Origin of mesenchymal stem cell

Size: px

Start display at page:

Download "Origin of mesenchymal stem cell"

Cameron Howard
5 years ago
Views:

1 Origin of mesenchymal stem cell Takumi Era Department of Cell Modulation, Institute of Molecular embryology and Genetics (IMEG) Kumamoto University

2 Differentiation pathways of in vitro ES cell culture Neuroepithelium Sox1 + Sox1 - PDGFRα - PDGFRα + Mesenchymal Stem Cell Adipocyte Osteocyte Chondrocyte PDGFRα + VEGFR2 + Immature Mesoderm PDGFRα + VEGFR2 - Paraxial Mesoderm Adipocyte Osteocyte Chondrocyte ES cell PDGFRα -- VEGFR2 + Lateral Mesoderm Blood cell Endothelial cell PDGFRα + VEGFR2 + Primitive Endoderm CXCR4 + ECD + Definitive Endoderm Hepatocyte Gut cell GSC + ECD + Mesendoderm Nishikawa and Era, Nature Rev. Mol. Cell. Biol., 27 CXCR4 + ECD - Axial Mesoderm Osteocyte

3 What is Mesenchymal stem cell (MSC)? Definition Fibroblastic morphology Adipocytes Self-renewal Chondrocytes Osteocytes Marshak, D et al, Science, 1999

4 Problems of MSCs Research Utilities Multipotency for differentiation Prevention from GVHD in transplantation Support for regeneration of cardiac muscles Problems Developemtal pathway is unclear Specific surface markers are undefined Transplantation methods are not established ES cell study are useful for understanding what MSC is Aims: To identify MSC progenitors and to define MSC differentiation pathway

VEGFR2 In vitro ES cell differentiation Paraxial mesoderm

5 Visualization of cell lineages by surface marker staining Paraxial mesoderm marker PDGFRα Lateral mesoderm marker VEGFR2 In vitro ES cell differentiation Paraxial mesoderm marker (PDGFRα) Mesoderm-like cells Lateral mesoderm marker (VEGFR2)

PDGFRα and VEGFR2 expressions during ES cell differentiation Paraxial mesoderm marker, PDGFRα Day 3 Day 3.

6 PDGFRα and VEGFR2 expressions during ES cell differentiation Paraxial mesoderm marker, PDGFRα Day 3 Day Day 4 Day Lateral Mesoderm marker, VEGFR2 1 day culture Double positive cells can give rise to single positive cells PDGFRα VEGFR2

7 PDGFRα single positive cells express paraxial mesoderm markers FACS Collagen IV coated mes cell PDGFRα + VEGFR2 + Monolayer culture PDGFRα and VEGFR2 staining Analyses 1.2 DP: Double positive Relative value.8.4 VEGFR2 PDGFRα Tbx6 Fst Mesp2 GATA2 VECD SCL PSP: PDGFRα single positive VSP: VEGFR2 single posive qpcr Paraxial mesoderm Markers Lateral mesoderm Markers

Two types of mesoderm-like cell in ES cell culture PSP: PDGFRα single

positive Vascular endothelial cell colonies Blood cells Mgk VEGFR2

Chondrocytes (VE-Cadherin staining) Erythroblast 1.2 Bone cell markers 1.

8 Two types of mesoderm-like cell in ES cell culture PSP: PDGFRα single positive Bone cells Cartilage cells PDGFRα VSP: VEGFR2 singel positive Vascular endothelial cell colonies Blood cells Mgk VEGFR2 Alizalin red staining for osteocytes Alcian Blue staining for Chondrocytes (VE-Cadherin staining) Erythroblast 1.2 Bone cell markers 1.2 Cartilage cell markers Vascular endothelial cell colonies Erythrocyte Fetal globin expression Bglap1 Bglap2 Col2a1 Cola1 3 2 βh1 globin expression PDGFRα+, VEGFR2- (PSP) (Q-PCR) PDGFRα-, VEGFR2+ (VSP) PSP VSP (Q-PCR) PSP VSP

Adipogenesis ES cells Monolayer Culture +RA Day On day 2 and 3 Day 5 Day 9 + Insulin,

9 Retinoic acid (RA) treatment at early phase enhances adipogenesis Mesoderm-like cell development Day Day 5 Day 9 + Insulin, dexamethasone, IBMX and troglitazone Day 18 Adipogenesis ES cells Monolayer Culture +RA Day On day 2 and 3 Day 5 Day 9 + Insulin, dexamethasone, IBMX and troglitazone Monolayer Culture Oil red O staining Dani C. and Austin S. et al, Development, 1997

10 RA treatment induces PDGFRα + mesenchymal cells No RA treatment Day 4 Day 5 Day % 8 2.4% % Mesodermal cells 4 2 Brachyury Mesoderm Markers Mesp2 Mesogenin OB-CAD RA treatment Mesenchymal Markers Vimentin PDGFRβ % % Mesenchymal cells % βactin RT-PCR Mesoderm and Mesenchymal Marker, PDGFRα

ES cell-derived PDGFRα + Mesenchymal stem cells Markers

Adiponectin 19 PDGFRα+ mesenchymal cells Fibroblastic

Cloning Osteocytes Alizalin red staining Osteopontin

11 ES cell-derived PDGFRα + Mesenchymal stem cells Markers Cell Growth Adipocytes Oil red O staining PPARγ Adiponectin 19 PDGFRα+ mesenchymal cells Fibroblastic morphology Cell number 14 9 PDGFRα+ mesoderm cells Cloning Osteocytes Alizalin red staining Osteopontin Osteocalcin Chondrocytes Alucian blue staining Col2a1 Passage Sox9 No RA RA x

