable results. Ultrastructural analysis
o at aspirates subjected to di erent centri ugation protocols did not reveal evident di erences. Both
3000 and 0,000 rpm–centri uged at aspirates showed adipocytes with clear signs o su ering and oc-
casional ruptures o the plasma membrane. In contrast, the requently present stromal cells appeared
morphologically intact and did not show evidence o cell su ering.
Fat Niche Transplantation: Postinjection Adipose-Derived Stromal Cell Survival
In the previously mentioned study by Rigotti et al,15 ultrastructural comparison o mammary radiole-
sions be ore and at di erent time points a er at gra ing clearly showed ongoing regeneration. A er
treatment, vasculature was “normalized” and cellular debris was progressively removed. T e most in-
teresting f nding pertained to evidence or the ormation o new adipocytes: at di erent time points
a er transplantation, there appeared a large number o precursors at di erent stages o di erentiation
toward the adipocyte phenotype. Be ore transplantation, almost all adipocytes to be transplanted were
seriously damaged by centri ugation; in the recipient radiodamaged tissue, neither mature adipocytes
nor di erentiating preadipocytes were observed. Reasonable interpretations o these f ndings are that ( )
the di erentiating preadipocytes observed a er treatment were adipocyte-committed ADSCs originally
embedded within transplanted at, or ( ) the di erentiating preadipocytes observed a er treatment were
adipocyte-committed (endogenous) ADSCs locally present be ore treatment and activated by the ectopi-
cally transplanted at. T e previously mentioned massive survival o ADSCs (CFU-F) in the transplanted
at, despite the procedures o liposuction and centri ugation, strongly support the f rst hypothesis.
However, the second hypothesis could be equally valid. It assumes that transplanted at, enriched with
ADSCs by centri ugation, could behave as an atypical ectopic niche that orchestrates tissue regeneration
by modulating endogenous tissue resources.

63
Chapter 3 • he Niche heory or Fat Gra t Survival
T e expression atypical ectopic niche was introduced by Martino and Pluchino18 in the context o neural
stem cells or neuroregeneration, but it could be adopted as a common paradigm o stem cell–based
therapies. T e concept is based on the idea o a “bystander” mechanism in which stem cells ectopically
transplanted into a generic site o lesion (such as a radio lesions, myocardial in arction, cerebral stroke,
and so on) do not replace tissue-specif c cells by direct di erentiation, but locally orm an atypical stem
cell niche that suppresses in ammation, promotes neoangiogenesis, and avors the activation o endoge-
nous stem cell precursors through the release o trophic actors, such as cytokines, proangiogenic mole-
cules, and growth actors. T e capability o undi erentiated ADSCs to modulate the immune system and
promote ormation o new vessels has been extensively documented.19,20
Fat Graf s: Niche Replacement
Expansion and differentiation
Lipoaspirate
Autologous transplant
Centrifugation
Stem cell isolation in vitro
Fig. 3-5
T e most pressing question that remains to be answered in the near uture is whether the capability o
ectopically transplanted ADSCs into a lesion site depends on the concomitant transplantation o their
original niche. In other words, it should be clarif ed whether transplantation o either ADSC-enriched
at or ADSCs isolated by culture could provide comparable results in terms o clinical outcome. T ere
are no data that could completely clari y this specif c question. However, in accordance with niche the-
ory, there is currently passionate debate as to whether the de ects or the aberrant plasticity o a tissue
altered by aging, by any kind o insult, or by neoplastic trans ormation are associated with changes o the
niche rather than o intrinsic properties o its stem cell reservoir.21,22 Aging, neoplastic trans ormation,
radiotherapy, and in arction o a given tissue not only induce direct and irreversible alterations o the
stem cells that reside therein, but also change their surrounding niche and reprogram the network o
signals that control the stem cell phenotype.
Autologous at gra s, in which at is moved rom a healthy region o the patient to an insult-damaged
region o the same patient, can ultimately be viewed as the replacement o a sick stem cell niche with
a healthy one and could be expected to be more e ective than transplantation o isolated ADSCs. T e
rejuvenation o hands and aces by at gra s could be seen as the replacement o an aged subcutaneous
at niche with another that is still young. T e liporegeneration o radiolesioned tissue could be regarded
as the replacement o an insult-damaged mammary at pad niche with another healthy one.

64
Part I • Principles and Basic Concepts
T is concept could also be extended to the context o cancer. It is currently accepted that tumors arise
rom trans ormed stem cells. In the light o niche theory, neoplastic trans ormation may be seen as
determined by aberrant signals rom an altered niche.22 In this view, i ectopically transplanted isolated
ADSCs could be recruited into the growing tumor,20 replacement o the tumorigenic niche with an en-
tire healthy niche could be expected to restore the physiologic network o signals and to correctly repro-
gram tissue homeostasis.
FAT GRAFTS: NEWS ABOUT NICHES
More accurate ultrastructural observation per ormed by the Sbarbati research group on human spec-
imens o adipose tissue harvested rom radiotreated, obese, and healthy patients has revealed a new
cellular component o regenerative niches.15 In these studies regenerative units, named niches, appeared
characterized by the presence o mature adipocytes, collagenic sca old and stem cells, but there is a
very interesting new aspect. In these samples o adipose tissue are o en observed some cellular ele-
ments o elongated shape and characterized by the presence o thin cytoplasm extensions and by the
presence o clusters o small lipid droplets.23 T ese elements could be def ned as being o adipose origin
because o the presence o basal membrane. In act, the simultaneous presence o lipid droplets and basal
membrane is su cient to def ne a cell as an adipose element. T ese cellular elements were described
in a study by Orci et al,24 but in subsequent publications we could not f nd urther descriptions o or
investigations regarding these cells, called postadipocytes. In an article by Sbarbati and colleagues,23 the
concept o postadipocytes was better described, using ultrastructural analysis, and a new theory about
the adipose cell cycle was ormulated. In the cycle o adipose cells there is a new phase, the postadipocyte
phase, in which the mature adipose cell loses its lipid charge. T is phase is not the cause o cell death but
represents a quiescent status o the cells. T is status is typical o stress-producing situations. When such
stress actors (interleukins, macrophage recruitment, mechanical or physical stimuli) are decremented,
postadipocytes are able to reactivate their lipid charge, becoming multilocular and f nally unilocular
adipocytes.24 Further analyses are necessary to establish whether the adipocyte observed a er the post-
adipocytic phase is the same as the adipocyte that was stressed and had lost its lipid charge.
C oncl usi on
aken together, the experimental f ndings and their interpretations discussed here shed a new light on
the biology, signif cance, and potential clinical applications o at gra ing. Our comprehension o all the
regeneration pathways a er at gra ing is still hazy, and several crucial aspects must be clarif ed be ore
we can ormulate a solidly demonstrable and unique theory.
Although the use o at gra ing in plastic and reconstructive surgery began more than a century ago,
mounting evidence over recent decades has completely revolutionized the sense o this practice— rom
“f ller gra ” to “gra o stem cells with their niche.” T e clinical potential inherent in this new view o at
gra s has still to be explored.
REFERENCES
. ran KV, Gealekman O, Frontini A, Zingaretti MC, Morroni M, Giordano A, Smorlesi A, Perugini J, De
Matteis R, Sbarbati A, Corvera S, Cinti S. T e vascular endothelium o the adipose tissue gives rise to both
white and brown at cells. Cell Metab : , .
. Galiè M, Rigotti G, Sbarbati A. T e plasticity o at: rom “civilization syndrome” to “therapeutical prom-
ise.” Adipocytes : 9, .
. Neuber F. Fett ransplantation. Chir Kong Verhandl Deutsch Gesellsch Chir : , 89 .
. Niechajev I, Sevćuk O. Long-term results o at transplantation: clinical and histologic studies. Plast Re-
constr Surg 9 : 9 , 99 .

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Chapter 3 • he Niche heory or Fat Gra t Survival
. Billings E Jr, May JW Jr. Historical review and present status o ree at gra autotransplantation in plastic
and reconstructive surgery. Plast Reconstr Surg 8 : 8, 1989.
. Schof eld R. T e relationship between the spleen colony- orming cell and the haemopoietic stem cell.
Blood Cells 4:7, 1978.
7. Calvi LM, Adams GB, Weibrecht KW, et al. Osteoblastic cells regulate the haematopoietic stem cell niche.
Nature 42 :841, 200 .
8. Yang M, Li K, Ng PC, et al. Promoting e ects o serotonin on hematopoiesis: ex vivo expansion o cord
blood CD 4+ stem/progenitor cells, proli eration o bone marrow stromal cells, and antiapoptosis. Stem
Cells 2 :1800, 2007.
9. Conover JC, Notti RQ. T e neural stem cell niche. Cell issue Res 1:211, 2008.
10. Gesta S, seng YH, Kahn CR. Developmental origin o at: tracking obesity to its source. Cell 1 1:242,
2007.
11. Galiè M, Pignatti M, Scambi I, Sbarbati A, Rigotti G. Comparison o di erent centri ugation protocols or
the best yield o adipose-derived stromal cells rom lipoaspirates. Plast Reconstr Surg 122:2 e, 2008.
12. Rigotti G, Marchi A, Sbarbati A. Adipose-derived mesenchymal stem cells: past, present, and uture. Aes-
thetic Plast Surg :271, 2009.
1 . Zannettino AC, Paton S, Arthur A, et al. Multipotential human adipose-derived stromal stem cells exhibit
a perivascular phenotype in vitro and in vivo. J Cell Physiol 214:41 , 2008.
14. Charles-de-Sá L, Gontijo-de-Amorim NF, Maeda akiya C, Borojevic R, Benati D, Bernardi P, Sbarbati A,
Rigotti G. Antiaging treatment o the acial skin by at gra and adipose-derived stem cells. Plast Reconstr
Surg 1 :999, 201 .
1 . Rigotti G, Marchi A, Galiè M, Baroni G, Benati D, Krampera M, Pasini A, Sbarbati A. Clinical treatment
o radiotherapy tissue damage by lipoaspirate transplant: a healing process mediated by adipose-derived
adult stem cells. Plast Reconstr Surg 119:1409; discussion 142 , 2007.
1 . Rigotti G, Charles-de-Sá L, Gontijo-de-Amorim NF, akiya CM, Amable PR, Borojevic R, Benati D, Ber-
nardi P, Sbarbati A. Expanded stem cells, stromal-vascular raction, and platelet-rich plasma enriched
at: comparing results o di erent acial rejuvenation approaches in a clinical trial. Aesthet Surg J :2 1,
201 .
17. Coleman SR. Hand rejuvenation with structural at gra ing. Plast Reconstr Surg 110:17 1, 2002.
18. Martino G, Pluchino S. T e therapeutic potential o neural stem cells. Nat Rev Neurosci 7: 9 , 200 .
19. Krampera M, Glennie S, Dyson J, et al. Bone marrow mesenchymal stem cells inhibit the response o
naive and memory antigen-specif c cells to their cognate peptide. Blood 101: 722, 200 .
20. Galiè M, Konstantinidou G, Peroni D, et al. Mesenchymal stem cells share molecular signature with mes-
enchymal tumor cells and avor early tumor growth in syngeneic mice. Oncogene 27:2 42, 2007.
21. Rando A. Stem cells, ageing and the quest or immortality. Nature 441:1080, 200 .
22. Li L, Neaves WB. Normal stem cells and cancer stem cells: the niche matters. Cancer Res :4 , 200 .
2 . Conti G, Benati D, Bernardi P, Jurga M, Rigotti G, Sbarbati A. T e post-adipocytic phase o the adipose
cell cycle. issue and Cell 4 : 20, 2014.
24. Orci L, Cook WS, Ravazzola M, et al. Rapid trans ormation o white adipocytes into at-oxidizing ma-
chines. Proc Natl Acad Sci U S A 101:20 8, 2004.

