Expert Opinion on Biological Therapy

ISSN: 1471-2598 (Print) 1744-7682 (Online) Journal homepage: http://www.tandfonline.com/loi/iebt20

Adipose tissue-derived stem cells in clinical

applications
Micha Pikua PhD, Natalia Marek-Trzonkowska VMD PhD, Anna Wardowska
PhD, Alicja Renkielska MD PhD & Piotr Trzonkowski MD PhD
To cite this article: Micha Pikua PhD, Natalia Marek-Trzonkowska VMD PhD, Anna Wardowska
PhD, Alicja Renkielska MD PhD & Piotr Trzonkowski MD PhD (2013) Adipose tissue-derived
stem cells in clinical applications, Expert Opinion on Biological Therapy, 13:10, 1357-1370, DOI:
10.1517/14712598.2013.823153
To link to this article: http://dx.doi.org/10.1517/14712598.2013.823153

Published online: 07 Aug 2013.

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Date: 15 February 2016, At: 10:21

Review

Adipose tissue-derived stem cells

in clinical applications
Micha Pikua, Natalia Marek-Trzonkowska, Anna Wardowska,
Alicja Renkielska & Piotr Trzonkowski

1.

Introduction

2.

Isolation and cell processing

before therapeutic application

3.

ASCs in clinical reconstructive

therapies and plastic surgery

4.

Immunoregulatory properties

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of ASCs
5.

Conclusion

6.

Expert opinion

Medical University of Gdansk, Department of Family Medicine, Gdansk, Poland

Introduction: In the past decade human adipose tissue has been identified as
a source of multipotent stem cells. Adipose tissue derived stem cells (ASCs) are
characterised by immunosuppressive properties and low immunogenicity.
Therefore, they can be used in regenerative medicine, as well as applied to
induce graft tolerance or prevent autoimmunity. ASCs can be easily harvested
with low morbidity, which is their main advantage over mesenchymal stem
cells (MSCs) derived from other sources.
Areas covered: The review focuses on reported clinical applications of ASCs
and discusses technical approaches of their isolation and processing. The differences in phenotype and differentiation preferences between ASCs and
other MSCs that may affect the choice of a particular cell type for the future
therapy are also described.
Expert opinion: ASCs seem to be the perfect tool for regenerative medicine
and immunosuppressive cellular therapies. Nevertheless, there are some tasks
that should be addressed by the future studies: i) ASCs require better characterisation; a set of markers determining ASCs should be clearly defined;
ii) there is need for more studies on safety of reconstructive therapies with
ASCs in cancer patients (e.g., after mastectomy); iii) release criteria should
be determined for freshly isolated and ex vivo expanded ASCs designed for
clinical applications.
Keywords: adipose tissue derived stem cells, cell processing, clinical application, phenotype,
safety, treatment outcome
Expert Opin. Biol. Ther. (2013) 13(10):1357-1370

1.

Introduction

The adipose tissue consists of mature adipocytes, preadipocytes, fibroblasts, vascular

smooth muscle cells, endothelial cells, pericytes, resident monocytes/macrophages
and lymphocytes [1-3]. In the past decade, human adipose tissue has been also identified as a source of adult multipotent adipose-derived stem cells (ASCs), which may
undergo adipogenic, chondrogenic, myogenic, osteogenic [1,4], cardiomyogenic [3]
and endothelial [5,6] differentiation in the presence of lineage-specific induction factors. In addition, it has been found that ASCs can give rise to early neurons, glia
cells [3,4,7-9], hepatocytes [10] and insulin-producing pancreatic-like clusters [11].
Owing to these properties -- ease of harvest of adipose tissue, low donor site morbidity and the available volume/amount of the collected tissue -- ASCs are perceived as
an attractive therapeutic tool.
Characteristics of ASCs
It was originally discovered that bone marrow is the source of mesenchymal stem
cells (MSCs) -- a self-renewing cell population capable of differentiation into various
mature tissues [12-15]. Nevertheless, soon it was observed that cells with the similar
properties can be isolated from other tissues [1,16-18]. Currently, it is widely accepted
that MSCs reside within connective tissue of all organs [19].
1.1

10.1517/14712598.2013.823153 2013 Informa UK, Ltd. ISSN 1471-2598, e-ISSN 1744-7682

All rights reserved: reproduction in whole or in part not permitted

1357

M. Pikua et al.

Article highlights.
.
.

ASCs seem to be the perfect tool for regenerative

medicine and immunosuppressive cellular therapies
Results of the first clinical studies with ASCs are very
promising and there are multiple ongoing clinical trials
that use ASCs for various applications
One of the most important questions regarding clinical
use of ASCs is their safety in the context of
tumorigenesis
urrently, the set of markers determining ASCs is not
clearly defined. Increasing number of reports suggests
that stem cells residing in adipose tissue are not a
homogenous, uniform population
There is a need to specify release criteria for ASCs/
lipoaspirates used for the clinical therapies to provide
standardization and reproducibility of the results

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This box summarises key points contained in the article.

ASCs are a stromal component of adipose tissues characterised by plastic adherence when plated in vitro. They predominantly reside within vessel walls and perivascular
niche where they provide signals for adipocyte and vascular
development/stabilisation, as well as participate in injury
repair [19-21]. They share many of the characteristics of
bone marrow-derived MSCs (BM-MSCs) including extensive proliferative potential, the ability to undergo multilineage differentiation, as well as regenerative and
immunosuppressive capabilities [3,22-24].
Nevertheless, there is still lack of uniform characterisation
of ASCs. According to Mesenchymal and Tissue Stem Cell
Committee of International Society for Cellular Therapy,
human MSCs, regardless the source of their origin, should
match the following criteria: i) they have to be plastic-adherent in standard culture conditions; ii) 95% of cells must
express CD105, CD90 and CD73 markers and must be negative ( 2% of positive cells) for CD45, CD34, CD14 or
CD11b, CD79a or CD19 and class-II major histocompatibility complex molecules (MHC class II); nevertheless cells positive for human leukocyte antigen class II DR (HLA-DR) may
still be termed MSCs if the expression was induced by
interferon-g (IFN-g) and they fulfil all the other criteria;
iii) the cells must differentiate into osteoblasts, adipocytes
and chondroblasts under standard in vitro differentiation
conditions [25].
In addition, several other markers are used in practice to
characterise MSCs derived from different sources, such as
CD29, CD44, CD146, CD166 and CD271 [26-28]. Another
important phenotypic feature of MSCs is lack of costimulatory molecules CD80, CD86, CD40 and CD40L
after stimulation with IFN-g [28-32].
ASCs fulfil all of these criteria, but there are some discrepancies in terms of CD34 expression. There are several
papers which show that ASCs are negative for CD34 [33,34],
while other studies present that a big fraction of ASCs is
1358

