Guided differentiation of ES cell into mesenchymal stem cell Takumi Era Division of Molecular Neurobiology Institute of Molecular Embryology and Genetics, Kumamoto University
Differentiation pathways of in vitro ES cell culture Neuroepithelium Sox1 + Sox1 - PDGFRα - PDGFRα + Mesenchymal Stem Cell Adipocyte Osteocyte Chondrocyte ES cell PDGFRα + VEGFR2 + PDGFRα + VEGFR2 + PDGFRα + VEGFR2 - Paraxial Mesoderm 1. To visualize the intermediates Immature New Mesoderm surface markers; PDGFRα and CXCR4 2. To establish the defined conditions PDGFRα -- Lateral Factors; Activin and VEGFR2 BMP4 + Mesoderm 3. To find the new molecules involved Primitive in Endoderm development Adipocyte Osteocyte Chondrocyte Blood cell Endothelial cell CXCR4 + ECD + Definitive Endoderm Hepatocyte Gut cell GSC + ECD + Mesendoderm Nishikawa and Era, Nature Rev. Mol. Cell. Biol., 27 CXCR4 + ECD - Axial Mesoderm Osteocyte
Gene expressions in the intermediates Expression Intensity Gene 1 Gene 2 Eomeso Sox17 Brachyury foregut Midgut E8.5 mouse embryo ES cell PDGFRα+ VEGFR2+ Mesoderm PDGFRα+ VEGFR2- PDGFRα- VEGFR2+ Day 1 Day 2 Day 3 Activin added Day 4 Day 5 Gsc +,E-cad + Endoderm differentiation Endoderm cell line ada and Era, Development, 25 akebe and Era, Dev. Biol. 26 ES cell-derived samples
Mixl1 expression restore the defect of endoderm by Eomeso KD Endoderm marker, Sox17-hCD25 4 3 2 1 Control 2 15 34 1 2 3 4 4 3 2 1 Eomesodermin Knock-down Vector only Vector only Mixl1-overexpressed Endoderm 3 4 31 1 2 3 4 1 2 3 4 Mesendoderm marker, Goosecoid-GFP 4 3 2 1 1 21 33 Eomesodermin Mixl1 Sox17 Izumi and Era, Stem Cells, 27
Differentiation pathways of in vitro ES cell culture Neuroepithelium Sox1 + Sox1 - PDGFRα - PDGFRα + Mesenchymal Stem Cell Adipocyte Osteocyte Chondrocyte ES cell PDGFRα + VEGFR2 + Immature Mesoderm PDGFRα + VEGFR2 - Paraxial Mesoderm 4. To answer an important question in developmental PDGFRα -- Lateral VEGFR2 biology + Mesoderm Adipocyte Osteocyte Chondrocyte 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
What is Mesenchymal stem cell (MSC)? Definition Fibroblastic morphology Adipocytes Self-renewal Chondrocytes Osteocytes Marshak, D et al, Science, 1999
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
Two distinct conditions induce adipocytes in in vitro ES cell culture Condition A (-RA) Day Day 5 Day 9 -RA + Insulin, dexamethasone, IBMX and troglitazone Day 18 Adipogenesis ES cells Monolayer Culture Sakurai, Era and Nishikawa et al, Stem Cells, 26 Condition B (+RA) +RA Day On day 2 and 3 Day 5 Day 9 + Insulin, dexamethasone, IBMX and troglitazone Monolayer Culture Dani C. and Austin S. et al, Development, 1997 Oil red O staining
Both condition induces the PDGFRα + adipocytic progenitors Condition A (-RA) Day 4 Day 5 Day 9 Adipogenesis 8 4.1% 8 2.4% 3 21.9% 6 6 2 4 4 2 2 1 2 3 4 1 2 3 4 1 2 3 4 TG:26 Condition B (+RA) 12 5 9 3.47 % 8 15.5 % 4 56.9 % 6 3 6 4 2 3 2 1 2 3 4 1 2 3 4 1 2 3 4 TG: 327 Mesoderm and Mesenchymal Marker, PDGFRα Oil red O staining
Condition B induces PDGFRα + mesenchymal cells Condition A (-RA) Day 4 Day 5 Day 9 8 4.1% 8 2.4% 3 21.9% 6 4 6 Mesodermal cells 4 2 Brachyury 2 1 2 3 4 2 1 2 3 4 1 2 3 4 Mesoderm Markers Mesp2 Mesogenin OB-CAD Condition B (+RA) Mesenchymal Markers Vimentin PDGFRβ 12 5 9 6 3.47 % 8 15.5 % Mesenchymal cells 6 4 3 56.9 % βactin 4 2 3 2 RT-PCR 1 2 3 4 1 2 3 4 1 2 3 4 Mesoderm and Mesenchymal Marker, PDGFRα