(Trang không có chữ)

67
Nomencl at ur e o t he St r omal
Vascul ar Fr act ion
Jacqueline Bliley, Damian Grybowski,
Lauren E. Kokai, Ernest Michael Meyer,
Albert D. Donnenberg, Kacey Gribbin Marra,
J. Peter Rubin
A g b g d g b y d , d
p p y d y p d , d p -d v d (ADSC ),
g d y. b 4-1 d w dw d Ap 201
z d ADSC d p v (SVF) p .
T SVF d ADSC g p p p y . Af
v g, b d ( G I ) d b w
“ ,” w SVF. T SVF b q y b d d py
p p y, d d p d w , d v
d . A g y v y g y SVF
g d , d d z d p d d z
b b d.
I p w w d b p d g SVF, v w
v b d w SVF , d d w SVF d z
y k d .
C h a p t e r 4
67

Part I • Principles and Basic Concepts
68
Table 4-1 op Clinical Indications for Adipose-Derived Stem Cell or Stromal Vascular
Fraction T erapies
Clinical Indication
Number of Trials Being
Performed Worldwide

Trial Location(s)
Osteoarthritis

7

United States (3), China (2),
South Korea (1), France (1)
Fat gra ing or so tissue
reconstruction
3

United States (3)
Acute ischemic stroke

2

Spain (1), China (1)
Kidney disease

2

Iran (1), United States (1)
Wounds, scars, so tissue de ects

2

Canada (1), Denmark (1)
Multiple degenerative or
in ammatory diseases
2

United States (2)
Degenerative disc disease

1

South Korea (1)
FSHD

1

Iran (1)
Liver cirrhosis

1

aiwan (1)
Diabetes mellitus type I

1

Jordan (1)
Ischemic cardiac disease

1

Denmark (1)
Gra versus host disease

1

Spain (1)
Ovarian cancer

1

United States (1)
ALS

1

Spain (1)
Spinal cord injury

1

Spain (1)
Scleroderma

1

France (1)
Pulmonary emphysema
1

Russia (1)
Perianal f stula and Crohn’s disease

1

France (1)
Reducing immunosuppression in
kidney transplantation
1

China (1)
ALS, Amyotrophic lateral sclerosis; FSHD, acioscapulohumeral dystrophy.
S troma V a scu a r F ra cti on a n d A d i p ose- D eri ved
S tem C el
C h a ra cteriza ti on
T e stromal vascular raction, or “raw isolate o adipose tissue,” is a heterogeneous population o cells,
including but not limited to, endothelial cells, macrophages, pericytes, and ADSCs. Despite the wide-
spread use o the SVF in therapeutics, there has been a lack o consensus on their characterization.
o characterize the SVF with ow cytometry be ore analysis, single cells are isolated, and cell clumps
and auto uorescence are removed. DAPI exclusion is used to identi y viable cells (top lef box). CD45
is subsequently used to exclude cells o hematopoietic origin (top right). Endothelial mature cells are
CD31+ CD34−, whereas progenitors are CD31+ CD34+ . T e pericyte proportion is represented by
CD31−CD14 + population, while ADSCs are CD31−CD14 −CD34+ .

Chapter 4 • Nomenclature o the Stromal Vascular Fraction
69
Fig. 4-1
CHARACTERIZATION OF SVF FLOW CYTOMETRY
raditionally, the most common way to distinguish cells, in both the laboratory and the clinic, is by ow
cytometry. Functional assays specif c to the cell type being studied are also used to assess the potency o
a particular cell population. Flow cytometry is a quantitative tool that allows the user to rapidly analyze
cellular populations and determine their viability, as well as the presence o cellular cluster o di erenti-
ation (CD) markers when used in conjunction with uorescent antibodies. Cluster o di erentiation or
CD markers are cell sur ace molecules that can be used to classi y cell populations. Viability should be
greater than 70% to allow or di erentiation o cellular populations.
Multicolorimetric ow cytometry or several sur ace antigens is indicated or particularly heterogeneous
cellular populations, including the SVF. T e use o numerous markers, both positive and negative, is
important because there is not one single marker that is entirely inclusive o all SVF subpopulations.
Common leukocyte antigen (CD 5), eliminates cells o hematopoietic origin, except red blood cells
(RBCs), which can represent 25% to 5% o the SVF depending on a number o actors, including the
donor site, patient characteristics, and isolation protocol used. o exclude red blood cells rom later anal-
ysis lysis with an ammonium chloride solution is occasionally advocated. However, it remains unclear
whether all RBCs are removed with the lysis procedure and how this solution a ects other cells during
the analysis. Instead, glycophorin A (CD235a) can be used as an exclusion marker or RBCs. o make
the analysis clear, additional markers should be used to eliminate other cells o hematopoietic origin,
including macrophages, which can be excluded based on their positivity or CD11b or CD1 . It should

70
Part I • Principles and Basic Concepts
be noted, however, that a large proportion o endothelial progenitors also express these markers. In this
case, a panel o sur ace markers, including stem progenitor markers (CD1 , CD9 , CD , and CD )
as well as endothelial markers (CD 1) should help to distinguish these populations.
Endothelial cells represent approximately 1 % to % o the SVF. o di erentiate endothelial cells and
their progenitors, CD- 1 (also known as PECAM-1 or endothelial cell adhesion marker) should be
used in concert with stem markers, including CD1 or CD . raditionally, CD 1+ CD + cells are
def ned as endothelial progenitors, whereas CD 1+ CD − antigens are associated with mature endo-
thelial cells.2 CD- 1 expression has also been used as criteria or separating cells with greater “stem-
ness” rom the SVF. Boquest et al3 ound that CD −CD + CD1 + CD 1− cells highly expressed
genes related to stemness and were able to di erentiate into bone, at, and neural-like tissue, whereas
CD −CD + CD1 + CD 1+ cells overexpressed endothelium and MHC-class II (HLA-DR) mark-
ers and were unable to proli erate in culture.
CD1 6 can delineate pericytic populations within the SVF; however, there are discrepancies in the liter-
ature on how these cells are additionally characterized. General consensus suggests that pericytes should
not express hematopoietic (CD ) nor endothelial (CD 1) markers, but should express stem progenitor
markers, including CD9 . T ey should be positive or certain stem progenitor markers, including CD9 .
Variable CD expression is also evident.2
Multiple stem-progenitor markers, including CD , CD9 , CD1 , CD11 , CD1 , and CD , can be
used in combination with the above markers to identi y tissue resident progenitors. CD9 and CD1
are also highly expressed on endothelial populations; thus multiple markers are needed to exclude and
separate each subpopulation within the SVF.
One o the most common progenitor markers used to identi y stromal stem cells is CD , which rep-
resents about % o the total SVF population. A high proportion ( % to 8 %) o stem progenitor
markers (including CD and CD1 ) are associated with the CD −CD 1−CD + cell population.4
Boquest et al5 noted that cells with a CD −CD + CD1 + expression prof le were able to pro-
li erate rapidly in culture and di erentiate into chondrogenic, adipogenic, and osteogenic lineages.
Similarly, CD should be used in conjunction with other stem markers to appropriately characterize
stromal progenitors with the SVF.
Table 4-2 Important CD Markers or Def ning the Stromal Vascular Fraction
Antigen

Signif cance
CD1 (Endoglin)

Stem/progenitor marker
CD11 (c-kit)

Stem/progenitor marker
CD1

Stem/progenitor marker
CD1 6 (MCAM)

Pericytic populations
CD a (glycophorin A)

Red blood cells
CD 1 (PECAM-1)

Endothelial cells and their progenitors
CD

Hematopoietic stem cells, stromal stem cells, and endothelial cells
CD (common leukocyte antigen) Cells o hematopoietic origin, except red blood cells
CD (L-VAP )

Stem/progenitor marker
CD9 (T y-1)

Stem/progenitor marker

71
Chapter 4 • Nomenclature o the Stromal Vascular Fraction
F i brob a stoi d C o ony– F ormi n g U
ni t A ssa y
In addition to employing ow cytometry to characterize the SVF, progenitor number can be estimated
using a f broblastoid colony– orming unit assay (CFU-F). T is procedure, which was originally adapted
or use in bone marrow–derived mesenchymal stem cells, allows the investigator to determine the
number o colonies derived rom an original progenitor within the SVF. Following isolation, the SVF
is seeded at a very low density (anywhere between 0 and 00 cells/cm2) and allowed to proli erate or
0 to days.2 Colonies containing 50 cells or greater are then counted. From this in ormation, the per-
centage o stromal progenitor cells within the SVF can be obtained as well as the rate o early population
doubling times. T e literature suggests that the progenitors represent approximately 0.5% to 5% o the
total nucleated SVF cell population.6
A d i p ose- D eri ved S tem C el
C a ssi f i ca ti on
Fig. 4-2
Immuno uorescence staining o adipose tissue shows CD3 + endothelial marker co-localized with al-
pha smooth muscle actin (alpha-SMA) on the blood vessel luminal wall. CD3 staining was visualized in
the supradventitial layer adjacent to the luminal endothelial wall. CD90 and CD3 were similarly located
in the supradventitial layer o vessels.
Characterization o cells can also be per ormed a er SVF culture expansion to obtain a more homo-
geneous ADSC population; however, approximately 5% o the cell populations will not adhere to
the culture plate and will be washed away. ADSCs are mainly categorized by their culture adherence
and dramatic decrease in CD 5+ (hematopoietic) cells. Sur ace markers can also be used to charac-
terize ADSCs. T e International Federation or Adipose T erapeutics and Science (IFA S) along with
the International Society or Cellular T erapeutics (ISC ) specif es that ADSCs should have less than

72
Part I • Principles and Basic Concepts
% contamination rom hematopoietic (CD45+ ) cells, and greater than 90% expression o stromal
stem/progenitor cell markers.7 A low proportion o endothelial cells (CD31+ ) should also be observed
in cultured ADSCs. Furthermore, to distinguish ADSCs rom contaminating bone marrow–derived
stem cells, many investigators have suggested using a combination o CD36 and CD106. ADSCs should
primarily express CD36, whereas bone marrow–derived cells should express CD106.7
When identi ying the multipotent mesenchymal ADSC population, stem/progenitor markers (CD 3,
CD105, CD13, CD90, and CD34) should also be used in conjunction with markers stated above.
However, it should be noted that the proportion o cells expressing these markers can change with cul-
ture conditions and continued passaging. T us analysis o these cells should be per ormed during the
same passage, in the same culture media, and at the therapeutic time-point. T is allows or a more accu-
rate representation o the cells being used clinically.
A d i p ose- D eri ved S tem C el
F uncti ona A ssa ys:
T ri i nea g e D i f f erenti a ti on
ADSCs are characterized by their capacity to di erentiate into adipose, cartilage, and bone. Di erentia-
tion protocols have been published and a number o kits are available rom commercial sources.
Fig. 4-3
ADIPOGENIC GENE EXPRESSION TIMELINE
MATURE ADIPOCYTE GENE EXPRESSION
Lipid droplet development
Day 0: Cells
seeded
at 100%
confluence
Day 1: Add
adipogenic
cocktail
Day 2:
Growth
arrest
Day 3