positive for CD34 [24,31,35,36]. In one of the studies which

compared MSCs derived from various sources,
Puissant et al. observed that 83.9 11.5% of ASCs were
CD34+, while BM-MSC were negative for CD34 [24]. More
recently, Rebeletto et al. reported that cells positive for
CD34 comprised 2.16 2.48%, 10.52 10.58% and
10.37 8.37% within MSCs derived from bone marrow,
umbilical cord blood and adipose tissue, respectively [37].
Our studies on ASCs confirm these results.
Therefore, there appears a question as to whether multipotent CD34+ ASCs characterised by regenerative and immunosuppressive capabilities comparable with BM-MSCs can
still be classified as MSCs? Recently, Suga et al. reported
that CD34+ and CD34- ASCs have distinct differentiation
preferences [38], which rather suggests heterogeneity within
MSCs derived from adipose tissue than negates that CD34+
ASCs are MSCs too. In addition, it has been reported that
expression of CD34 by ASCs decreases during the culture in
vitro [31]. It is noteworthy that some of the studies reporting
lack of expression of CD34 by ASCs presented the surface
marker analysis of cultured ASCs [34]. Thus, lack of
CD34 could result from changes that occurred during the culture in vitro. Nevertheless, immunophenotype alone, including
CD34 expression, cannot be used to distinguish between
MSCs and more differentiated mesenchymal progenies. Functional assays demonstrating clonogenicity and in vitro multilineage differentiation are always required for full characterisation of
the expanded cell population.
Nomenclature
Recent discovery of MSCs and continuous development of
the knowledge at the field of their biology result in a confusion regarding nomenclature and characteristics of MSCs
derived from various sources. Thus, progenitor cells derived
from adipose tissue stroma are called adipose tissue-derived
stromal/stem cells (ADSCs/ASCs), adipose stroma vascular
cell fraction (SVF), adipose-derived regenerative cells, multipotent adipose tissue-derived mesenchymal stem cells, adipose-derived adult stem/stromal cells, adipose mesenchymal
stem cells, lipoblasts, preadipocytes and processed lipoaspirate cells [15,39]. To prevent the confusion, the International Fat Applied Technology Society decided to adopt
the term adipose-derived stem cells (ASCs) to identify the
isolated, plastic-adherent, multipotent cell population [15,39].
1.2

ASCs -- the choice for clinical therapies

Various proportions of CD34+ and CD34- cells within MSCs
derived from different sources suggest that expression of
CD34 can have functional consequences that may influence
the choice of MSC source for the particular clinical application. The choice may be also affected by other factors such
as amount of tissue available for MSCs isolation. Clinical
therapies require large quantities of stem cells for infusion or
co-transplantation. In contrast to bone marrow, adipose tissue
can provide sufficient number of stem cells for each patient.
1.3

Expert Opin. Biol. Ther. (2013) 13(10)

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ASCs in the clinic

Lipectomy is also associated with much lower donor site morbidity than aspiration of bone marrow. In addition, in adipose
tissue, the frequency of MSCs is ~ 2%, while in bone marrow,
the frequency of MSCs is assessed to be 1:25,000 to
1:100,000 cells [3,8,40,41]. In addition, BM-MSC yield strongly
depends on donor age, body mass index and tissue harvest
site [3]; thus, the required number of these cells cannot be
obtained from each patient. Furthermore, there are studies
reporting that cultured ASCs display higher proliferative
potential in vitro than BM-MSCs. Therefore, clinically effective cell dose of ASCs can be reached earlier than the same
number of BM-MSCs [3,24,42]. In total, 1 g of adipose tissue
can give rise to 5000 colony-forming units (CFU), while
1 ml of bone marrow can be a source of 100 -- 1,000 CFU [3].
In addition, there are animal and human studies which
show that MSCs derived from adipose tissue, bone marrow,
umbilical cord blood (UB-MSCs) and cartilage (C-MSCs)
vary in differentiation preferences [37,43]. It has been shown
by Peng et al. that in rats ASCs have higher proliferative
potential than BM-MSCs and C-MSCs. Despite the fact
that ASCs were capable of osteogenic, adipogenic and chondrogenic differentiation, they had the highest adipogenic
potential among analysed MSCs, while BM-MSCs were characterised by the highest osteogenic capacity. In addition,
ASCs were the most sensitive to oxidative stress and the least
prone to apoptosis due to serum deprivation [43]. Concomitantly, human studies showed that BM-MSCs and ASCs have
similar adipogenic, osteogenic and chondrogenic potential
and that adipocytes derived from BM-MSCs are even more
mature than those from treated cultures of ASCs [24,37]. In addition, Puissant et al. reported that, in contrast to BM-MSCs,
human ASCs were capable of in vitro differentiation into endothelial cells [24]. These data underline heterogeneity of MSCs
derived from various sources and emphasize significance of preclinical studies on human material, as mechanisms of ASC regulation are still poorly understood.

Isolation and cell processing before

therapeutic application

2.

