Documenti di Didattica
Documenti di Professioni
Documenti di Cultura
Review
1.
Introduction
2.
3.
Immunoregulatory properties
of ASCs
5.
Conclusion
6.
Expert opinion
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
1357
M. Pikua et al.
Article highlights.
.
.
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
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.
2.
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.
1359
M. Pikua et al.
3.1
1360
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
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
15
Enrolment/
estimated
enrolment
Intervention/
procedure
Rheumatoid arthritis
Osteoporotic fractures
Osteoarthritis
Application
1361
1362
Perianal fistulas
Enterocutaneous,
recto-vaginal or
complex perianal fistula
Recto-vaginal fistula
Enterocutaneous fistula
Complex perianal fistula
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
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.
Renal failure
Ulcerative colitis
Crohns disease
Application
M. Pikua et al.
Reconstruction, contour
irregularities, volume
insufficiency
Deformities
post-lumpectomy with/
without radiation
With peripheral
circulatory disorders
ischemic
Ischemic or
haemorrhagic
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
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.
I and II
10
II
I and II
10
20
I and II
10
48
I and II
48
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.
Intervention/
procedure
Myocardial infarction
Multiple sclerosis
Stroke
Diabetes
mellitus type II
Breast
reconstruction/
modelling
Systemic sclerosis
Application
1363
M. Pikua et al.
1364
4.
1365
M. Pikua et al.
5.
Conclusion
Expert opinion
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,
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.
Bibliography
Papers of special note have been highlighted as
either of interest () or of considerable interest
() to readers
1.
2.
3.
4.
5.
6.
7.
Acknowledgement
N Marek-Trzonkowska and M Pikua equally contributed to
this study.
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/
NZ3/00262. The funding organisations are Polish national
institutions supporting the science and had no impact on
the points of views presented in the manuscript. The authors
are employees of Medical University of Gdansk, which is a
public medical academic institution. The authors state no
conflict of interests and have received no payment for
preparation of this manuscript.
9.
10.
11.
12.
13.
Dhanasekaran M, Indumathi S,
Harikrishnan R, et al. Human omentum
fat-derived mesenchymal stem cells
transdifferentiates into pancreatic
islet-like cluster. Cell Biochem Funct
2013; published online 12 January 2013;
doi: 10.1002/cbf.2948
Friedenstein AJ, Petrakova KV,
Kurolesova AI, et al. Heterotopic of bone
marrow. Analysis of precursor cells for
osteogenic and hematopoietic tissues.
Transplantation 1968;6:230-47
Caplan AI. Mesenchymal stem cells.
J Orthop Res 1991;9:641-50
14.
15.
Sanz-Ruiz R, Fernandez-Santos E,
Domnguez-Munoa M, et al. Early
translation of adipose-derived cell therapy
for cardiovascular disease.
Cell Transplant 2009;18:245-54
16.
17.
Salingcarnboriboon R, Yoshitake H,
Tsuji K, et al. Establishment of
tendon-derived cell lines exhibiting
pluripotent mesenchymal stem cell-like
property. Exp Cell Res
2003;287:289-300
18.
19.
20.
1367
M. Pikua et al.
21.
22.
23.
24.
25.
26.
27.
28.
31.
32.
33.
29.
30.
1368
34.
35.
36.
37.
38.
..
39.
40.
41.
42.
43.
44.
45.
46.
..
47.
..
48.
49.
50.
51.
52.
53.
54.
55.
56.
Dhanasekaran M, Indumathi S,
Rajkumar S, et al. Characterization of
human adipose tissue derived
hematopoietic stem cell, mesenchymal
stem cell and side population cells.
Int J Biol 2010;2:71-8
Lin K, Matsubara Y, Masuda Y, et al.
Characterization of adipose tissue-derived
cells isolated with the Celution system.
Cytotherapy 2008;10:417-26
Yamamoto T, Gotoh M, Kato M, et al.
Periurethral injection of autologous
adipose-derived regenerative cells for the
treatment of male stress urinary
incontinence: report of three initial cases.
Int J Urol 2012;19:652-9
Yoshimura K, Sato K, Aoi N, et al.
Cell-assisted lipotransfer for cosmetic
breast augmentation: supportive use of
adipose-derived stem/stromal cells.
Aesthetic Plast Surg
2008;32:48-55; discussion 56-7
McIntosh K, Zvonic S, Garrett S, et al.
The immunogenicity of human
adipose-derived cells: temporal changes in
vitro. Stem Cells 2006;24:1246-53
This paper shows that long-term
culture decreases immunogenicity and
increases homogeneity of ASCs. In
addition, time of culture and number
of passages potentiate
immunosuppressive properties of
ASCs. These results are of great
importance for the future
immunosuppressive therapies
with ASCs.
Di Bella C, Farlie P, Penington AJ. Bone
regeneration in a rabbit critical-sized
skull defect using autologous
adipose-derived cells. Tissue Eng Part A
2008;14:483-90
Bodle JC, Hanson AD, Loboa EG.
Adipose-derived stem cells in functional
bone tissue engineering: lessons from
bone mechanobiology. Tissue Eng Part
B Rev 2011;17:195-211
Mizuno H, Zuk PA, Zhu M, et al.
Myogenic differentiation by human
processed lipoaspirate cells.
Plast Reconstr Surg
2002;109:199-209. discussion 210-11
Wu L, Cai X, Zhang S, et al.
Regeneration of articular cartilage by
adipose tissue derived mesenchymal stem
cells: perspectives from stem cell biology
and molecular medicine. J Cell Physiol
2013;228:938-44
57.
58.
59.
Garca-Olmo D, Garca-Arranz M,
Herreros D, et al. A phase I clinical trial
of the treatment of Crohns fistula by
adipose mesenchymal stem cell
transplantation. Dis Colon Rectum
2005;48:1416-23
60.
61.
62.
63.
64.
65.
66.
67.
69.
70.
71.
72.
73.
74.
75.
76.
77.
78.
1369
M. Pikua et al.
80.
81.
Trottier V, Marceau-Fortier G,
Germain L, et al. IFATS collection:
using human adipose-derived stem/
stromal cells for the production of new
skin substitutes. Stem Cells
2008;26:2713-23
82.
83.
84.
85.
86.
87.
88.
1370
89.
90.
91.
92.
93.
94.
95.
96.
97.
99.
Affiliation
Micha Pikua1 PhD,
Natalia Marek-Trzonkowska2 VMD PhD,
Anna Wardowska1 PhD,
Alicja Renkielska3 MD PhD &
Piotr Trzonkowski4 MD PhD