Day 4

Day 5
CEP-beta
CEP-delta
PPAR-gamma
CEP-alpha
FABP4, Adiponectin
GLUT4, Lipoprotein Lipase
Perilipin, Fatty Acid Transporter
Adipogenic di erentiation is induced by incubating cells in a cocktail o actors that includes insulin,
insulin-like growth actor (IGF), isobutylmethylxanthine (IBMX), indomethacin, or hydrocortisone. A -
ter approximately 3 days, ADSCs will begin accumulating lipid droplets within their cytoplasm, which
can be quantif ed using an Oil Red O stain. Expression o certain adipogenic genes, including lipopro-
tein lipase (LPL), peroxisome proli erator activated receptor-gamma (PPAR-gamma), atty acid binding
protein 4 (FABP4), CCAA /enhancer–binding protein alpha/beta/delta (C/EBP-alpha/beta/delta), will
also be signif cantly elevated a er induction.
Di erent cell types (muscle and liver cells) can store lipids under pathogenic conditions without ac-
tivation o the adipogenic gene expression program; thus it is imperative to analyze both protein and
relevant gene expression parameters.

73
Chapter 4 • Nomenclature o the Stromal Vascular Fraction
A
B
Fig. 4-4
T ese Brightf eld images (20× ) o ADSCs were cultured in A, normal media, compared with B, adipo-
genic media 1 days a er induction. Lipid vacuoles are present within the cytoplasm o di erentiated
ADSCs.
Chondrogenic di erentiation is per ormed through the use o a pellet or micromass culture system al-
lowing or a three-dimensional environment akin to cartilage condensation during embryonic devel-
opment. ADSCs can be maintained within a hydrogel, like alginate, or by maintaining approximately
250,000 cells in a pellet and incubated with trans orming growth actor beta- ( GF beta- ), GF beta-
1, ascorbate 2-phosphate, L-proline, and dexamethasone to induce cartilaginous extracellular matrix
deposition. A er 1 or 2 weeks o culture, ADSCs will begin producing extracellular matrix proteins o
cartilage, including aggrecan, and collagen types II and VI.
Table 4-3 Early and Late Markers o Cartilage Dif erentiation
Mesenchymal
Dif erentiation Subtype Early Marker(s) Late Marker(s)
Chondrogenic

SOX9

Collagen II, collagen IV, aggrecan, sul ated proteoglycans
Fig. 4-5
T ese Alcian blue stain pelleted ADSCs were subjected to chondrogenic di erentiation. Abundant sul-
ated proteoglycans are evident (blue) and suggest the ormation o a mature cartilaginous matrix. Alcian
blue stain can be used to detect sul ated proteoglycans, which are characteristic o a cartilaginous matrix.
Alternatively, ADSCs can be di erentiated into osteoblast-like cells by incubating cells in a mixture o
beta-glycerophosphate, ascorbate, vitamin D , and dexamethasone.

74
Part I • Principles and Basic Concepts
Fig. 4-6
Alizarin red staining di erentiated these ADSCs at day 21. Alizarin red staining detects calcium deposi-
tion ollowing osteogenic di erentiation. A er 2 to weeks in these culture conditions, ADSCs will
start producing a calcium phosphate mineral, which can be quantif ed with Alizarin red or Von Kossa
staining. Di erentiated ADSCs will also express alkaline phosphatase (ALP), osteopontin (OPN), runt-
related transcription actor-2 (RUNX2), bone morphogenetic protein 2 and (BMP-2 and ), and os-
teocalcin (OCN).
Table 4-4 Early and Late Markers o Osteogenic Dif erentiation
Mesenchymal Dif erentiation Subtype Early Marker(s)

Late Marker(s)
Osteogenic

RUNX2, alkaline phosphatase Osteocalcin, collagen I
Using these di erentiation protocols allows investigators to quanti y the therapeutic potential o isolated
ADSCs. T ese parameters can subsequently be correlated to expected clinical outcomes.
S troma V a scu a r F ra cti on a n d A d i p ose- D eri ved S tem
C el
C h a ra cteriza ti on a n d C i ni ca T ra n s a ti on
SVF derived rom di erent human subjects displays variable proportions o stromal progenitors and
subpopulations and this can contribute to the variability observed in the clinical success o SVF thera-
pies. For instance, the number o CD3 + progenitors present within the SVF ranges rom .2% to 36. %
o the total number o nucleated cells.7 Sample processing during isolation and harvesting can also con-
tribute to variability observed in the clinical isolate. Selection or certain sur ace markers or phenotypes
may be help ul in predicting clinical success in ADSC and SVF therapies. However, limited research has
been per ormed to determine what aspects o the SVF and ADSCs are necessary or good outcomes.
T us it is essential or clinicians to appropriately characterize the cells being transplanted and correlate
these actors to clinical outcomes. In the ollowing section, SVF isolation variables and their contribu-
tion to cell unction and sur ace marker variability will be discussed. Finally, studies in which cellular
sur ace markers and unction have been predictive in estimating therapeutic success will be highlighted.

75
Chapter 4 • Nomenclature o the Stromal Vascular Fraction
G enera l I sol a ti on
Several SVF and ADSC isolation methods have been described, but most commonly include a procedure
described by Zuk et al.8 T is technique involves collagenase digestion, centri uging to separate the cell
raction rom mature adipocytes and connective tissue components, and subsequent red blood cell lysis.
Fat
Collagenase
digestion and
centrifugation
RBC lysis,
centrifugation,
tissue culture, plating
ADSCs in culture
Oil layer
Aqueous layer
SVF cell pellet
Fig. 4-7
However, modif cations o the Zuk protocol also exist with di erent collagenase concentrations, diges-
tion times, and centri ugation speeds. It is important to note that these discrepancies can alter the po-
tency o the isolated material; the proportions o cellular subpopulations obtained, and associated clin-
ical outcomes. For instance, di erent concentrations o collagenase (ranging rom . % to . %) as
well as digestion times (ranging rom to hours) can reduce SVF cell viability. Faustini et al9 ound
cell viability was signif cantly increased with a -hour, . % collagenase digestion. However, increased
digestion times (more than hour) decreased the proportion o viable cells isolated.
In addition to collagenase, other proteases have been employed to extract cells rom adipose. Pilgaard
et al10 compared six enzyme blends with equal collagenase activity but variable protease concentration
(Blendzyme , ,3, liberase H , and a crude collagenase mixture). Blendzyme and the crude collagenase
mixture yielded more viable cells per gram o tissue at all time-points. However, Blendzyme and the
crude collagenase mixture produced a higher proportion o colony- orming units. Interestingly, signif -
cantly more progenitors (CD + and CD3 +) were isolated with increasing incubation time suggesting
the diverse location o stem cells throughout adipose tissue.
Despite the disseminated presence o progenitors throughout adipose, investigators have used nonenzy-
matic isolation methods to shorten the operation time and provide a sa er alternative to bacteria-derived
collagenase. T ese procedures usually consist o a simple centri uging step combined with erythrocyte
lysis. Yoshimura et al11 compared these lipoaspirate uid–derived cells (LAF) to the SVF obtained rom
standard collagenase digestion. As expected, less-adherent ADSCs were obtained rom the lipoaspirate
uid. However, culture expansion yielded cells with similar CD markers, osteogenic and adipogenic di -
erentiation capacity when compared with ADSCs derived rom the collagenase digestion.
Rather than directly isolating rom the lipoaspirate uid, investigators have also placed adipose rag-
ments in culture, allowing migration o the stromal stem cells into the culture asks. ADSCs derived
rom this procedure maintain multipotent di erentiation potential, immunomodulatory activity, proli -
eration, and sur ace marker expression when compared with ADSCs derived rom enzymatic digestion.
However, it appears this procedure is unable to produce high cellular yields without lengthy culture
times.
Mechanical disruption o the lipoaspirate has also been per ormed to enhance cell yield rom adipose
tissue. Using vibroassisted liposuction, Baptista et al12 increased cell yields when compared with stan-
dard liposuction, and cultured cells expressed typical mesenchymal stem cell markers. However, enzy-
matic digestion yielded signif cantly more adherent cells.

76
Part I • Principles and Basic Concepts
H a rvesti n g L i p oa sp i ra te f or th e S troma l V a scul a r
F ra cti on
Numerous studies have investigated the e ects o harvesting technique on SVF yield and subsequent
unctionality. Standard liposuction can be used to harvest the lipoaspirate but there is some concern on
how negative aspirating pressures may a ect SVF cell yield. Mojallal et al investigated three harvesting
procedures with di erent aspirating pressures: including hand-held manual aspiration with a 0 cc sy-
ringe (the Coleman technique), traditional pump suction at – 0 mm Hg and – 00 mm Hg, and power
assisted liposuction at – 0 mm Hg and – 00 mm Hg. T e authors ound increased SVF viability with
a – 0 mm Hg aspirating pressure with power-assisted and traditional liposuction yielding more SVF
per milliliter o lipoaspirate.13 Conversely, Aguena et al14 ound no di erence in the viable SVF yield and
the requency o mesenchymal progenitors within the SVF when comparing – 0 mm Hg lipoaspiration
to the hand-held aspiration. However, these authors investigated only live cell yield and did not urther
investigate cellular unction a er being exposed to negative aspirating pressures, which may help in de-
termining the clinical e ectiveness o the isolate.
Schreml et al15 compared SVF yield and di erentiation potential a er standard liposuction without tu-
mescence and resection. T e number o viable SVF cells was signif cantly greater in the liposuction
harvest group when compared with resection. However, human ADSCs derived rom liposuction har-
vest were less able to di erentiate towards osteogenic and chondrogenic lineages. Oedayrajsingh-Varma
et al16 also compared SVF isolation rom at samples that had been obtained via resection, tumescent
liposuction, or ultrasound-assisted liposuction. T e investigators ound no di erence in SVF viability
with harvesting technique. Yet lipoaspirate harvested with ultrasound-assisted liposuction had signif -
cantly decreased numbers o adherent cells in culture when compared with resection. In culture, these
cells also displayed longer population doubling times. Interestingly, no unctional di erences between
proli eration as well as chondrogenic and osteogenic di erentiation potential were observed between
the resection and tumescent liposuction group, suggesting that tumescence does not signif cantly a ect
cellular unction.
With large variations in results and very ew studies per ormed in the literature, it is evident that more
research needs to be conducted to understand the e ect o harvesting technique on SVF viability, unc-
tion, and subsequent clinical outcomes. Furthermore, because harvesting is user and technique depen-
dent, it may be necessary to characterize cells a er each harvest to ensure quality control and reproduc-
ibility.