There are various isolation and cell processing protocols for

therapeutic use of ASCs described in literature. Most of the
isolation protocols are based on the method previously
described by Zuk et al. [1]. The harvested tissue is extensively
washed with phosphate-buffered saline to remove blood
which can inhibit enzymatic digestion. Then, the samples
are treated with collagenase at 37
C for 30 -- 60 min. Enzyme
activity is usually neutralised with culture medium supplemented with 10% foetal bovine serum. Subsequently, the
digestion product is filtered through 40 -- 200 m nylon
mesh filters and centrifuged [15,44,45]. In some protocols, at
this point, cells are plated in a complete culture medium [33,46],
while in the others, filtration is preceded [1] or followed by
erythrocyte lysis with NH4Cl. The alternative for red blood
cell lysis is centrifugation in Ficoll gradient [47]. In our

laboratory, we have tested both: NH4Cl lysis and Ficoll gradient centrifugation, and we had finally chosen the first method,
as it results in higher final cell number and is less time-consuming. Nevertheless, it has to be taken under consideration
that the isolation method may affect the final ratio of certain
progenitor cell type. Al Battah et al. reported that ASCs
isolated with a step of Ficoll gradient centrifugation had
higher neurogenic and lower osteogenic potential than
those obtained via the procedure with erythrocyte lysis [47].
There are also reports that Ficoll gradient centrifugation can
be used instead of filtration step when longer digestion time
(60 -- 120 min) is applied [48].
To facilitate the work and reduce time of isolation and risk
of contamination, a cell separation system has been elaborated. Celution SystemTM (Cytori Therapeutics Inc., San
Diego, CA, USA) is an automatic cell isolation system which
enables processing of 250 ml of lipoaspirate for ~ 1 h. The
Celution System uses enzymatic digestion to separate a single
cell suspension, which is washed to remove adipocytes and
matrix fragments from the desired cell fraction containing
adult mesenchymal-like stromal cells, endothelial progenitor
cells and other adipose tissue stromal cells [15,49,50].
There are also differences between protocols of cell processing before therapeutic application. Usually, ASCs are used
i) immediately after isolation [50,51] or ii) after expansion [44].
Both approaches have their advantages and disadvantages.
The main advantages of use of freshly isolated ASCs in comparison with expanded cells are: i) shorter time of the procedure, ii) lower costs and iii) lower risk of contamination as
cell manipulation is significantly reduced. Nevertheless, plastic adherence and subsequent expansion has been reported
to result in relatively homogeneous cell population based on
immunophenotype. In contrast to cultured ASCs, freshly
isolated SVF is characterised by relatively high prevalence
of cells positive for hematopoietic-associated markers
(CD11a, CD14, CD45, CD86 and HLA-DR) [52]. Therefore, cell purity (percentage of ASCs within the cells used
for the treatment) is always lower when freshly isolated cells
are applied. In addition, in some clinical applications an
immunosuppressive effect of ASCs may be important. It
has been reported that late passage cultured ASCs, but not
freshly isolated SVF, had significantly suppressed T-cell proliferation in mixed lymphocyte reaction (MLR) [52]. Another
important issue is the availability of therapeutic cell dose.
Expansion enables to reach the target dose which might be
much higher than that obtained just after cell isolation.
These observations suggest that ASCs expanded in vitro are
better choice for the clinical applications than freshly isolated SVFs. First human trials which compared effectiveness
of both approaches support this point of view [44]. Thus,
future studies will probably focus on expansion of ASCs.
Nevertheless, each cell culture for clinical application
requires not only additional work and time but also strict
environment of good laboratory practice (GLP) that
increases the costs and limits availability of the method.

Expert Opin. Biol. Ther. (2013) 13(10)

1359

M. Pikua et al.

ASCs in clinical reconstructive therapies

and plastic surgery
3.

ASCs are currently under investigation for a variety of clinical

therapeutic applications. The research areas include ischemia
revascularisation, cardiovascular tissue regeneration, bone/
cartilage repair, treatment of urethral sphincteric deficiency,
tracheomediastinal and enterocutaneous fistulas, breast reconstruction and wound healing. ASCs seem to be also the future
of therapies for neurological deficits.
Treatment of musculoskeletal disorders
One of the first clinical applications of ASCs was performed
in 2004. Lendeckel et al. used combination of autologous
ASCs, bone grafts and fibrin glue to treat a 7-year-old girl
after severe head injury with multi-fragment calvarial fractures [45]. ASCs were used when the first skull reconstruction
failed due to the progressive and disseminated calvarial bone
resorption. In this study, ASCs were applied together with
cancellous milled bone from the ilium to enhance the regeneration process limited by the restricted amount of cancellous
bone. ASCs used for the therapy were isolated from adipose
tissue of gluteal region harvested during the procedure of
bone graft collection from the dorsal iliac crest. ASCs were
kept in place with autologous fibrin glue application. Three
months after the treatment, marked ossification in the defect
regions was observed. Follow up had shown symmetrical
calvaria contour and no neurological deficits [45].
Few years later, Di Bella et al. showed in animal model that
autologous ASCs can be successfully used to treat criticalsized skull defects even without concomitant bone graft
administration. Nevertheless, the best clinical effect was
observed when fibronectin-coated scaffold was applied and
ASCs were pre-cultured in osteogenic medium. Undifferentiated ASCs had significantly lower regenerative potential in the
analysed model than osteo-induced ASCs [53]. Other animal
studies showed that ASCs are also effective in cartilage, muscle
and tendon regeneration that make them an useful tool for
treatment of various disorders of human musculoskeletal
system [1,54-57].

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3.1

Treatment of enterocutaneous fistulas

Next clinical studies showed that ASCs can be safely used to
treat enterocutaneous fistulas which are a challenging problem in Crohns disease. The lesions usually do not respond
to standard treatment and their recurrence significantly
decreases the quality of life of affected individuals [58]. In
the Phase I clinical trial, Garca-Olmo et al. administered
autologous ASCs in five patients with Crohns disease. The
cells were isolated from lipoaspirate and used for the therapy
at passage 3 or earlier. After 8-week follow up, 75% of the
treated fistulas were considered healed. No adverse effects
were reported in any patient during the follow up
(12 -- 30 months) [59]. As the results were very encouraging,
3.2