12 What kinds of cells are progenitors for MSCs? +RA Mesenchymal stem cells ES cells?? PDGFRα + adipocytes chondrocytes osteocytes

13 RA treatment induces neuro-epithelial cells Condition A Condition B -RA +RA Mesoderm Day Brachyury Mesogenin Endoderm Foxa2 GATA4 Ectoderm Otx2 Sox1 Neurogenin1 β-actin RT-PCR

14 Visualization of neuroepithelium as Sox1-GFP + cells Neuroepithelium Sox1 Expression 8.5dpc 9.5dpc Neural Tube ISH Pevny et al. Development, 1998 Sox1-GFP Knock-in ES cells Sox1 Promoter GFP J. Aubert et al. PNAS, 23

15 Neuro-epithelium is a origin of ES cell-derived MSC With RA Day 4 6 days after purification Adipogenesis Mesenchyme marker PDGFRα.8.5 Sox1 + Purification 48 PDGFRα Sox1-GFP Neuroepithelium marker Sox1-GFP

16 Differentiation pathway from ES cell to MSC Neuro-epithelial cells Mesenchymal stem cells ES cells +RA Sox1+ PDGFRα- Sox1- PDGFRα+ adipocytes chondrocytes osteocytes

17 4N tetraploid embryo Tetraploid chimera Only ES cells can contribute to embryo. 2N ES cell carrying GFP marker E9.5 x Review by Tam, PP. and Rossant, J. Development, 23 Sox1-GFP Tetraploid chimera Sox1/Nestin/TO-PRO

18 Visualization of Sox1 + cells in mouse embryos Adipogenesis Sox1-GFP tetraploid chimera FACS analysis 41 4 Trunk region PDGFRα Mesoderm Neuroepithelium Osteogenesis 31 Cloning E9.5 Mouse embryo Sox1-GFP Neuro-epithelium is the earliest origin of MSCs. Chondrogenesis

19 Tracing of Sox1 + neuro-epithelial and P + neural crest cells Sox1-Cre Knock-in mouse Cre gene is specifically expressed in Sox1 + neuro-epithelium cells P-Cre transgenic mouse Cre gene is specifically expressed in P + neural crest cells Rosa26-STOP-YFP mouse Rosa-p loxp Cre recombinase Rosa-p STOP YFP loxp YFP YFP marker are constitutively expressed during mouse development

20 YFP expression in Sox1-Cre and P-Cre YFP mice Sox1-Cre P-Cre x

21 YFP expression in Sox1-Cre and P-Cre embryos 21 1 Sox1-Cre Neuroepithelium) P-Cre (Neural crest) FACS Analysis PDGFRα PDGFRα 13 YFP E14.5 embryos YFP

+ Sox1/YFP PDGFRα + - - - - + P/YFP + + - PDGFRα + - - - + Neuroepithliumderived Neural

22 Neuroepithelium-derived MSCs in mouse embryos Colonies of MSCs, Colony Forming Unit-Fibroblast (CFU-F) Sox1-Cre (neuroepithelium-derived) P-Cre(Neural crest-derived) + + Sox1/YFP PDGFRα P/YFP PDGFRα Neuroepithliumderived Neural crestderived Neuroepithelium- and other tissue-derived PDGFRα + MSCs are present in E14.5 mouse embryos

23 E14.5 Sox1 + -derived PDGFRα + cells shows a multipotency and a sustained growth in vitro Cell Morphology:Fibroblastic Gene expression PDGFRα Single cell sorting βactin PDGFRα OBCAD PDGFRβ Vimentin Slug Snail Passage 18 RT-PCR Sox1-Cre, Rosa-YFP Differentiation Potential of clonal cell lines Adipogenesis Osteogenesis Chondrogenesis

24 New differentiation pathway of MSCs Neuro-epithelium Neural crest Sox1 - PDGFRα + MSCs Sox1 + PDGFRα - P + Neural and pigment cells Takashima, Era and Nishikawa, Cell, 27

25 MSCs activity of neonate Sox1 + -derived PDGFRα + cells FACS pattern of Femur bone (from P to P7) 3 CFU-F assay YFP + YFP - YFP - YFP + PDGFRα + PDGFRα + PDGFRα - PDGFRα - PDGFRα 2 1 Single cell Sorting Sox1-Cre YFP Fibroblastic Morphology.3 Differentiation activity of clonal cell lines Adipogenesis Osteogenesis Chondrogenesis Passage 18

26 NE-derived MSC are reduced after birth Proportion of PDGFRα + population %.15 4-week old Neonates. P<.1.5 YFP + PDGFRα + YFP + PDGFRα -

27 Two developmental waves of MSCs Neuro-epithelium supplies the earliest and transient wave of MSCs Neuroepithelium origin Unknown origin Mesoderm? MSC activity Embryo After Birth Development

Biology Group Yasuhiro Takashima Shin-Ichi Nishikawa Animal Resources and

28 Acknowledgements Department of Cell Modulation Institute of Molecular Embryology and Genetics (IMEG) Kumamoto University RIKEN CDB Stem Cell Biology Group Yasuhiro Takashima Shin-Ichi Nishikawa Animal Resources and Genetic Engineering Kazuki Nakao Genta Tsuzuki Cambridge University Austin Smith

Nature Biotechnology: doi: /nbt Supplementary Figure 1. Generation of paraxial mesoderm from the H7 hesc line.

Nature Biotechnology: doi: /nbt Supplementary Figure 1. Generation of paraxial mesoderm from the H7 hesc line. Supplementary Figure 1 Generation of paraxial mesoderm from the H7 hesc line. H7 hescs were differentiated as shown in Figure 1a. (a) Flow cytometric analyses of the proportion of CD56+, PDGFRα+, and KDR+