77
Chapter 4 • Nomenclature o the Stromal Vascular Fraction
P a ti ent D emo g ra ph i c F a ctors ( S ex, A g e, B od y
M
a ss I n d ex) a n d A n a tomi c R e g i on s
It is well known that with increasing age the regenerative potential o various tissues throughout the
body declines. Several studies have noted a decline in SVF yield in aged individuals. Dos-Anjos Vilaboa
et al17 ound a negative correlation between increasing age and SVF concentration and suggested that
this could be the result o a number o di erent actors, including the normal aging process, as well as
hormonal considerations (as all o the subjects were emale in this study). T eir work suggests that larger
SVF doses may be required rom older individuals to obtain a similar therapeutic outcome. However, a
major weakness o their study is that the unction o aged cells was not evaluated.
Schipper et al18 ound no signif cant di erence in apoptosis susceptibility, di erentiation potential, and
proli eration o ADSCs with increasing age. T is suggests that despite declining cellular yields,17 ASC
unction remains intact. T ese results were later conf rmed by Kokai et al19 regarding the impact o aging
on ADSC unction rom the same patients a er aging - years.
Table 4-5 Example Findings on the Impact of Donor Age on SVF and ADSC Properties
Age Consideration Finding

Reference
Female Age
Viable SVF per gram o tissue

Age
Osteogenic potential

Age in mice
Attachment
Proli eration
PCNA
Adipogenesis

Age in mice
No di erence in neuroregenerative actor secretion

Di erent anatomic at depots also vary with respect to their metabolic utilization and immunomod-
ulatory capacity, suggesting that the cells derived rom these locations may also di er. For instance,
Jurgens et al20 noted no di erence in the number o nucleated SVF cells derived rom the abdomen and
thigh region, but did observe a signif cant decrease in the number o adherent ADSCs rom the thigh
region. Other authors have noted anatomic region di erences in males. Faustini et al9 ound that there
was no signif cant di erence in SVF yield based on depot in emales; however, males had signif cantly
higher SVF yield rom the abdominal depot.9 Sachs et al21 urther investigated the unctionality o cells
derived rom di erent depots based on CD marker and pluripotent gene expression (OC , SOX , and
NANOG), as well as di erentiation potential. T e authors ound no di erence in mesenchymal di er-
entiation ability with cells derived rom the abdomen, arms/legs, breast, and buttocks. No di erence in

78
Part I • Principles and Basic Concepts
pluripotent gene expression was observed between di erent depots; however, an increased proportion
o CD positive ADSCs were ound in the abdomen. Similarly, Schipper et al18 ound no di erence in
di erentiation potential o ADSCs derived rom f ve di erent subcutaneous adipose depots, including
upper arm, medial thigh, trochanteric, superf cial and deep abdominal. However, the arm depot did dis-
play a signif cant increase in native PPAR-gamma receptor expression, a requirement or adipogenesis,
and increased lipolytic activity compared with the other depots studied. Compared with the abdomen,
choukalova et al22 ound a greater proportion o early-di erentiated (aP + CD6 –) preadipocytes were
located within the emoral depot. Femoral preadipocytes also displayed less di erentation and higher
resistance to apoptosis when compared with preadipocytes derived rom the abdominal depot.
Table 4-6 Current Research Findings on the Impact of Adipose Anatomic Depots on SVF
and ADSC Properties
Anatomic Depots Studied

Finding

Reference
Abdomen, thigh
Adherent ADSCs derived rom thigh

Back, abdomen, knee, breast, thigh
SVF yield rom abdominal depot in males only
9
Abdomen, arms/legs, breast, and
buttocks
CD + proportion o ADSCs derived rom
abdomen

Upper arm, medial thigh, trochanteric,
superf cial abdominal, deep abdominal
PPAR-gamma receptor expression and lipolytic
unction in the arm depot

Femoral, abdomen
Early di erentiated (aP + CD6 −) preadipocytes in
emoral depot
Adipogenesis and apoptosis in the emoral depot

Mouse epididymal and inguinal at
MHC class II expression in inguinal derived SVF
Ability to di erentiate into bone and cardiomyocytes
in inguinal derived ADSCs

Much o the regenerative a ect o ADSCs and the SVF has been attributed to their ability to secrete para-
crine actors; thus analysis o growth actor secretion is also important to assess potential e cacy. Sowa
et al23 ound no di erence in the ability o ADSCs derived rom separate anatomic depots to secrete
neuroregenerative actors in young and old mice. Baglioni et al24 also observed no signif cant di erence
in the ability o human visceral and subcutaneous ADSCs to secrete proangiogenic and immunomod-
ulatory cytokines. However, heterogeneous CD marker expression within SVF was observed between
these two sources, suggesting that culturing can lead to a more homogeneous stem cell product. In
an e ort to analyze di erences in SVF rather than in the more homogeneous culture–derived ADSCs,
Prunet-Marcassus et al25 isolated SVF rom two distinct at depots within a mouse model: the epididy-
mal and inguinal at pad. T e direct SVF isolate showed increased variability in CD marker expression
with increased MHC class II expression in inguinal derived SVF.
While there have been many studies per ormed on the impact o patient demographics and depots on
SVF and ASC unction, very ew studies have ocused on how these di erences contribute to meaning ul
clinical impacts in vivo. With this in mind, it is di cult to discern what actors (enhanced proli eration,
di erentiation, or proportion o a cellular marker) may contribute to a success ul therapy. For this rea-
son, it is strongly suggested that clinicians using these therapies correlate their clinical outcomes to a
number o predictors, including ow cytometry, di erentiation capacity, proli erative potential, and so
orth.

79
Chapter 4 • Nomenclature o the Stromal Vascular Fraction
C l i ni ca l C orrel a tes: H ow S troma l V a scul a r F ra cti on
C h a ra cteriza ti on M
a y A f ect O utcomes
Stromal vascular raction and adipose stem cell isolations contain many variables that can impact the
cell product. T us it is not surprising that many researchers have sought to determine what impact these
actors have on clinical results and how sur ace marker expression and stem cell unctionality contribute
to outcomes.
Many authors have correlated CD marker expression with stem cell unction. For instance, Philips et al26
noted a wide variation in the number o CD3 + progenitors within the adipose tissue o eight human
subjects. T e researchers also ound a signif cant correlation between the proportion o CD3 + progen-
itors present within adipose samples and resulting at gra retention suggesting CD3 may be a positive
predictor o gra take. Indeed, many authors have investigated the proangiogenic potential o ADSCs
and their ability to migrate and survive within nonvascularized adipose or an extended period o time,
suggesting a possible vascular mechanism or CD3 prediction o retention.
Recent work with single cell genomics has also made it possible or the characterization o highly puri-
f ed stem cell populations. For instance, single-cell genomics analysis has helped researchers understand
underlying pathologies, such as that observed in lymphedema trans ormed adipose tissue ollowing
breast reconstruction. Levi et al27 showed a signif cant decrease in expression o stemness marker KLF ,
a decrease in vasculogenesis, and an increase in adipogenic potential in the stromal vascular raction
derived rom lymphedema associated adipose tissue.
Table 4-7 Example Ef ects o Stem Cell Dif erentiation Capacity in Cell Populations Sorted
or Stem Cell CD Markers
CD Marker Expression

Ef ect
KLF
Adipogenic potential in lymphedema associated
adipose tissue
CD3
Fat gra retention
CD105
Osteogenesis
CD 0
Osteogenesis
CD 0+ CD105
Osteogenesis
Bone morphogenetic protein receptor type
IB (BMPR-IB)
Osteogenesis
Single-cell genomics has also helped researchers isolate purif ed stem cell populations with increased
clinical potential. Levi et al28 isolated a population o CD105-depleted cells (CD105lo) that displayed
enhanced osteogenesis compared with CD105hi and unsorted cells in a mouse calvarial de ect model.
Similarly, Chung et al29 identif ed that selecting or a CD 0+ subpopulation demonstrated enhanced os-
teogenic potential based on gene expression as well as in vivo bone ormation in a mouse calvarial de ect
model. Interestingly, CD 0+ cells outper ormed unsorted cells, as well as the enhanced osteogenesis