1360

a Phase II multicenter, randomised controlled trial was

launched to investigate effectiveness of the treatment of
complex perianal fistulas in Crohns and non-Crohns disease. The patients were treated with fibrin glue or fibrin
glue plus 20 million autologous ASCs. The second dose of
fibrin glue or fibrin glue plus 40 million ASCs was administered if healing was not observed 8 weeks after the first treatment. Fistula healing was reported in 71% of the patients
who received ASCs and fibrin glue, while only 16% of individuals were cured in group who were treated with fibrin
glue alone. The clinical outcome was comparable in Crohns
and non-Crohns subgroups. Lesion recurrence after 1 year
observation in the patients treated with ASCs was estimated
to be 17.6% [60]. Subsequently, the same group reported that
use of ex vivo expanded ASCs offers much better results than
freshly isolated cells [44].
Treatment of urinary system disorders
Nevertheless, recently autologous freshly isolated ASCs have
been used with good clinical results to treat male stress urinary
incontinence in three patients with urethral sphincteric deficiency after radical prostatectomy and holmium laser enucleation of the prostate. In this study, ASCs were isolated from
250 ml of abdominal adipose tissue harvested during liposuction and injected transurethrally into the rhabdosphincter. In
addition, mixture of ASCs and adipose tissue was administered into submucosal space of the urethra. The clinical
improvement had been observed 2 weeks after ASC administration and had been maintained for the whole period of follow up (6 months). Treated patients were characterised by
decreased leakage volume, frequency and amount of incontinence (one patient achieved a total continence). There was
also an increase in maximum urethral closing pressure, functional profile length and increase in blood flow through area
of the injection. No significant adverse events have been
reported [50].
3.3

Treatment of cardiovascular disorders

ASCs seem to be a very useful tool for the future therapies of
cardiovascular disorders. It has been reported that both animal
and human ASCs spontaneously differentiate into cardiomyocytes in vitro. It was confirmed by phenotypic analysis, expression of the cardiac-specific markers such as troponin-I and
myosin light chain 2 [61-63], as well as functional tests which
revealed pacemaker activity of these cells. It has been also
reported that cardiomyocytes derived from ASCs responded
to adrenergic and cholinergic agonists with increased and
decreased beating rate, respectively [62,63]. In addition, ASCs
are a source of mediators which act in a paracrine fashion
enhancing angiogenesis, reducing cell apoptosis rates [64] and
promoting neuron sprouts in damaged myocardium. ASCs
also increase an electrical stability of the injured heart [65].
Administration of both cultured and freshly isolated ASCs
has been shown to improve cardiac function in experimentally
induced myocardial injury in animal models. Intramyocardial
3.4

Expert Opin. Biol. Ther. (2013) 13(10)

Expert Opin. Biol. Ther. (2013) 13(10)

Autologous ASCs;
intrahepatic arterial
administration
Autologous ASCs ; i.a.z
(vertebral artery) and i.v.
Autologous ASCs; i.v.

Liver cirrhosis

I and II

I and II

10
10

Not provided

I and II

II

10
18

I and II

I and II

I and II

II

I and II
I and II

60

15

20

290

120
500

I and II

I and II

Phase

Recruiting

Enrolling
by
invitation
Recruiting

Recruiting

Ongoing

Recruiting

Ongoing

Recruiting

Recruiting

Ongoing
Recruiting

Recruiting

Recruiting

Recruiting

Status

Tijuana, Mexico

Tijuana, Mexico

Seoul, Republic of
Korea
Kanazawa, Japan

Madrid, Spain

Seoul, Republic of
Korea
Louisville, United
States, Kentucky

Panama City, Panama

Basel, Switzerland

Shanghai, China
Aventura, United
States, Florida

Seoul, Republic of
Korea
Seoul, Republic of
Korea
Seoul, Republic of
Korea

Location

NCT01501461

NCT01453803

NCT01062750

2012-000290-23
(HULPVAS-2011-01)
NCT01302015

NCT01305863

NCT01643655

NCT01885819

NCT01532076

NCT01809769
NCT01739504

NCT01643681

NCT01769872

NCT01624779

Identification
number of
the trial*

The table presents ongoing interventional clinical trials using ASCs. The information was obtained from ClinicalTrials.gov and EudraCT websites. Phase III and Phase IV clinical trials are bolded. As discussed in the text,
there is some confusion regarding nomenclature of ASCs. Therefore, the term ASCs is used throughout this table regardless of whether the cells are cultured or administered immediately after the isolation. The term
expanded was added in cases where it was specified in available online protocol.
*Sponsor protocol numbers are present in brackets for trials registered in EudraCT.
i.a.: Intra-articular; i.a.z: Intra-arterial; i.c.: Intracoronary; i.l.: Intralesional; i.m.: Intramuscular; i.mi.: Intramyocardial; inj.: Injection; i.t.: Intrathecal; i.v.: Intravenous.

Frailty syndrome

Parkinsons disease

Buergers disease

Expanded
polytetrafluoroethylene
vascular graft with lumen
coated with autologous
ASCs; implanted as a
peripheral bypass graft
Autologous ACSs
(expanded); i.m.
Autologous ASCs; i.m.

15

Autologous ASCs; i.v. and

i.t.
Autologous ASCs; inj.
into lumbar intervertebral
disc
Autologous ASCs; i.a.
Autologous
ASCs + autologous
platelet rich plasma; i.a.
(ASCs suspended in PRP)
Autologous ASCs + fibrin
gel + hydroxyapatite
microgranules; i.l.
Autologous ASCs
(expanded)
Autologous ASCs; i.l.
8

15

Enrolment/
estimated
enrolment

Autologous ASCs; i.t.

Intervention/
procedure

Lower limb ischemia

Avascular necrosis of the femoral head

Rheumatoid arthritis

Osteoporotic fractures

Osteoarthritis

Lumbar intervertebral disc degeneration

Spinal cord injury

Application

Table 1. Ongoing clinical trials using ASCs.

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ASCs in the clinic

1361

1362

Perianal fistulas

Enterocutaneous,
recto-vaginal or
complex perianal fistula
Recto-vaginal fistula
Enterocutaneous fistula
Complex perianal fistula

Expert Opin. Biol. Ther. (2013) 13(10)

Autologous ASCs; i.a.z

(renal artery) and i.v.
Autologous ASCs
(expanded); i.v.

Autologous ASCs
(expanded) vs ASCs
(expanded) + fibrin
adhesive vs fibrin
adhesive; i.l.
Autologous ASCs
(expanded); inj. into
gastrointestinal tract
Autologous ASCs
(expanded); i.l.
Autologous adipose tissue
enriched with ASCs;
periurethral inj.
Autologous ASCs
(expanded); i.l.
Autologous ASCs;
periurethral inj.

