Faizal Drissa Hasibuan

Spesialis Penyakit Dalam-Konsultan

Hematologi & Onkologi Medik
Bagian Ilmu Penyakit Dalam
FK Universitas YARSI
Jakarta
2019
 INTRODUCTION
 HALLMARKS OF MSC
 ISOLATION
 MARKER
 PROCESSING & FREEZING
 APPLICATION
 Mesenchymal stem cells (MSCs) are
fibroblast-like cells isolated from bone
marrow, adipose, and other tissues -
including cord blood, peripheral blood,
fetal liver, skeletal muscle,
placenta,amniotic fluid and synovium.
 These are all vascularized tissues, and
accumulating evidence suggests that
MSCs are pericytes which closely
encircle endothelial cells in capillaries
and microvessels of multiple organs.
The International Society for Cellular Therapy
position paper:
•Defined the minimal criteria for defining
multipotent mesenchymal stromal cells.
•Plastic-adherent cells expressing CD105,
CD73 and CD90, but not CD45, CD34, CD14,
CD11b, CD79alpha, CD19 or HLA-DR.
•MSC must differentiate to osteoblasts,
adipocytes and chondrocytes in vitro.
Van Pham P . Stem Cell Processing ; 2016
stemcells.nih.gov/info/Regenerative_Medicine/2006
 ESCs
Potency Totipotent: zygote – morula
Pluripotent: inner cell mass of
blastocyst
Major sources Inner cell mass of blastocyst
Cell surface markers hESC lines: SSEA-4, Tra 1-60, Tra 1-
81
mESC lines: NANOG, OCT4, SOX2,
SSEA-1
Potential clinical application in cardiac • Yield a variety of cardiomyocyte-atrial,
regeneration ventricular and sinus-nodal like cells
• Isolation of pure ventricular
cardiomyocyte population using
adenovirus vectors
Advantages Differentiates into three germ layers:
ectoderm, mesoderm, endoderm
Limitations • Availability of cell lines for federally
funded research
• Risk of producing teratomas from
transplanting undifferentiating stem
cells
Ethical concerns • Involves human blastocyst
• Consent for blastocyst/egg donation
is required
ESC embryonic stem cell, HSC hematopoietic stem cell, iPSC induced pluripotent stem cell, MSC mesenchymal stem cell, VEGF vascular endothelial growth
factor
iPSCs
Pluripotent
Reprogramming of adult cells
Cell surface antigenic markers
expressed on ESCs, e.g. SSEA-3 in
human, SSEA-1 in mouse
Generation of cardiomyocyte sheet
along with endothelial cells using
angiogenic. factors (VEGF)
Produced using adult cells, hence
avoids ethical issues
• Generation and safe delivery of iPSC-
derived cardiomyocytes is strenuous
• Tumour formation possible
None specifically
HSCs
Pluripotent
Bone marrow, peripheral blood,
umbilical cord blood
CD34, CD133+
No transdifferentiation into cardiac cells
in ischaemic tissues
Proliferation and migration to site of
injury
• Insufficiency in the DNA repair system
caused by ageing, thereby limiting the
function of HSCs
• Insufficient information on signalling
pathway
• Possibility of gonadal dysfunction and
infertility
• Need for clinical parity
• Consideration required for cure of
children withess severe sickle cell
disease
MSCs
Multipotent
Bone marrow, adipose tissues,
umbilical cord matrix
CD70+, CD90+, CD105+
CD34–
• Improves heart function
• Increase in augmented angiogenesis
• Reduction in fibrosis
• Allogenic grafting possible without
immunosuppressive agents
• Limited inclination towards mutation
• Insufficient information on which MSC
source to be used for the therapeutic
strategy concerning a disease
• Route of administration is uncertain
for different diseases
None specifically
 Self-Renewal
 Differentiation
 Migration
 Apoptosis
Risitano, et al. Current Stem Cell Research & Therapy 2(1):39-52
 MSCs have ability to self-renew, differentiate into
bone, adipose and cartilage tissue the expression of
CD105, CD73 and CD90, and the lack of expression
of CD45, CD34, CD11b and HLA-DR.
 Nevertheless, to date MSCs are widely defined as a plastic-
adherent cell population that can be directed to differentiate
in vitro into cells of osteogenic, chondrogenic, adipogenic,
myogenic, and other lineages (Pittenger et al.,1999, Javazon
et al., 2004, Prockop, 2009).
 As part of their stem cell nature, MSCs proliferate and give
rise to daughter cells that have the same pattern of gene
expression and phenotype and, therefore, maintain the
“stemness” of the original cells.
 Self-renewal and differentiation potential are two criteria
that define MSCs as real stem cells, however, these
characteristics have only been proved after in vitro
manipulation, in bulk and at single-cell level, and there is
 No clear description of the characteristics displayed by
unmanipulated MSCs in vivo (Javazon et al., 2004, Yoshimura
et al., 2006, Lee et al., 2010a).
Autologous versus Allogeneic

•Autologous cells considered safer because

there are no issues with immune rejection of graft
versus host
•Autologous MSCs require a minimum of 5 weeks
for isolation, expansion and release. This limits
their application
 Liquid phase storage
 Short term  UCB / PBSC = 4 0C
 < 4  crystal formation and make cell
death, however actual freezing point of
human plasma is -0,8 0C
 Matsumoto Study (2002) : -2 0C in
University of Wisconsin medium without
DMSO for 72 hours. Superior outcome
compared to control ( -80 0C in DSMO)
 DMSO ( Dymethylsulfoxide) :
- Small amphiphatic molecule
- Penetrates into stem cell
- Frequently side effects : GI + Cardiovascular  nausea + abdominal
cramping (70%), Vasovagal reaction ie bradycardia, hypotension
- Recommended DSMO concentration : 5% or 10% ( 2% DSMO :
inferior cell integrity )
• HES ( Hydroxyethyl Starch) :
- Hydrophilic macromolecule
- Stays outside the cell
- 5 % DSMO + 6% HES  superior than 10% DSMO
• Sucrose/ Trehalose :
- Do not penetrate cell
- Preserve cell membrane integrity
- Combination with DSMO can reduce DSMO to 2,5%
• Caspase inhibitors, Alpha Tocopherol etc
Wang X, et al. BD Biosciences, (1) : 2012
Verena Börger Int. J. Mol. Sci. 2017, 18(7), 1450
Fig. 1
Mechanisms of action of MSCs for cardiac regeneration. (a) miR-133a
downregulates the expression of Apaf-1 and caspase 3 and 9, leading to
attenuated fibrosis. ECs producing growth factors such as VEGF-A help in
recruiting the peripheral stem cells, along with coordinating the differentiation
of MSCs into endothelial cells, thereby leading to vascularization. BMP7
expressed by MSCs lead to inhibition of fibrosis on counteraction of TGF-β
secreted by macrophages. 5-azacytidine induces differentiation of MSCs into
cardiomyocyte, thereby mitigating cardiac contractibility. (b) PLGF-induced
macrophage polarization from M1 to M2 promotes neovascularization.
CardioChimeras are mono-nucleate fusion of CSCs and MSCs which have
exclusive growth kinetics, and have proven to be superior to the parent
precursors. (c) MSCs pretreated with various compounds show cryoprotective
effects along with enhanced cardiomyogenesis and improved heart function.
bFGF basic fibroblast growth factor, CSC cardiac stem cell, EC endothelial cell,
HGF hepatocyte growth factor, LV left ventricular, MSC mesenchymal stem
cell, PLGF platelet-derived growth factor, TGF tumor growth factor, VCAM
vascular cell adhesion molecule, VEGF vascular endothelial growth factor