80
Part I • Principles and Basic Concepts
seen in the CD1 5lo population that was previously described. McArdle et al30 also identif ed and en-
riched a population o ADSCs expressing the bone morphogenetic protein receptor type IB (BMPR-IB).
T ese cells displayed enhanced osteogenic potential in vitro and enhanced calvarial de ect bone orma-
tion compared with ADSCs that did not express this receptor.
T ese studies providing intriguing data that certain subpopulations within the SVF and ADSCs can be
tailored to clinical indications. However, more research needs to be per ormed to determine what mark-
ers and unctional assays predict clinical success. T us it is necessary or the clinician using SVF and
ADSC therapies to appropriately characterize the cells obtained through the methods described in this
chapter and ultimately, correlate their f ndings to clinical outcomes.
REFERENCES
1. Open end clinical trials on adipose derived stem cells and stromal vascular raction. Available at www.
clinicaltrials.gov.
2. Zimmerlin L, Donnenberg VS, P ei er ME, et al. Stromal vascular progenitors in adult human adipose
tissue. Cytometry Part A 77:22, 2 1 .
. Boquest AC, Shahdad ar A, Fronsdal K, et al. Isolation and transcription prof ling o purif ed uncultured
human stromal stem cells: alteration o gene expression a er in vitro cell culture. Mol Biol Cell 16:11 1,
2 5.
4. Maumus M, Peyraf tte JA, D’Angelo R, et al. Native human adipose stromal cells: localization, morphol-
ogy and phenotype. Int J Obes (Lond) 5:1141, 2 11.
5. Boquest AC, Shahdad ar A, Brinchmann JE, et al. Isolation o stromal stem cells rom human adipose
tissue. Methods Mol Biol 25: 5, 2 6.
6. Peroni D, Scambi I, Pasini A, et al. Stem molecular signature o adipose-derived stromal cells. Exp Cell
Res 14:6 , 2 .
7. Bourin P, Bunnell BA, Casteilla L, et al. Stromal cells rom the adipose tissue-derived stromal vascular
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tional Federation or Adipose T erapeutics and Science (IFA S) and the International Society or Cellular
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. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells rom human adipose tissue: implications or cell-
based therapies. issue Eng 7:211, 2 1.
9. Faustini M, Bucco M, Chlapanidas , et al. Nonexpanded mesenchymal stem cells or regenerative medi-
cine: yield in stromal vascular raction rom adipose tissues. issue Eng Part C Methods 16:1515, 2 1 .
1 . Pilgaard L, Lund P, Rasmussen JG, et al. Comparative analysis o highly def ned proteases or the isolation
o adipose tissue-derived stem cells. Regen Med :7 5, 2 .
11. Yoshimura K, Shigeura , Matsumoto D, et al. Characterization o reshly isolated and cultured cells de-
rived rom the atty and uid portions o liposuction aspirates. J Cell Physiol 2 :64, 2 6.
12. Baptista LS, do Amaral RJ, Carias RB, et al. An alternative method or the isolation o mesenchymal stro-
mal cells derived rom lipoaspirate samples. Cytotherapy 11:7 6, 2 9.
1 . Mojallal A, Auxen ans C, Lequeux C, et al. In uence o negative pressure when harvesting adipose tissue
on cell yield o the stromal-vascular raction. Biomed Mater Eng 1 :19 , 2 .
14. Aguena M, Fanganiello RD, issiani LA, et al. Optimization o parameters or a more e cient use o adi-
pose-derived stem cells in regenerative medicine therapies. Stem Cells Int 2 12: 61 , 2 12.
15. Schreml S, Babilas P, Fruth S, et al. Harvesting human adipose tissue-derived adult stem cells: resection
versus liposuction. Cytotherapy 11:947, 2 9.
16. Oedayrajsingh-Varma MJ, van Ham SM, Knippenberg M, et al. Adipose tissue-derived mesenchymal
stem cell yield and growth characteristics are a ected by the tissue-harvesting procedure. Cytotherapy
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Chapter 4 • Nomenclature o the Stromal Vascular Fraction
7. Dos-Anjos Vilaboa S, Navarro-Palou M, Llull R. Age in uence on stromal vascular raction cell yield
obtained rom human lipoaspirates. Cytotherapy 6: 092, 20 .
. Schipper BM, Marra KG, Zhang W, et al. Regional anatomic and age e ects on cell unction o human
adipose-derived stem cells. Ann Plastic Surg 60:53 , 200 .
9. Kokai LE, raktuev DO, Zhang L, et al. Adipose stem cell unction maintained with age: an intra-subject
study o long-term cryopreserved cells. Aesthet Surg J 37: 5 , 20 7.
20. Jurgens WJ, Oedayrajsingh-Varma MJ, Helder MN, et al. E ect o tissue-harvesting site on yield o stem
cells derived rom adipose tissue: implications or cell-based therapies. Cell issue Res 332: 5, 200 .
2 . Sachs PC, Francis MP, Zhao M, et al. Def ning essential stem cell characteristics in adipose-derived stro-
mal cells extracted rom distinct anatomical sites. Cell issue Res 3 9:505, 20 2.
22. choukalova YD, Koutsari C, Votruba SB, et al. Sex- and depot-dependent di erences in adipogenesis in
normal-weight humans. Obesity (Silver Spring) : 75, 20 0.
23. Sowa Y, Imura , Numajiri , et al. Adipose-derived stem cells produce actors enhancing peripheral
nerve regeneration: in uence o age and anatomic site o origin. Stem Cells Dev 2 : 52, 20 2.
2 . Baglioni S, Francalanci M, Squecco R, et al. Characterization o human adult stem-cell populations iso-
lated rom visceral and subcutaneous adipose tissue. FASEB J 23:3 9 , 2009.
25. Prunet-Marcassus B, Cousin B, Caton D, et al. From heterogeneity to plasticity in adipose tissues: site-spe-
cif c di erences. Exp Cell Res 3 2:727, 2006.
26. Philips BJ, Grahovac L, Valentin JE, et al. Prevalence o endogenous CD3 + adipose stem cells predicts
human at gra retention in a xenogra model. Plast Reconstr Surg 32: 5, 20 3.
27. Levi B, Glotzbach JP, Sorkin M, et al. Molecular analysis and di erentiation capacity o adipose-derived
stem cells rom lymphedema tissue. Plast Reconstr Surg 32:5 0, 20 3.
2 . Levi B, Wan DC, Glotzbach JP, et al. CD 05 protein depletion enhances human adipose-derived stromal
cell osteogenesis through reduction o trans orming growth actor beta ( GF-beta ) signaling. J Biol
Chem 2 6:39 97, 20 .
29. Chung M , Liu C, Hyun JS, et al. CD90 (T y- )-positive selection enhances osteogenic capacity o human
adipose-derived stromal cells. issue Eng Part A 9:9 9, 20 3.
30. McArdle A, Chung M , Paik KJ, et al. Positive selection or bone morphogenetic protein receptor type-IB
promotes di erentiation and specif cation o human adipose-derived stromal cells toward an osteogenic
lineage. issue Eng Part A 20:303 , 20 .

(Trang không có chữ)

83

C h a p t e r 5
O
g g ud h h v d u b h z u h u h v
v d x . I h 2000 w u d h h d d
h h d , ud g u u v .1 T w d
h g f u b , w b u u , d h d u -
g d . S h , h b g z d d ud d h u
du u .
Fig. 5-1
Ad / g u g z d u d v u h . Top:
CD 4+ w d g ; d h g h u v , d
u dv d (SA-ASC, d ) u g u d h v .
Lef : CD 46+ (green) d u u d h v . Right: CD90+ ( d
SA-ASC, red) ju b d h dv , k d b h - h u (αSMA).
Ant iin l ammat or y St em Cel l
Pr incipl es
Mayara N.A. Silva, Vera S. Donnenberg, J. Peter Rubin,
Ludovic Zimmerlin, Albert D. Donnenberg
83

84
Part I • Principles and Basic Concepts
Fig. 5-2
T e perivascular orientation o adipose-resident leukocytes is shown. Immunohistochemical staining
was per ormed on a Cryostat section o whole at. Cells were stained with peroxidase-labeled anti-CD 5
antibody. Antibody binding hematopoietic cells (lymphocytes and macrophages) are revealed by oxida-
tion o , ′-diaminobenzidine (dark brown). Adipocytes (light brown) stain nonspecif cally. T e tissue is
counterstained with hematoxylin and eosin, revealing the vascular cells (blue nuclei).
Associated with the microvasculature are mesenchymal and endothelial progenitor cells and their
lineage-committed transit-ampli ying progeny. Four distinct progenitor populations are organized
around the small vessels o adipose tissue in an anular ashion.1 Luminal endothelial progenitor cells
are CD 5−/CD + /CD + , pericytes (CD 5−/CD −/CD 6+ ) are closely associated with αSMA
cells on the sur ace o the vessels. Supraadventitial adipose stromal cells (CD 5−/CD −/CD 6−/
CD + ), sometimes called preadipocytes, surround the vessels like a sheath. T e ourth population,
detectable by ow cytometry but too rare to be seen by tissue staining, is a highly proli erative CD 6+
CD + intermediate between pericytes, supraadventitial stromal cells (SA-ASC) and endothelial pro-
genitor cells, suggesting the pericyte as a common progenitor.2,3 CD 5+ hematopoietic cells ( cells and
macrophages) are distributed throughout adipose tissue in loose association with the vasculature. T ese
cells have weaker intercellular adhesion and survive collagenase digestion and mechanical disaggrega-
tion ar better than vascular or stromal cells. Immune cells are there ore grossly overrepresented in the
stromal vascular raction (SVF) compared with their prevalence in native adipose tissue.4 Despite their
prevalence in the SVF, the immune components and their relevance to adipose transplant biology have
not been systematically addressed. In this chapter we will present new data on the cell and macrophage
components o the SVF, summarize the immunomodulatory properties o the mesenchymal component,
and discuss the clinical implications o these f ndings.

85
Chapter 5 • Antiin lammatory Stem Cell Principles
Box - Glossary of Immunology and Stem Cell Biology Terms
Adipose-derived stromal cells (ADSC): Stem cells ound in adipose tissue. Many o them can be isolated rom whole or lipoas-
pirated at by f ltration, collagenase digestion, and centri ugation steps to obtain the cellular raction used or regenerative
medicine.
Antiglobulin: An antibody raised against another (usually cross-species) antibody. In immunohistostaining and ow cytometry,
antiglobulins are o en conjugated to a detection molecule such as a uorochrome or enzyme.
APCH7: Allophycocyanin-H7 is a tandem uorochrome used in multicolor ow cytometry. It is o en conjugated directly to
antibodies recognizing cellular proteins.
CD: Cluster o di erentiation re ers to the CD nomenclature adopted by the International Workshop and Con erence on Human
Leukocyte Di erentiation Antigens in 1982. It is still used to classi y the specif city o monoclonal antibodies. More than 330
unique CD clusters have been identif ed.
DAMPs: Damage-associated molecular patterns are host molecules that, when released by damaged cells, can induce an
in ammatory response.
ECD: Energy-coupled dye. A proprietary tandem uorochrome (phycoerythrin coupled to exas Red) used in ow cytometry.
Endothelial progenitor cell (EPC): A cell capable o di erentiating into mature endothelial cells that line the blood vessels.
Fc-gamma receptor: A receptor or the tail region o immunoglobulin G (called the Fc or fraction crystallizable). Fc receptors
are used by macrophages and other cell types to arm themselves with antibodies, con erring the specif city o the bound
antibodies.
Fluorochrome: A uorescent dye used to label biological materials. Fluorochromes, either conjugated to monoclonal antibodies
or antiglobulins, are used in ow cytometry and immuno uorescent staining to detect cellular proteins and other constituents.
HLADR: Human leukocyte antigen−antigen D related was identif ed as a major target o allogra rejection. Its unction is to
present peptide antigens to CD4+ helper cells.
Mesenchymal stromal cells (MSCs): MSCs are multipotent stromal cells that can di erentiate into a variety o cell types
including adipocytes, osteoblasts, chondrocytes and myocytes.
MHC class I: Major histocompatibility complex class I molecules are expressed on the sur ace o almost all somatic cells. T eir
unction is to present peptide antigens to CD8+ cytotoxic cells.
MHC class II: T e class o major histocompatibility molecules to which HLADR belongs.
PAMPs: Pathogen-associated molecular patterns are molecules associated with groups o pathogens that are recognized by cells
o the innate immune system.
PECY5: Phycoerythrin-cyanine 5 is a tandem uorochrome used in multicolor ow cytometry.
PECY5.5: Phycoerythrin-cyanine 5.5 is a tandem uorochrome used in multicolor ow cytometry.
Pericyte: Pericytes are cells that wrap around the endothelial cells o capillaries and venules throughout the body. T ey are
recognized as multipotential progenitor cells.
PGE2: Prostaglandin E2 is a member o the prostaglandin amily. It is involved in resolution o in ammation and suppression o
cell signaling.
Stromal vascular fraction (SVF): A heterogeneous cellular raction resulting rom the disaggregation o adipose tissue into a
single cell suspension.
Supraadventitial adipose stromal cells: CD45−/CD31−/CD34+ cells that orm a ring above the adventitia o small blood
vessels. T ese cells are also known as preadipocytes because they are committed to di erentiate into adipocytes.
T 1: -helper 1 cells are a subset o CD4+ cells triggered by IL-12, IL-2. When stimulated the major cytokine that they secrete
is inter eron-gamma. T ey promote e ector responses by macrophages, CD8 cells and IgG B cells.
T 2: -helper 2 cells are a subset o CD4+ cells triggered by IL-4. When stimulated the major cytokines that they secrete are
IL-4, IL-5, IL-9, IL-10 and IL-13. T ey promote e ector responses by eosinophils, basophils, and mast cells.
T 17: -helper 17 cells are a subset o CD4+ cells distinct rom T 1 and T 2 cells. When stimulated, the major cytokines
secrete a host o cytokines, including interleukin 17 (IL-17). T 17 cells are localized primarily in the skin and mucosa.
olerogenic: Inducing immunologic tolerance, an unresponsive state to oreign antigens, particularly transplantation antigens.
oll-like receptors ( LRs): LRs are a class o receptor proteins that play a key role in the innate immune system. LR are
expressed in sentinel cells such as macrophages and dendritic cells that recognize structurally conserved molecules derived rom
microbes (PAMPs) or damaged host tissues (DAMPs).
ransit-amplifying progenitor cell: ransit-ampli ying cells are a class o progenitor cells that arise rom stem cells, proli erate a
f nite number o times, and then di erentiate.