10
10
156

Autologous ASCs; i.l.

Autologous ASCs; i.l.
Autologous ASCs
(expanded); i.l.
Allogeneic ASCs
(expanded); i.l.
Autologous ASCs
(expanded) + fibrin glue
vs fibrin glue; i.l.

40

10

I and II

I and II

I and II

I and II

10
12

I and II

II

I and II

III

III

III

I and II
I and II
III

I and II

Phase

12

10

207

80

208

15

Enrolment/
estimated
enrolment

Autologous ASCs

Intervention/
procedure

Ongoing

Enrolling
by
invitation
recruiting

Enrolling
by
invitation
Recruiting

Ongoing

Ongoing

Ongoing

Recruiting

Ongoing

Recruiting
Recruiting
Ongoing

Ongoing

Status

Madrid, Spain

Tijuana, Mexico

Moscow, Russian
Federation

Madrid, Spain

Moscow, Russian
Federation

Madrid, Spain

Madrid, Spain

Spain, Netherlands,
Italy, Germany, Austria
Madrid, Zaragoza,
Salamanca,
Velencia,
Pamplona, Spain
Tres Cantos (Madrid),
Spain

Madrid, Spain
Madrid, Spain
Spain and Netherlands

Pamplona, Spain

Location

2011-006254-85
(CeTMad/ELA/2011)

NCT01453816

NCT01889888

NCT01804153

2010-024331-16
(HULPURO-2010-01)
NCT01850342

2010-023798-20
(HLPDIG-2010-01)

2006-003370-95
(Cx401/FATT1)

NCT01548092
NCT01584713
2008-004286-25
(CX-401/FATT2)
2011-006064-43
(Cx601-0302)
NCT01803347

NCT01157650

Identification
number of
the trial*

The table presents ongoing interventional clinical trials using ASCs. The information was obtained from ClinicalTrials.gov and EudraCT websites. Phase III and Phase IV clinical trials are bolded. As discussed in the text,
there is some confusion regarding nomenclature of ASCs. Therefore, the term ASCs is used throughout this table regardless of whether the cells are cultured or administered immediately after the isolation. The term
expanded was added in cases where it was specified in available online protocol.
*Sponsor protocol numbers are present in brackets for trials registered in EudraCT.
i.a.: Intra-articular; i.a.z: Intra-arterial; i.c.: Intracoronary; i.l.: Intralesional; i.m.: Intramuscular; i.mi.: Intramyocardial; inj.: Injection; i.t.: Intrathecal; i.v.: Intravenous.

Amyotrophic lateral sclerosis

Renal failure

Urethral strictures in males

Stress urinary incontinence

Urinary incontinence in women due to strain

Ulcerative colitis

Complex perianal fistulas not associated with

inflammatory bowel disease

Crohns disease

Application

Table 1. Ongoing clinical trials using ASCs (continued).

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M. Pikua et al.

Reconstruction, contour
irregularities, volume
insufficiency
Deformities
post-lumpectomy with/
without radiation
With peripheral
circulatory disorders

Expert Opin. Biol. Ther. (2013) 13(10)

ischemic

Ischemic or
haemorrhagic

Autologous ASCs; i.mi.

and i.v.

Non-ischemic congestive heart failure

10

36

10

I and II

I and II

II

I and II

Recruiting

Ongoing

Ongoing

Ongoing

Recruiting

Ongoing

Recruiting

Recruiting

Recruiting

Ongoing

Ongoing

Recruiting

Recruiting

Status

Copenhagen,
Denmark; Rotterdam
and Utrecht,
Netherlands; Madrid,
Spain
Tijuana, Mexico

Spain

Netherlands

Tijuana, Mexico

Madrid, Spain

Tijuana, Mexico

Tijuana, Mexico

Tijuana, Mexico

Sevilla, Spain

Spain

Sao Paulo, Brazil

Marseille, France,

Location

NCT01502501

2010-022153-42
(ADVANCE)
2007-000339-24
(APOLLO_01)
NCT00426868
PRECISE

2011-001393-26
(EC-INC-09-01)
NCT01453764

NCT01453829

NCT01453777

NCT01453751

2010-019774-33
(CeTMMoTa/ICPDI/
2010)

2007-007956-33
(RESTORE-2)

NCT01771913

NCT01813279

Identification
number of
the trial*

The table presents ongoing interventional clinical trials using ASCs. The information was obtained from ClinicalTrials.gov and EudraCT websites. Phase III and Phase IV clinical trials are bolded. As discussed in the text,
there is some confusion regarding nomenclature of ASCs. Therefore, the term ASCs is used throughout this table regardless of whether the cells are cultured or administered immediately after the isolation. The term
expanded was added in cases where it was specified in available online protocol.
*Sponsor protocol numbers are present in brackets for trials registered in EudraCT.
i.a.: Intra-articular; i.a.z: Intra-arterial; i.c.: Intracoronary; i.l.: Intralesional; i.m.: Intramuscular; i.mi.: Intramyocardial; inj.: Injection; i.t.: Intrathecal; i.v.: Intravenous.

Autologous ASCs; inj.

into left ventricle

I and II

10

II

I and II

10

20

I and II

10

48

I and II

48

Autologous ASCs; i.c.

IV

70

216

II

Not provided

Phase

24

13

Enrolment/
estimated
enrolment

Autologous: mononuclear
cells of bone marrow,
CD133+ endothelial
progenitor cells or ASCs;
i.a.z
Autologous ASCs; intrapancreatic inj. + i.v. vs i.v.
only
Autologous ASCs; i.az
(internal carotid artery)
and i.v.
Autologous ASCs ; i.a.z
(internal carotid artery)
and i.v.
Allogeneic ASCs
(expanded); i.l.
Autologous ASCs ; i.v.
and i.t.
Autologous ASCs; i.c.

Autologous ASCs; s.c.

(into hands)
Autologous adipose tissue
graft enriched in ASCs vs
adipose tissue graft
Autologous ASCs; s.c.

