Nasser Aghdami MD., PhD

Transcription

1 CONTRIBUTON OF HUMAN INDUCED PLURIPOTENT STEM CELL DERIVED ENDOTHELIAL CELLS IN VASCULAR REGENERATION OF BLEOMYCIN-INDUCED SCLERODERMA MOUSE MODEL Nasser Aghdami MD., PhD Department of Regenerative Medicine Royan Institute 18 th ISCT annual Meeting Seattle 2012

2 Scleroderma Systemic sclerosis (SSc) is characterized by vascular alterations, activation of the immune system and tissue fibrosis Vascular insufficiency manifests early in the disease, and although there is evidence of an active repair process, capillaries deteriorate and regress Trojanowska, M. Nat. Rev. Rheumatol. 2010

3 Raynaud s Phenomenon Raynaud M. De l'asphyxie locale et de la gangrène symétrique des extrémités. Doctoral thesis, published February 25, 1862.

4 Vascular Repair Hematopoietic stem cells Myeloid precursor Monocyte CD45 CD14 VEGF2r CD34 CD13 3 VEGF2r CD34 CD45 CD14 Endothelial Progenitor Cells Mesenchymal Stem Cells CD16 6 CD10 5 Tissue injury, hypoxia CD73 Tissue resident stem cells Cipriani P, et al. Autoimmun Rev. 2011

5 EPCs in years of disease Del Papa N et al. Arthritis Rheum, 2006

6 EPCs reduction in SSC Kuwana M et al. Lancet, 2004

7 EPCs dysfunction in SSc Kuwana M et al. Lancet, 2004

8 MSC dysfunction in SSC There are several evidences that in SSc there is a complex impairment in the BM microenvironment, involving not only the endothelial compartment but also the MSC. Hypothesize that alteration in this cell population may also contribute to the defective vasculogenesis in SSc Del Papa N, et al. Arthritis Rheum 2006 & Cipriani P, et al. Arthritis Rheum 2007

9 Embryonic stem cells (ESC) Induced pluripotent stem cells (ips)

10 Questions Can hipscs efficient differentiate into Endothelial Cells? Can hipsc-ecs contribute in vasculogenesis in Bleomycin induced scleroderma disease model?

11 Levenberg et al,.nature(2010) Differentiation protocol 3 Step Differentiation protocol for Endothelial Cells(EC) RH5&RhiPSC1 S1(D4 ) Matrigel Coll IV (DMEM-F12+bfgf) (α-mem+fbs10%, 6 days) (S1) (S2) Coll I (EGM2+FBS10%) (S3) Coll I (EGM2+FBS10%)

12 Posetive cells% Results Fluorescence microscopy (CD31+ cells After FACS 2) CD31 VWF RH5 Cell line R-hiPSC1

13 Mean Colony Count Dil-ac-ldl uptake % R-hiPSC1 Results CD31+ cells After FACS RH5 R-hiPSC1 * * D7 D14 D21 Time(Day) RH5 R-hiPSC RH5 Cell line R-hiPSC1

14 Results Formation of tubular structures is time dependent 19

15 Results d0(dmem-f12+bfgf) d2,6(α-mem+fbs10%) d8,12,16(egm-2+fbs10%)

16 Summary RH5 & R-hiPSC1 can differentiate into endothelial progenitor cells & endothelial cells in vitro RH5 & R-hiPSC1 are the same in differentiation to Endothelial progenitor cells and endothelial cells such as: Morphology, surface markers and functionality With using FACS sorting we can gain homogen population with high purity (above 97%) in various differentiation stages

17 Animal Model BALB/C (6 weeks/female) gr weight Bleomycin subcutaneously injection (100µgr/100µL) for 4 weeks (Daily) Yamamato et al., Rheumatology (2004)

18 Results Light microscopy MT staining group Data are expressed: Mean±SD

19 Vascular changes in BLM Model Yamamoto T et al. Int J Rheumatol. 2011

20 Cell Transplantation BLM ג, 100µgr/ml,100,every day,sc CsA 20mgr/Kg,every day,ip Day Groups 1: BLM+ CsA+ Cell 2: BLM+ CsA+ PBS (ctrl1) 3: BLM+ PBS (sham or ctrl2) 4: Normal Cell Transplantation Sample Taking (SC) Sample Taking

21 TGF-β1 concentration (ngr/ml) Results MT staining Normal Ctrl 1 Cell transplanted Ctrl Time after transplantati

22 Results TB staining

23 Summary Reduction of amount of fibrosis BUT not significant difference in transplanted and CsA treated group Decreas of degranulated number of Mast Cells BUT not significant difference in transplanted and CsA treated group The cause of this improvement can not be attributed solely to the effects of endothelial cells However We are looking for contribution of cell

24 Homing DiI labeled cells in tissue section 1 week after transplantation (Ctrl-)

25 Homing DiI labeled cells in tissue section 4 week after transplantation

26 Lung Skin Results DiI labeled cells in tissue section 4 week after transplantation

27 Summary DiI labeled transplanted ECs were able to migrate in SSc skin and homing around micro vessels Survival and cell division of DiI labeled cells were observed one months after transplantation The assessment of immunohistochemistry for intracellular antigen VWF were positive for transplanted cells

28 Patient Specific induced Pluripotent Stem Cells (PSiPS)

29 Future Direction ips from patients with Scleroderma Endothelial differentiation in 2D and 3D Functional study of Endothelial differentiated cells from PSiPS Study in Animal model

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