86
Part I • Principles and Basic Concepts
M
a teri a l a n d M
eth od s
IMMUNOHISTOSTAINING
Our methods or immunostaining have been previously published.5 Brie y, we roze small pieces o at
in liquid nitrogen. Sections were then cut on a Cryostat and slides were prepared. For immuno uores-
cence, sections were stained with antibodies specif c or CD34, CD90, CD14 and αSMA. Nuclei were
visualized by staining with the uorescent DNA intercalator DAPI. For immunohistochemical staining,
sections were stained with hematoxylin and eosin and then counterstained with antibodies to CD4
(recognizing hematopoietic cells). Cells positive or CD4 were detected by staining with an antiglobulin
conjugated with the enzyme horseradish peroxidase, ollowed by incubation with an enzymatic substrate
that stains cells brown only when they have peroxidase on their sur ace.
FLOW CYTOMETRY
C
D
1
4
A
P
C
H
7
CD45 PECy5.5
A: 5.9%
CD3 PECy5
[B]
S
S
C
-
A
C: 18.7%
[B]
CD19 ECD
S
S
C
-
A
D: 0.9%
Clean events
Fig. 5-3
Detection o macrophages, T cells, and B cells is demonstrated in disaggregated adipose tissue. Lef to
right: CD4 + /CD14+ events (macrophages, A gate: pink), and CD4 + /CD14− events (B gate: green)
are identif ed among “clean events.” Here clean events are def ned as singlet cells with 2N to 4N DNA
content, as measured by staining with DAPI (4′, -diamidine-2′-phenylindole dihydrochloride, not
shown). T cells (center) are identif ed as CD4 + /CD14−/CD3+ events (C gate: turquoise) with low side
scatter area (SSC-A). B cells (right) are identif ed as CD4 + /CD14−/CD19+ (D gate: magenta) events
with low side scatter area (SSC-A). Percentages are percent o nucleated cells (lef panel), or percent o
CD4 bright cells (center and right panels).

87
Chapter 5 • Antiin lammatory Stem Cell Principles
CD90 APC

CD105 FITC
97.8 ± 0.7%
93.3 ± 3.7%
CD90 APC

CD105 FITC
60.5 ± 10.2%
29.6 ± 6.9%
CD90 APC

CD105 FITC
95.5 ± 1.2%
94.4 ± 3.2%
CD90 APC

CD105 FITC
71.9 ± 7.4%
53.1 ± 7.6%
ENDOTHELIAL PROGENITOR
SA-ASC PREADIPOCYTE
PERICYTE
TRANSIT AMPLIFYING
C
D
7
3

P
E
Fig. 5-4

T e expression o mesenchymal stem cell markers on our stem/progenitor populations o the adipose
stromal vascular raction are illustrated. Coexpression o mesenchymal-associated markers CD90,
CD 3, and CD10 are shown or the our stem/progenitor populations that were identif ed as previously
reported. T e percentages shown within the histogram regions display the mean ± standard error or
eight independent samples studied.

88
Part I • Principles and Basic Concepts
Flow cytometry is a power ul single-cell technique by which a heterogeneous mixture o cells, such as
SVF, is stained with multiple antibodies (each conjugated to a di erent uorescent dye). T e antibod-
ies are usually directed against specif c proteins o interest, many o which are identif ed by their “CD”
number. For example, a protein that is expressed only on the sur ace o lymphocytes is designated
CD3. T e cytometer interrogates cells one at a time and or each, measures the uorescence intensity o
each dye. In this way, quantitative measurements o the expression o multiple proteins are made on each
individual cell. Although the data are multidimensional, the results ( uorescence associated with a given
protein) are usually plotted in two-dimensional histograms.
Single-cell suspensions were prepared rom whole at tissue and lipoaspirates and stained as previously
described.5,6 Cells were f rst simultaneously prestained in bulk with a backbone panel containing an-
tibodies to CD4 -PECy . (antibody-dye), CD3-PECy , CD1 -ECD and CD14-APCH . Populations
o interest or this report were identif ed by sur ace expression o CD4 + /CD3+ ( cells), CD4 + /
CD1 + (B cells) and CD4 + /CD14+ (tissue macrophages). Bulk-stained cells were then added to the
BD FACSCAP Lyoplate system (BD Biosciences, Mat. No. BP 03 4), a single -well plate containing
uorochrome-conjugated antibodies (three per well) covering 242 di erent cell-sur ace proteins. In this
way we were able to determine the degree to which each protein was expressed on cells, B cells, and tis-
sue macrophages. Four independent samples were analyzed. Summarized results are given as arithmetic
mean ± SEM to indicate the precision with which mean values are known, or as mean ± SD to indicate
intersample heterogeneity.7
R esul t s
A detailed description o the hematopoietic components o the SVF has not been previously reported.
o address this, we used the FACS CAP Lyoplate system to survey the cell-sur ace proteome o adipose
resident cells, B cells, and macrophages in SVF rom the abdominal at o our healthy subjects. CD3+
cells represented . ± 3. % (mean SD) o all nucleated single cells. CD14+ macrophages ( . ±
. %) and CD1 + B cells (0.2 ± 0.1%) compose the remainder o immune cells in the SVF. T e high
ratios o cells to B cells (30 to 1) and macrophages to B cells (32 to 1) are ar di erent than expected
in normal peripheral blood (approximately :1 and 2:1, respectively), suggesting that the majority o
cells and macrophages detected are o tissue origin, and the contribution o contaminating peripheral
blood is minimal.
ADIPOSE-RESIDENT T CELLS
Adipose tissue cells and macrophage subsets are highly selected compared with those in the peripheral
circulation. Adipose cells display a unique phenotype partially resembling skin cells,8 especially with
respect to expression o CD1 2, a leukocyte adhesion molecule also known as cutaneous lymphocyte-
associated antigen. Like skin cells, the majority ( 2.4 ± 1 .3%) are CD4+ , or helper cells, the type that
directly interact with MHC-class II proteins (HLA-DR) and peptide antigens presented by macrophages
and dendritic cells to initiate immune e ector responses. Only .1 ± 4.0% o the cells are CD + . T e
combination o markers usually associated with “experience” and unction (naïve, memory, e ector) are
present in a unique combination not observed in peripheral or lymph node cells, with the majority
o cells expressing the RB iso orm o CD4 (conventionally a naïve cell marker), but in the absence o
CD 2L (def nitively present on naïve cells). High expression o adhesion molecules (CD44, CD11a,
CD1 ) and markers associated with motility (CD1 2, CD1 1) is consistent with the act that these
cells have extravasated into the adipose tissue, where they most likely participate in immune surveil-
lance, interacting with tissue macrophages and conditioning their polarization and unctionality in the
process. able -1 gives a more complete phenotypic description o adipose resident cells, with the ca-
veat that expression o single markers was determined on CD4 + /CD3+ cells. T is precludes def nitive
identif cation o subsets def ned by the expression pattern o two or more markers. T e overall prof le
suggests tissue resident antigen-experienced-memory or e ector-memory cells.9

89
Chapter 5 • Antiin lammatory Stem Cell Principles
Table 5-1 Phenotypic Marker Expression on CD45+ /CD3+ Adipose-Resident T Cells
Reagent
Percent Positive
Function
Mean

SD
Subset

CD
CD
In erred CD4−/
CD8−
.
.
.
.
.
Recognizes antigen in context o MHC Class II
Recognizes antigen in context o MHC Class I
Innate immune response
Naïve/memory
CD RA
CD RB
CD RO
CD
CD L
CD
CD
CCR
CLA
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
.
Protein tyrosine phosphatase, RA iso orm
Protein tyrosine phosphatase, RB iso orm
Protein tyrosine phosphatase, RO iso orm
Hyaluronic acid receptor
Lymphocyte-endothelial cell interactions
Dipeptidyl peptidase-4, growth actor cleavage
Immune checkpoint, signal transduction
Skin tropism
Skin tropism
Activation

CD a
CD
CD
HLADR
.
.
.
.
.
.
.
.
Cellular adhesion, costimulatory signaling
IL2 receptor
Early lymphocyte activation
Peptide presentation to CD4 cells
ADIPOSE-RESIDENT MACROPHAGES
T e great majority o adipose macrophages ( . % ± . %) express HLA-DR, the histocompatibility
marker essential or presenting peptide antigens to CD + cells. T ey are mixed concerning M (proin-
ammatory, tumor resistant, microbicidal) and M (antiin ammatory, matrix deposition, tissue remodel-
ing) polarization markers. A sizable proportion o adipose macrophages express the cell costimulatory
molecule CD and/or the Fc-gamma receptor CD , characteristic M macrophages, whereas an ap-
proximately equal proportion express CD , CD , and/or CD , receptors associated with M macro-
phages. About a third express the toll-like receptor LR- , allowing them to respond to danger signals in
the environment. Recent thinking concerning macrophage polarization emphasizes their ability to change
their phenotype and unction in response to cell signaling and the extracellular cytokine milieu.10 able -
gives a more complete phenotypic description o adipose-resident macrophages, again with the caveat that
they were measured one marker at a time on CD + /CD + cells.
Interestingly, the nuclear receptor PPAR-gamma plays an essential role in the regulation o both adipose
tissue and tissue macrophages. In adipose tissue, PPAR-gamma activation in perivascular mesenchymal
stromal cells (by endogenous ligands) coordinates the expression o the genes responsible or adipose
dif erentiation.11 In tissue macrophages, PPAR-gamma activation occurs as part o a two-way interaction
with resident T cells, which secrete the cytokines IL- and IL- . PPAR-gamma activation primes
tissue macrophages or M polarization, inducing CD expression in the process.12 CD , a scavenger
receptor also known as atty acid translocase, is involved in atty acid uptake,13 and is highly expressed by
adipose-resident macrophages (see able - ).
T e complexity o the adipocyte-macrophage- cell axis is suggested by the observation that obesity is
an in ammatory condition characterized by an accumulation o tissue macrophages.14 Not only does the
number o macrophages decrease, but also their polarization shi s rom M to M when the adipose
burden is reduced by gastric bypass.15 T us it appears that in obesity, adipose is proin ammatory. T is
apparent conundrum is explained by the recently proposed paradigm that, like macrophages and cells,
MSCs (and presumably ADSCs) are susceptible to polarization and may promote or inhibit in amma-
tion.16 In addition to tissue remodeling and lipid metabolism,17 M -polarized macrophages have also

90
Part I • Principles and Basic Concepts
been shown to play a role in promoting angiogenesis.18 All o these processes are highly active in adipose
tissue, which as a readily expandable and contractible source o stored energy must continually remodel
and revascularize.
Table 5-2 Phenotypic Marker Expression on CD45+ /CD14+ Adipose-Resident
Macrophages
Reagent
Percent Positive
Function
Mean

SD
HLADR

.