Intervention/
procedure

Coronary artery disease not amenable for

revascularisation

Myocardial infarction

Multiple sclerosis

Stroke

Brain lesions (general)

Diabetes
mellitus type II

Breast
reconstruction/
modelling

Systemic sclerosis

Application

Table 1. Ongoing clinical trials using ASCs (continued).

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M. Pikua et al.

or intracoronary injection of ASCs was found to decrease

the negative cardiac remodelling and to preserve ventricular
function after a myocardial infarction [65-67].
ASCs hold a great promise for the treatment of cardiovascular diseases in human. Nevertheless, till now there are no
data available regarding clinical use of ASCs in cardiologic
patients. However, the studies on administration of human
ASCs into immunocompromised animals are very encouraging [15,65,68,69]. In addition, no cases of arrhythmia or tumorigenesis were reported after myocardial treatment with
ASCs [65].
Currently, there are four ongoing clinical studies, PRECISE, APOLLO, ADVANCE and NCT01502501 (Table 1),
which are testing the safety, feasibility and efficacy of ASCs in
cardiovascular disorders. The first three studies are prospective, double-blind, randomised, placebo-controlled trials,
using ASCs freshly isolated with Celution system. The PRECISE project has enrolled patients with end-stage coronary
artery disease not amenable for revascularisation and with
moderate-to-severe left ventricular dysfunction; APOLLO
has recruited patients with acute myocardial infarction
and left ventricular ejection fraction impairment, while
ADVANCE is dedicated to patients with ST-elevation
myocardial infarction after percutaneous coronary intervention with stent. The last of the four mentioned projects
(NCT01502501) is an open-label, non-randomised, multicentre, patient-sponsored study, recruiting individuals with
non-ischemic congestive heart failure. As the previously
described trials, this study also aims to use freshly isolated,
not expanded ASCs. Results of these studies will shed light
on the safety of ASCs in the treatment of cardiovascular diseases in human. Nevertheless, experience from human studies using ASCs in different applications suggests that
isolation and expansion of ASCs may result in much better
clinical outcome than application of a mixture of freshly
isolated cells which contains only around 2% of real ASCs
and are a very heterogeneous population [44,70]. Therefore,
objective assessment of the safety and effectiveness of
ASCs for the treatment of heart diseases requires studies
with defined numbers of pure ASCs, which is feasible only
after their expansion.
Tissue augmentation and reconstruction
ASCs have also been successfully used to improve clinical
results of soft tissue augmentation and reconstruction.
Yoshimura et al. used lipoaspirate enriched in freshly isolated
ASCs for cosmetic breast augmentation. The technique
resulted in a smaller size of the breasts than in cases where
implants of the same size were used. However, all patients
treated with lipoaspirate were satisfied with more natural contour and softness (with 1 exception per 60 patients) of the
breasts without any palpable nodules for a 6-month follow
up. The reported adverse effects related to the treatment were
fibrous breast tissue and fibrosis on the sternum in one patient
at 6 months, cyst formation (< 12 mm) observed in two
3.5

1364

patients and microcalcification in two patients at 24 months [51].

Nevertheless, recent studies suggest that ASCs should be used
with caution for breast reconstruction in patients previously
treated for cancer. In vitro and in vivo animal studies showed
that in presence of cancer cells, ASCs may promote the tumour
growth by secreting proangiogenic factors and present
enhanced differentiation into myofibroblasts [71].
Treatment of nervous system disorders
ASCs secrete pro-survival and repair-inducing trophic
factors which may promote nerve healing [72]. It has been
reported that ASCs can differentiate into Schwann, glia and
neuron-like cells in vitro [7-9,73]. In addition, intra-cerebral
transplantation of human ASCs was observed to improve
the neurological deficits after cerebral ischemia in rats. It is
noteworthy that the transplanted cells migrated to various
parts of the brain with predilection to the injured cortex [73].
These observations suggest that ASCs can be used in the
future for treatment of peripheral and central nervous system
injuries in human.
3.6

Healing of chronic wounds

Wound healing is a complex biological process. Its defects
may have serious clinical implications. It is estimated that
about 0.2 -- 1% of the population of developing countries suffers from chronic wounds. The problem mainly affects older
people [74,75]. Wound healing comprises of three basic stages:
inflammation, proliferation and remodelling. Stem and progenitor cells are predominantly involved in the first two stages
of wound healing. The main role is played by epidermal stem
cells. Nevertheless, it was reported that bone marrowderived stem cells, as well as ASCs residing in the subcutaneous layer, may also participate in this process [76-78]. The
animal studies showed that ASCs are extremely potent stimulators of wound healing in the absence of hypertrophic
changes in acute, chronic and irradiated wounds [78,79]. In
addition, it has been found that under the influence of certain
growth factors (e.g., fibroblast growth factor 2 [FGF2] and
epidermal growth factor [EGF]) ASCs give rise to keratinocytes, fibroblasts and endothelial cells [6,80].
Recent in vitro studies with human ASCs suggest that the
cells will probably play a prominent role in the future treatment of chronic wounds and other skin lesions in humans.
Trottier et al. produced functional skin substitutes, applying
keratinocytes on stromal compartment reconstructed by
human ASCs. After 14-day culture of both cell types at the
air--liquid interface, a formation of stratified epidermis such
as in normal human skin has been observed. Furthermore, terminally differentiated corneocytes formed a thick stratum corneum that is known to provide the skins barrier function. In
addition, K19+ epithelial stem cells were detected in the basal
layer of the produced skin substitutes where they can provide
long-term renewal of epithelium. Overall, the successful
reconstruction of this new type of skin substitute, good epidermal proliferation and differentiation were maintained in
3.7