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 4 2 3 Passage Condition A-derived Condition B-derived Chondrocytes Alucian blue staining x Col2a1 Sox9
What kinds of cells are progenitors for MSCs? +RA Mesenchymal stem cells ES cells?? PDGFRα + adipocytes chondrocytes osteocytes
RA treatment induces neuro-epithelial cells Condition A Condition B -RA +RA Mesoderm Day 3 4 5 3 4 5 Brachyury Mesogenin Endoderm Foxa2 GATA4 Ectoderm Otx2 Sox1 Neurogenin1 β-actin RT-PCR
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
Neuro-epithelium is a origin of ES cell-derived MSC Condition B (+RA) Day 4 6 days after purification Adipogenesis Mesenchyme marker PDGFRα.8.5 Sox1 + Purification 48 PDGFRα 3 4.5 17 Sox1-GFP Neuroepithelium marker Sox1-GFP
Differentiation pathway from ES cell to MSC Neuro-epithelial cells Mesenchymal stem cells ES cells +RA Sox1+ PDGFRα- Sox1- PDGFRα+ adipocytes chondrocytes osteocytes
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
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 Takashima, Era and Nishikawa Cell, 27
Development of Neural Crest Cells They appear in the border between nuero-epithelium and epidermis and migrates from dorsal to ventral regions. Bone, cartilage and neuron in head, and synpathetic neurons and pigment cells in trunk Neuroepithelium Neural crest cells Gilbert, Developmental Biology
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
YFP expression in Sox1-Cre and P-Cre embryos 21 1 Sox1-Cre euroepithelium) P-Cre Neural crest) FACS Analysis PDGFRα PDGFRα 13 YFP 3 9 5 E14.5 embryos YFP
Neuroepithelium-and neural crest-derived MSCs in mouse embryo Colonies of MSCs, Colony Forming Unit-Fibroblast (CFU-F) Sox1-Cre (neuroepithelium-derived) P-Cre(Neural crest-derived) + + ox1/yfp PDGFRα + - - - - + P/YFP + + - PDGFRα + - - - + Neuroepithliumderived Neural crestderived Neuroepithelium- and neural crest-derived PDGFRα + MSCs are present in the trunk of E14.5 mouse embryos
E14.5 Sox1 + -derived PDGFRα + cells shows a multipotency and a sustained growth in vitro 4 21.1 1.27 Cell Morphology:Fibroblastic Gene expression PDGFRα 3 2 1 Single cell sorting βactin PDGFRα OBCAD PDGFRβ Vimentin Slug Snail 47.9 29.8 1 2 3 4 Passage 18 RT-PCR Sox1-Cre, Rosa-YFP Differentiation Potential of clonal cell lines Adipogenesis Osteogenesis Chondrogenesis
MSCs activity of neonate Sox1 + -derived PDGFRα + cells FACS pattern of Femur bone (from P to P7) 3 CFU-F assay 1 2 3 4 4 49.3 YFP + YFP - YFP - YFP + PDGFRα + PDGFRα + PDGFRα - PDGFRα - PDGFRα 2 1 Single cell Sorting 3 1 2 3 4 Sox1-Cre YFP Fibroblastic Morphology.3 Differentiation activity of clonal cell lines Adipogenesis Osteogenesis Chondrogenesis Passage 18
NE-derived MSC are reduced after birth Proportion of PDGFRα + population %.15 4-week old Neonates. P<.1.5 YFP + PDGFRα + YFP + PDGFRα -
New differentiation pathway of MSCs Neuro-epithelium Neural crest MSCs Sox1 + PDGFRα - P + Sox1 - PDGFRα + Takashima, Era and Nishikawa, Cell, 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
Acknowledgements RIKEN CDB Stem Cell Biology Group Yasuhiro Takashima Shin-Ichi Nishikawa Cambridge University Austin Smith Animal Resources and Genetic Engineering Kazuki Nakao