.
Peptide presentation to CD cells
CD 63
.

.5 Hemoglobin-haptoglobin receptor (M )
CD 6

36.6

3.7 Mannose receptor (M )
CD36
6 .7

.
Fatty acid endocytosis
CD 6
3 .

6.
B7- (costimulation)
CD6
.
.3
FcγR (antibody-mediated phagocytosis)
CD

3 .

.
LR2 (receptor or pathogen- and damage-associated molecular pattern
molecules, PAMPs and DAMPs)
aken together, adipose-resident cells and macrophages probably play a role not only in immune
surveillance, but also in regulating a milieu conducive to tissue remodeling. When concentrated in the
SVF in cell-augmented lipotrans er products, they may positively contribute to the autologous adipose
gra product by promoting immune surveillance a er surgical intervention, acilitating wound healing,
angiogenesis and gra stability, and countering scar ormation.
THE MESENCHYMAL NATURE OF ADIPOSE STROMAL CELLS
T e stem/progenitor populations resident in adipose tissue are now recognized as belonging to the class
o adult tissue stem cells known as mesenchymal stromal cells.4 Mesenchymal stromal cells (MSCs) are
multipotent cells that can di erentiate into a variety o cell types, including osteoblasts, chondrocytes,
myocytes and adipocytes, depending on environmental cues. Originally described as a very minor com-
ponent o the bone marrow that could be readily expanded in culture,19 MSC can be identif ed in asso-
ciation with the vasculature o virtually every organ,20 and are prevalent in highly vascularized adipose
tissue.1 Immuno uorescent staining has been a key technology or identi ying MSCs in adipose tissue;
von Willebrand actor (vWF) and αSMA serve as landmarks, the ormer marking vascular endothelial
cells and the latter marking the outer layer o the vessel. Pericytes are CD 6+ /vWF negative. CD is
coexpressed with vWF on capillary endothelial cells, but is absent in the luminal cells o larger vessels.
T e mesenchymal marker CD73 is present on the pericytic ring o small vessels, where it is coexpressed
with CD 6. CD and CD73 are both expressed in the supraadventitial ring surrounding capillaries
and small vessels.4
Such histologic landmarks acilitate the interpretation o ow cytometric evaluation o the SVF. T e
advantage o ow cytometry is that it allows multiple markers to be simultaneously measured on sin-
gle cells, painting a more complete phenotypic picture o the SVF and allowing it to be interpreted
in the context o the intact adipose tissue rom which it was prepared. In a series which we reported
previously,4 the composition o the nonhematopoietic component o the SVF consisted o endothelial

91
Chapter 5 • Antiin lammatory Stem Cell Principles
progenitors ( . ± . %, mean ± standard error), pericytes ( .0 ± . %), CD + /CD + transit
ampli ying cells (0. ± 0. ) and supraadventitial stromal cells (SA-ASC, preadipocytes, .0 ± 0.0%).
T e expression o mesenchymal markers CD , CD 0, and CD 0 on each o the our stem/progenitor
populations is illustrated in Fig. - . Percentages shown within histogram regions display the mean ±
standard error or the eight patients studied. Described rom luminal sur ace outward, mature endothe-
lial cells (CD + /CD −) did not signif cantly express mesenchymal markers (not shown), whereas
about hal o adipose endothelial progenitor cells ( . ± . %) coexpressed CD and CD 0 , and
. ± . % expressed CD 0. Pericytes were the least mesenchymal o the our subpopulations ( . ±
. % CD + /CD 0 + , 0. ± 0. % CD 0+ ). However, the CD + subset o CD + pericytes, hy-
pothesized to be transit ampli ying progenitor cells leading to the generation o SA-ASC and endothelial
progenitors rom pericytes, were uni ormly mesenchymal ( . ± . % CD + /CD 0 + , . ± 0. %
CD 0+ ). T e most prevalent and most adipose-committed subpopulation, SA-ASC (or preadipocytes),
were also highly mesenchymal ( . ± . % CD + /CD 0 + , . ± . % CD 0+ ). T e perivascular
orientation o mesenchymal cells is not unique to adipose tissue and has been observed in other tissues
with rich microvascular beds,21 including the dermis.22
IMMUNOMODULATORY EFFECTS OF CULTURED
ADIPOSE-DERIVED STROMAL CELLS
Mesenchymal stromal cells are def ned by the International Society o Cellular T erapy by their ( ) abil-
ity to adhere to plastic, rom which they can be expanded in culture in serum containing medium, ( )
cell sur ace immunophenotype (positive or CD , CD , CD 0, CD 0 and HLA-Class I, and negative
or CD , CD b, CD , CD , CD ), and ( ) ability to di erentiate into mesenchymal-derived tissue
(adipogenesis, chondrogenesis and osteogenesis).23 Phenotypically, native preadipocytes, also termed su-
praadventitial adipose stromal cells, diverge slightly rom this pattern, expressing CD , a marker absent
on both cultured bone marrow−derived MSCs and adipose-derived stromal cells (ADSCs).4 In addition
to their properties as multipotent progenitor cells, MSC exert potent immunomodulatory e ects on
their environment. Un ortunately, almost all available in ormation concerning the immunomodulatory
nature o MSC, both in vitro and in vivo, has been obtained using culture-expanded cells, or in the world
o adipose biology, cultured ADSCs. It is understandable that virtually no in ormation is available con-
cerning the immunomodulatory properties o native (unpassaged) bone marrow−derived MSC, since
they are exceedingly rare, constituting about 0.0 % o nucleated bone marrow cells.24 However, def ning
the immunologic properties o MSC-like cells within unpassaged adipose SVF is not methodologically
insurmountable, given their prevalence and our ability to sort-puri y SVF subpopulations.25 T at it has
not been done to date represents a signif cant gap in our understanding o adipose biology, especially
since these cells are a native component o therapeutic adipose-derived cellular products and are inten-
tionally augmented in progenitor-enriched adipose tissue products.26
Nevertheless, the immunomodulatory properties o cultured MSCs and ADSCs are o signif cance, not
only to model the e ects o their native counterparts in vivo, but also because they are currently being
evaluated or their utility as cellular therapeutics. T e immunomodulatory e ects o MSC have been
primarily characterized as immunosuppressive, antiin ammatory and tolerogenic. cell proli eration
stimulated by mitogens, recall antigens and alloantigens is reduced by coculture with MSCs or their con-
ditioned culture media. Both in vitro and in vivo experiments have demonstrated that ADSCs increase
number and action o regulatory cells.27 Whether MSC also modulate the unction o B cells and
natural killer cells is controversial.27 Like MSC, ADSCs stimulated by inter eron-gamma, or conditioned
medium rom these cells can reduce proli eration o peripheral blood lymphocytes. T is e ect is accom-
panied by reduced secretion o proin ammatory cytokines.28 ADSCs are not constitutively immunosup-
pressive. T ey require activation by in ammatory mediators such as inter eron-gamma, NF-alpha, and
IL- -beta. Recently, in keeping with the concept o MSC polarization, it was suggested that ADSCs too
can be driven to either a proin ammatory or an antiin ammatory phenotype, depending on how they
are stimulated.29

92
Part I • Principles and Basic Concepts
Fig. 5-5
1
5
3
2
3
4
4
DAMPs
PAMPs
IL-10
TNF-α
IL-6
IL-1
sIL-1r
PGE2
Preadipocyte
MΦ
T cell
T e in ammatory/antiin ammatory macrophage, cell, and mesenchymal axis in the adipose tissue are
shown. ( ) Macrophages are activated by damage-associated molecular patterns (DAMPs) and pathogen-
associated molecular patterns (PAMPs), ( ) activated macrophages (M-phi) secrete proin lamma-
tory cytokines, ( ) mesenchymal supraadventitial adipose stromal cells (preadipocytes) are activated and
cells are polarized (T ) by proin ammatory cytokines, and (4) activated preadipocytes secrete antiin-
ammatory PGE and the decoy receptor sIL- , resulting in the release o the potent antiin ammatory
cytokine IL- , inhibition o macrophage-secreted proin ammatory cytokines, and reconditioning o
the cell milieu. T e dashed lines indicate secretion; solid lines indicate the e ect o a soluble mediator;
red lines indicate inhibition. T is illustration is greatly simplif ed and shows only some o the known
mediators that regulate expansion and contraction o adipose tissue, wound healing, and antimicrobial
responses. Recent evidence indicates that toll-like receptor ( LR)−positive MSCs can also be stimulated
by PAMPs and DAMPs, driving them to a proin ammatory state analogous to the macrophage M state.
In vivo animal models o autoimmunity have been used to evaluate the antiin ammatory properties o
human MSCs, and improvement in symptoms has been noted a er system administration in models
o acute lung in ammation,30 in ammatory bowel disease, multiple sclerosis, diabetes, myocardial in-
arction, cerebrovascular disease, in ammatory lung disease, and gra versus host disease (GVHD).27 In
vitro and in vivo, cultured MSCs appear to counterbalance the e ects o proin ammatory macrophages
that have been activated by sterile tissue damage or in ectious agents (Fig. 5-5). T e same macrophage-
produced cytokines (IL- -alpha, IL- -beta, NF-alpha) which initiate the in ammatory cascade also ac-
tivate MSC, which through short-range acting mediators (IL- r-alpha, prostaglandin E [PGE ], and
NF-alpha−stimulated gene/protein 6 [ SG-6]) in uence local macrophage polarization as well as
dampen the downstream e ects o proin ammatory cytokines.31 In particular, PGE secreted by activated
MSCs induces tissue macrophages to secrete the potent antiin ammatory cytokine IL- , which in turn
inhibits macrophage release o proin ammatory cytokines NF-alpha and IL-6.32 IL- also exerts direct
inhibitory e ects on CD4 cell activation through inhibition o IL- production.33 MSCs, and particu-