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ASCs in the clinic

vivo after engraftment to athymic mice. These data suggest

that ASCs can be applied for the wound therapies instead of
previously used fibroblasts [81]. Another advantage of ASCs
is that they can be easily harvested and transplanted in the
autologous system directly from one part of the body to
another (wound bed) and can serve to create various forms
of skin equivalents for cell therapies.
In a pilot clinical study in humans, Rigotti et al. reported
that injection of autologous lipoaspirates containing ASCs
can be effectively used to treat patients with progressive
lesions after radiation therapy. Application of ASCs led to
dramatic improvement of symptoms observed in all the
patients. ASCs induced radical structural changes and
transformed damaged tissue into normal tissue. The beneficial
effects have been particularly evident in patients with
cutaneous ulcers and osteoradionecrosis [82].
The regenerative effect of ASCs seems to result from their
multidirectional way of action. It was reported that ASCs
stimulated dermal fibroblasts towards proliferation, migration
and production of extracellular matrix (ECM) elements in
direct cell-to-cell interactions and via paracrine stimulation
with soluble factors [83,84]. In addition, production of vascular
endothelial growth factor (VEGF) by ASCs stimulates angiogenesis which is vital for a proper wound healing [79] [85].
ASCs are also a source of anti-inflammatory factors (e.g.,
TGF-b and IL-10) and increase the activity of superoxide dismutase and glutathione peroxidase which scavenge apoptosis
inducing reactive oxygen species [86-88]. Nevertheless, ASCs
not only influence oxidation of the tissues but also respond
to this process. It has been reported that hypoxia increases
regenerative potential of ASCs. At low oxygen concentration,
ASCs were found to upregulate growth factors such as FGF2,
hepatocyte growth factor (HGF), TGF-b and VEGF [39,64].
Thus, ASCs seem to be the perfect tool for treatment of
chronic wounds, as hypoxia is one of the factors limiting their
healing. Besides hypoxia, platelet-rich plasma (PRP) seems to
be another factor that can augment ASC effectiveness in
wound-healing process [89].
Nevertheless, one of the most important factors required
for fast and complete wound healing by ASCs in experimental
settings seems to be an appropriate scaffold for their administration. Scaffold not only creates a niche for ASCs, prevents
their loss during administration, but also facilitates infiltration
of the graft with native cells such as fibroblasts and endothelial
cells [79]. This accelerates wound healing, because as it was
mentioned before, ASCs are not only a source of new cells
but they also engage neighbouring cells in wound healing
process via secreted growth factors.
In terms of scaffold production, the most attention
has been paid to the components of ECM. An interesting
approach has been presented by Vermette et al. who used
ascorbic acid to induce production of ECM by ASCs. Thus,
human ASCs produced a fully biocompatible scaffold for
themselves [90].

4.

Immunoregulatory properties of ASCs

It is well established that BM-MSCs have immunosuppressive

properties [91-93] and are not immunogenic [94]. In addition,
the first clinical studies in human showed that intravenous
administration of BM-MSCs is safe [95] and can be used
for the control of graft versus host disease (GVHD) [96,97].
Interestingly, the therapeutic effect was comparable for
HLA-identical, haploidentical and third-party BM-MSCs [97].
Similar conclusions were derived from studies on immunoregulatory properties of MSCs isolated from other sources.
Keyser et al. compared mice MSCs obtained from omentum,
muscle, adipose tissue and bone marrow. Experiments in vitro
revealed that ASCs were the strongest suppressors of allogeneic MLR [98]. These results suggest that ASCs can be an alternative to BM-MSCs for suppression of GVHD or to induce
tolerance to allografts.
Nevertheless, there are significantly less studies on immunoregulatory potential of human ASCs than BM-MSCs.
Puissant et al. confirmed that human ASCs share most of
the immunosuppressive properties with BM-MSCs. Both
cell types were found not to provoke response of allogeneic
lymphocytes and were effective suppressors of MLRs as well
as lymphocyte proliferation in response to mitogen stimulation. The experiments with cell-separating transwells revealed
that direct cell-to-cell contacts are required for the maximal
immunosuppressive effect of both BM-MSCs and ASCs.
Both cell types were also found to secrete immunosuppressive
HGF, IL-10 and TGF-b, but the cytokines seem not to be
implicated in stem cell suppressive effect in MLRs [24]. These
data suggest that despite fine phenotypic differences, ASCs
share main immunosuppressive properties with BMMSCs and thus can be an alternative to BM-MSCs.
In the first clinical study, Fang et al. used 1  106/kg of
ASCs to treat six patients with steroid-refractory acute
GVHD (grades III -- IV) which developed after allogeneic
haematopoietic stem cell transplantation. ASCs used for
the therapy were derived from haploidentical family donors
or from unrelated mismatched donors. ASCs were isolated
from subcutaneous abdominal adipose tissue obtained during
lipectomy. Cells that were infused were positive for subsequent markers: foetal liver kinase, CD166, CD105, CD44,
CD29 and HLA class I, and negative for: CD34, CD45,
CD14 and HLA class II. There were no side effects related
to ASCs infusion. The significant clinical improvement was
observed in five among six treated patients. Four of the
patients have been alive and in good clinical condition after
a median follow up of 40 months (range: 18 -- 90 months)
since the initiation of the therapy. The clinical results were
not affected by the ASC donor/recipient gender and were
comparable for ASCs obtained from haploidentical and
unrelated donors [23]. These data indicate that ASCs can be
effectively used for immunoregulatory therapies in human
even across HLA.

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5.

Conclusion

Currently, human adipose tissue is considered to be the best

known source of adult stem cells. It can be easily harvested
from subcutaneous fat during small surgical or liposuction procedures with low morbidity and transplanted in the autologous
or allogeneic system. Furthermore, the frequency of MSCs in
adipose tissue is estimated to be ~ 2%, while in bone marrow,
it is between 1:25,000 to 1:100,000 cells [3,8,40,41].
These advantages, ability to differentiate into multiple
types of adult cells, as well as stimulation of tissue renewing
in a paracrine mode, make ASCs a useful tool for regenerative
medicine [1,7-9,45,50,51,54-59,61-63,65-67,73,76-79,81,82,86-89]. In addition, their low immunogenicity and production of antiinflammatory factors put ASCs in the centre of interest of
immunologists [23].
Results of the first clinical studies with ASCs are very
promising. It has been reported that ASCs exerted beneficial
effects in treatment of musculoskeletal disorders [45], enterocutaneous fistulas [59,60], urinary system disorders [50] and
chronic wounds [81,82]. ASCs seem to be also a perfect tool
for tissue augmentation/reconstruction [51] and a promise for
the future therapies of neurodegenerative diseases. These findings are expressed by ongoing multiple clinical trials all over
the world (Table 1).
We now understand that in reconstructive therapies ASCs
are not only a source of new cells but are also able to coordinate function and proliferation of neighbouring cells, as well
as protect them from harmful factors, preventing their
apoptosis [64,84-88].
Recently, it has been showed that ASCs slightly differ from
MSCs derived from other sources [24,31,35-37,98] and seem to be
a heterogonous cell population [38,47]. Further studies will elucidate if various subsets of ASCs exist. As these studies will be
continued, we will be able to choose the most appropriate
type of ASC for a particular clinical therapy.
Good accessibility of adipose tissue and low morbidity of
the harvesting procedure makes ASCs the best candidates for
the future clinical studies on adult stem cells and attractive
tool for regenerative medicine. Owing to these features, as
well as high regenerative potential and immunosuppressive
properties, ASCs seem to become the most extensively studied
stem cells.
6.

