骨髓间质干细胞体外分化为

RT PCR analysis of expression of cardiomyocyte specific genes : ANP
RT PCR analysis of expression of cardiomyocyte specific genes : β MHC
DISCUSSION

Immunohistochemical methods indicated the expressions of MHC and cTnT respectively
MHC positive staining （× 100）
cTnT positive staining
（× 100）
Negative comparison （×100）
To inverstigate the immunologic properties of MSC after differentiation

Methods

The cultured cells were induced into cardiomyocytes by 5-AZA in vitro
Desmin, cTnT, Streptavdinperoxidasebiotin ( SP)
5-AZA 24h
Restrain T cell proliferation
?
cardiomyocytes
Liquid Scintillation Analyzer : c p m
MSC
RT PCR
ANP, β MHC

Reverse transcriptase RT-PCR of cardiomyocyte-specific genes, including atrial natriuretic peptide (ANP) and MHC
PCR primers used in this study


Internal Lane Standard
Induction of Marrow Mesenchymal Stem Cells to Cardiomyocytes in vitro and immunologic properties of differentiated mesenchymal stem cells
Postgraduate： WANG Qinghai Tutor： ZENG Qiutang
Research ex vivo
Methods-Mixed Cell Cultures Scheme
Grouping
Single reactive cells group (Lp) Mixed group (Lp+Ls) Co-incubating with MSCs group (MSCs+Lp)


5- AZA play the determining role in the cardiomygenic differatiation of MSC The induced effect depended on the amount of 5-AZA

DNA demethvlating agent
Cells culture

Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Paque (1.077g/mL, Invitrogen) density gradient centrifugation
Mixed lymphocyte culture
Introduction




MSC have multipotentiality to be induced MSC were induced into cardiomyocytes in vitro MSC can modulate immune responses Can differentiated MSC modulate immune responses ?
glyceraldehyde-3-phosphate-dehydroge-nase, (GAPDH ) 5′-CAT CAC CAT CTT CCA GGA GCC-3 5′-TGA CCT TGC CCA CAG CCT TG-3′ 443 bp

ANP primer
5′-GGC TCC TTC TCC ATC ACC AA-3′ 5′-TGT TAT CTT CGG TAC TGG CG-3′ Nhomakorabea
MSC were treated with 10µ mol/l of 5azacytidine for 24 h to induce cell differentiation The shape of MSC were observed by Inverted-type phase-contrast video microscope
of Marrow Mesenchymal Stem Cells to Cardiomyocytes in vitro
Flow chart
MHC cTnT,
Streptavdinperoxidase-biotin ( SP)
5-AZA 24h
cardiomyocytes
MSC
RT PCR
ANP, β MHC
Questions

It has been proved that MSC can be induced into cardiomyocytes in vitro

Can differentiated MSC immune responses ?
modulate
Part two
To inverstigate the immunologic properties of MSC after differentiation and the feasibility of treatmen of myocardialinfarction by exogenous stem cells


After 5-azacytidine (10 micromol/L ) treatment (4 weeks), the cultured cells were evaluated by immunohischemistry on the expression of cardiac-specific cardiac troponin T and MHC
Question:
Can differentiated cardiomyocytes from
MSC modulate immune responses ?
Objective

To further substantiate the multipotentiality of Mesenchymal stem cells(MSC), the cultured cells were induced into cardiomyocytes
5-AZA
MSC
cardiomyocyte
Bartholomew confirmed that MSC failed to elicit a proliferative response of allogeneic lymphocytes in 2002
msc
allogeneic lymphocytes
327 bp (+4~+461 bp)
β MHC
primer
5′-ATC CTG CCC TCT GCC TTT 3 ′ 5′-GTG AAG GTG GGC AAC GAA 3′
538 bp (+6~+452 bp)
Results

Phase-contrast photographs of MSC after 5-azacytidine treatment. Some of MSC became spindle-like cells
The cultured cells were evaluated by immunohistochemical staining for, cardiac-specific troponin T(cTnT) and myosin heavy chain(MHC) The MSC differentiation was also observed by RT-PCR
lymphocytes

Lately, it has been proved that the cultured cells induced into fat, bone and gristle can’t express MHCII molecule and failed to elicit a proliferative response of allogeneic lymphocytes in mixed lymphocyte reaction
Materials and methods
Bone marrow cells were isolated from adult Wistar rats leg bone Then cells were washed, counted and plated at 2.0×105 cells/cm2 onto flasks in low-glucose Dulbecco’s modified Eagle’s medium (DMEM,)supplemented with 10% fetal bovine serum (FBS, Hyclone,) Medium was replaced and the nonadherent cells were removed at 72 hours of initial culture and every three or four days thereafter