93
Chapter 5 • Antiin lammatory Stem Cell Principles
larly their adipose-derived counterparts, can also directly inhibit di erentiation o dendritic cells,34 down-
modulating cell costimulatory molecules and inhibiting T polarization.35
Inhibition o macrophage-derived NF-alpha and IL- also a ects cell polarization, because NF-
alpha is a potent promoter o cytotoxic cell activity,36 and because IL- is a central inducer o the
proin ammatory IL- , IL- , H- axis.37 MSCs can also directly blunt cell responses by produc-
tion o indoleamine-pyrrole , -dioxygenase (IDO),38 an enzyme that degrades the essential amino acid
tryptophan, a requirement or cell activation and proli eration. ADSCs and bone marrow−derived
MSCs appear to exert similar immunomodulatory e ects39; thus it is reasonable to hypothesize that the
proin ammatory cascade initiated in a surgical wound by macrophages, through interaction o DAMPs
with their toll-like receptors, could be signif cantly modif ed by increasing the autologous MSC content
o an adipose gra product as is currently done in progenitor-enriched tissue products. Since immuno-
modulatory e ects are mediated by activated but not resting ADSCs,28 it ollows that their therapeutic
e ect could potentially be enhanced by preconditioning the SVF cells by short exposure to inter eron-
gamma,28,40 NF-alpha, and/or IL- be ore adding them to the adipose gra .
THE CONUNDRUM OF INTRAVENOUSLY ADMINISTERED
MESENCHYMAL STROMAL CELLS
T e migration and biodistribution o systemically administered MSCs is a major concern, because it
relates directly to sa ety and clinical outcomes. Un ortunately, neither experimental models nor clinical
studies have provided def nitive results. For clinical studies per ormed to date, intravenous adminis-
tration has been the avored route o MSC delivery, because it is convenient, minimally invasive, and
accommodates a broad range o cell doses. Antiin ammatory e cacy, as demonstrated in numerous
animal studies and clinical trials,29 provides indirect support or the notion that injected MSCs circulate
and reach the site o in ammation. However, direct experimental evidence suggests that this process is
very ine cient, i it occurs at all. Cell tracking studies in a murine model o liver ischemia-reper usion
injury revealed that intravenously in used MSCs were trapped by the lungs, where they persisted as
viable cells or only hours, never migrating to the site o injury.41 In contrast, a murine cutaneous
burn injury model revealed intravenously administered MSCs at the margins o the burn site rom day
a er in usion through day .42 Identi ying which actors are crucial to MSC survival and homing is o
critical importance to ensure that intravenously administered therapeutic cells will reach their intended
target organs. It is still unclear why the e ects o in used MSCs on in ammatory mechanisms last longer
than their presence. T e only cogent hypothesis to explain the e cacy o intravenously administered
MSCs, albeit unsupported by experimental evidence, is that they initiate immunomodulatory e ects
that are propagated by resident cells such as macrophages, cells, and stromal cells. It should be noted
that lung trapping, tra cking, and homing concerns do not apply when adipose SVF is admixed directly
with a lipotrans er preparation. T ey can also be signif cantly mitigated when MSCs are injected or
applied directly into an a ected tissue, especially i the therapeutic cells are immobilized in a biocompat-
ible matrix such as f brin.43
AUTOLOGOUS VERSUS ALLOGENEIC CELLULAR PRODUCTS
Autologous therapies are usually assumed to be superior to allogeneic materials because they do not
elicit a gra rejection response. However, harvesting and expanding autologous cells or each individual
patient is ar more labor and resource intensive than creating a well-characterized cell bank to be used
or allogeneic MSC therapy. Furthermore, the therapeutic potential o autologous MSCs harvested rom
individuals with chronic in ammatory or autoimmune diseases44,45 or aged individuals is unknown. On
the other hand, allogeneic MSCs are reported to stimulate no or minimal innate and adaptive immune
responses. T is may be explained by low MHC class I antigen expression and the absence o MHC class
II and costimulatory molecules CD , CD , and CD . A er activation, MSCs increase the expression
o MHC class I and express MHC class II, but they do not elicit alloreactive cell responses in vitro.46
Despite these e ects, re erring to MSCs as immunoprivileged exaggerates the case. Evidence in animal
models points to the act that an attenuated but nevertheless e ective immune response to allogeneic
MSCs may be responsible or the observed rapid clearance, but again, persistence does not appear to be a

94
Part I • Principles and Basic Concepts
prerequisite to a therapeutic e ect. ADSCs di er somewhat, since they constitutively express high levels
o MCH class I, and in some cases express antigen-presenting cell-associated molecules CD , CD58, and
CD 0.47
CURRENT CLINICAL APPLICATIONS OF NATIVE AUTOLOGOUS
STROMAL VASCULAR FRACTION AND CULTURE-EXPANDED
ALLOGENEIC MESENCHYMAL STROMAL CELLS
T erapeutic options or in ammatory and autoimmune diseases have incorporated ADSCs because o
their proangiogenic, antif brotic, and antiapoptotic e ects. Randomized control trials are by ar the best-
suited designs to address therapeutic e cacy; 532 clinical trials related to mesenchymal stem cells have
been registered to date on clinicaltrials.gov. T ese trials are in various stages o completion (recruiting,
active, completed). T e indications or therapy are enumerated in able 5-3. Narrowing the search to
adipose derived stem cells, there were 1 3 randomized controlled trials, most o which are recruiting or
active, but without published results.
Table 5-3 Current Randomized Controlled rials of Mesenchymal Stromal Cell T erapy
Category

Indications
Cardiovascular Dilated cardiomyopathy
Chronic ischemic myocardiopathy
Myocardial in arction
Chronic limb ischemia
Respiratory

Acute respiratory distress syndrome
Bronchopulmonary dysplasia
Pulmonary f brosis
Pulmonary emphysema
Chronic obstructive pulmonary disease
Neurologic

Duchenne muscular dystrophy
Multiple sclerosis
Parkinson disease
Amyotrophic lateral sclerosis
Epilepsy
Cerebral ischemia
Ataxia
Encephalopathy
Alzheimer disease
raumatic

Fractures
Knee cartilage injuries
Spinal cord injuries
Burns
Amputation stump so tissue reconstruction
Cranio acial reconstruction
Congenital

Polycystic kidney disease
Cle lip and palate
Osteogenesis imper ecta
Familial hypercholesterolemia
Orthopedic

Osteoarthritis
Osteonecrosis o emoral head
Lumbar intervertebral degenerative disc disease
Neoplastic

Ovarian cancer
Autoimmune

ype I diabetes mellitus
Aplastic anemia
Gra versus host disease
Sjögren syndrome
Scleroderma
Crohn’s disease
Ulcerative colitis
Lupus nephritis
Rheumatoid arthritis
Primary biliary cirrhosis
Ankylosing spondylitis
Autoimmune hepatitis
Miscellaneous
Cirrhosis
Maxillary cysts
In ertility
Glaucoma
Erectile dys unction
Fecal incontinence
Psoriasis
Cytomegalovirus in ection
Diabetic oot ulcer

95
Chapter 5 • Antiin lammatory Stem Cell Principles
AUTOLOGOUS LIPOTRANSFER IN CANCER
umor resection in neoplastic diseases such as head and neck cancer and breast cancer is o en disf g-
uring. In such cases, tissue reconstruction using at gra ing presents an attractive but still controversial
option, largely because o the ear that signals provided by the regenerating tissue in combination with
the MSC-like immunosuppressive properties o ADSCs will create an environment permissive to the
growth o persistent occult neoplastic cells. Currently, the cancer-promoting potential o human MSCs
and ADSCs remains a theoretical concern that is diminishing with clinical experience. Experimentally,
MSCs and ADSCs have been success ully used in vitro and in vivo to reduce tumor proli eration and
invasion, either based on their secretory activity or as treatment vectors capitalizing on their ability to
home to tumor sites.48,49
Most experimental data relating to the interaction o MSCs with neoplastic cells are based in coculture
and human/murine xenotransplantation studies. T e use o tumor-derived cell lines in vitro and in vivo
as models or cancer addresses a particular question: How does interaction with MSCs a ect the behav-
ior o rapidly proli erating trans ormed cells? T ese models ail to take into account tumor heterogeneity
and the importance o occult and dormant tumors in the natural history o neoplastic disease and disease
recurrence. T e question most relevant to the possible e ects, either positive or negative, o MSCs in au-
tologous tissues used or reconstruction a er cancer surgery is: How do MSCs and the wound-healing
environment associated with surgical reconstruction a ect the behavior o occult tumor that has per-
sisted a er therapy? T is is a much more di cult problem to model. It is there ore unsurprising that
MSC/cancer interaction studies have been controversial and at times contradictory. For the same type o
cancer and the same source o MSCs there are studies that show tumor-enhancing and tumor-inhibitory
responses.50,51 T e MSC polarization paradigm may explain these apparent contradictions.16,52 Subtle
di erences in assay conditions may determine MSC polarization and there ore the direction (protumor
or antitumor) o MSC-mediated modulatory e ects.
In bladder cancer, in vitro studies demonstrated reduction in proli eration, induction o apoptosis, sup-
pression o migration, and cell cycle arrest in the presence o ADSCs, both through juxtacrine and para-
crine interactions.53 In melanoma there is evidence, both in vitro and in vivo, that ADSCs can mediate
a therapeutic e ect. As in bladder cancer, ADSCs reduced proli eration, induced apoptosis, and caused
cell cycle arrest. Both MSC migration to the tumor site and reduction o tumor growth were observed in
this murine model.54
In breast cancer, the use o at gra ing, especially SVF-enriched at gra ing, or reconstruction a er
breast-conserving surgery or mastectomy raises similar concerns relating to the e ect on cancer reacti-
vation exerted by resident or intentionally added mesenchymal cells. It should be noted that reconstruc-
tion is per ormed a er completion o therapy, presumably in the absence o active disease. However,
the high prevalence o dormant occult cancer cells can be in erred rom the incidence o local recur-
rence a er breast-conserving surgery. Estimates vary, but Fisher et al,55 reporting on a -year ollow-up
or the National Surgical Adjuvant Breast Project, observed % ipsilateral recurrence in patients with
lumpectomy alone versus % in women who had lumpectomy plus breast irradiation. T e same group
reported recurrence rates o . % and . %, respectively, a er -year ollow-up.9 T e extent to which
reconstructive surgery (either with or without at gra ing) and the associated wound-healing response
in uence the behavior o such occult breast cancer cells is currently unknown. As in other cancers, the
proli eration o experimental data, both in vitro and in vivo, has not helped to resolve this concern. Our
own studies were per ormed using sort-purif ed unpassaged clinical breast cancer isolates in a murine
xenogra model in which injected cells take or more months to orm a palpable tumor. T ey support
the notion that ADSCs enhance the tumorigenicity o actively proli erating tumor cells, as well as the in
vitro growth o unselected reshly isolated tumor cells. However, ADSCs did not in uence, positively or
negatively, the tumorigenicity o the resting mesenchymal-like population that has been described as the
breast cancer stem cell.51

96
Part I • Principles and Basic Concepts
C oncl usi on
Although preclinical studies have provided much evidence concerning the immunomodulatory prop-
erties o MSCs, caution is needed to interpret these results rom a clinical perspective. It is important to
consider that the protective e ects o culture-expanded MSCs decrease with passage number,56 and their
regenerative potential depends on a high proli erative rate and an open chromatin status.57 Regarding
their immunosuppressive properties, reduction in cell proli eration is an in vitro phenomenon not
directly correlated with specif c therapeutic e ects. Further, the immunomodulatory e ects o MSCs
are conditioned by the local environment and the presence o danger signals (PAMPs and DAMPs) as
well as other interacting cell types. In vivo, MSCs may inhibit or promote in ammation, depending on
environmental cues.
Nevertheless, mesenchymal stromal cells, either culture-expanded rom bone marrow or rom adipose
tissue, or reshly prepared as an adipose SVF, are promising therapeutic modalities or reconstructive
surgery as well as or a wide variety o in ammatory and autoimmune disorders. Autologous SVF is o
value as an adjunct to at gra ing, not only because o its regenerative and angiogenic properties, but
because o its potential to condition the immunologic milieu o the gra in a way that promotes wound
healing and counters in ammation and scar ormation. As evidence generated in preclinical studies
reveals more about the nature and mechanisms o the regenerative/immune system axis and the cells
regulating in ammation and mediating tissue maintenance and repair, new therapeutic interventions
will suggest themselves and be translated into clinical practice.
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