Expert opinion

Human adipose tissue seems to be the perfect source of adult

multipotent stem cells capable of adipogenic, chondrogenic,
myogenic, osteogenic, cardiomyogenic, neurogenic, hepatogenic and endothelial differentiation. ASCs are characterised
by low immunogenicity and thus can be transplanted in
both auto- and allogeneic settings (Table 1), which makes
them an attractive therapeutic tool for commercial companies.
In addition, ASCs have been identified as potent immune
1366

suppressors that can be potentially used to induce graft tolerance or prevent excessive immune activation. Thus, they are
found to be in the centre of interest of immunologists.
The key findings of the clinical trials with ASCs so far are
that the cells are safe and well tolerated. No side effects related
to the therapies have been reported with the exception of
breast augmentation. Nevertheless, the frequency and severity
of the changes observed in these studies (e.g., < 12 mm cyst
formation, microcalcification and fibrous tissue formation)
are much lower than those reported for the routinely used
methods of breast augmentation. In addition, injection of
lipoaspirate results in more natural shape and structure
of the breasts. Taking into consideration the accessibility of
adipose tissue and economical aspects of its use (lipoaspirate
obtained during liposuction has been previously treated as
waste material), ASCs seem to be the future of plastic surgery.
One of the most important questions regarding clinical
use of stem cells is their safety in the context of tumorigenesis. Till date, no malignant transformation of injected ASCs
has been reported in human trials. Also conclusions from
animal studies suggest that ASCs are not more prone to carcinogenesis than differentiated adult cells. Nevertheless, at
this point we have to mention about immunosuppressive
properties and secretory activity of ASCs. Even if ASCs do
not transform into malignant cells, they may promote
tumour growth secreting proangiogenic and immunosuppressive factors. Therefore, ASCs should be used with caution for breast reconstruction in patients previously treated
for cancer. They can be a good option for mastectomized
patients after complete tumour removal, but in other cases
they can potentially accelerate growth of the residual cancer
cells. Nevertheless, a question appears: is it possible to
undoubtedly find out at any time point that all neoplastic
cells have been removed?
The first clinical studies with ASCs are very promising, but
the cells have been investigated since nearly 12 years and there
is still need for their better characterisation. GLP demands
wider knowledge regarding mechanisms that regulate biology
of ASCs, self-renewal, differentiation, as well as their interactions with the cell niche. Currently, the set of markers
determining ASCs is not clearly defined. In addition, the
increasing number of reports suggests that stem cells residing
in adipose tissue are not a homogenous, uniform population.
Therefore, future studies should address the question as to which
ASCs (e.g., CD34+ or CD34-) would be more appropriate for
a particular application.
Another important task for the future regards release criteria for ASCs/lipoaspirates used for clinical therapies. In multiple studies, including human trials, freshly isolated
lipoaspirates with undefined number and phenotype of
ASCs have been used. Administration of such a product not
only eliminates the time-consuming culture but also precludes
standardisation of the method as unknown number of ASCs is
injected to the patient. Thus, reproducibility of the clinical
results is questionable when lipoaspirate is used. Therefore,

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ASCs in the clinic

for safe and effective therapy with ASCs, this topic needs
more attention in future.
Nevertheless, the therapeutic properties and potential clinical
applications of ASCs are impressive. In our opinion (not surprisingly), one of the main directions of the future clinical studies with ASCs will be use of these cells with the aim to accelerate
tissue regeneration. Application of ASCs into chronic wounds
(e.g., in diabetic patients) or co-administration of ASCs with
graft tissues (e.g., bone fragments or skin fragments in
burned patients) is a relatively simple procedure which can
and probably will revolutionised this field of medicine.
Our research team is also interested in immunosuppressive
properties of ASCs. The main advantage of ASCs over other
types of suppressor cells, such as regulatory T cells [99,100], is
that they can be harvested in very high numbers during liposuction procedure and easily expanded ex vivo for a long time
with no loss of immunosuppressive function. Therefore, we
can expect that another important direction of future studies
on human ASCs will be immunoregulation in transplanted
patients and autoimmune diseases.
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N Marek-Trzonkowska and M Pikua equally contributed to
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Declaration of interest
The study was supported by funds for science from Polish
national budget for years 2012--2014 (grant of the Polish
Ministry of Science no. IP2011 033771) and funds of the
Polish National Science Centre granted to N MarekTrzonkowska based on the decision no. DEC-2011/01/D/
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Affiliation
Micha Pikua1 PhD,
Natalia Marek-Trzonkowska2 VMD PhD,
Anna Wardowska1 PhD,
Alicja Renkielska3 MD PhD &
Piotr Trzonkowski4 MD PhD

Author for correspondence

1
Medical University of Gdansk,
Department of Clinical Immunology and
Transplantology, ul. Debinki 7,
80-210 Gdansk, Poland
2
Medical University of Gdansk,
Department of Family Medicine,
ul. Debinki 2, 80-210 Gdansk, Poland
Tel: +48 58 349 15 92;
Fax: +48 58 349 91;
E-mail: natalia.marek@gumed.edu.pl
3
Professor,
Medical University of Gdansk,
Department of Plastic Surgery,
ul. Debinki 7, 80- 210 Gdansk, Poland
4
Professor,
Medical University of Gdansk,
Department of Clinical Immunology and
Transplantology, ul. Debinki 7,
80-210 Gdansk, Poland