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Directed Differentiation of Human Embryonic Stem Cells

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Directed Differentiation of Human Embryonic Stem Cells DirectedDirected differentiationdifferentiation ofof humanhuman embryonicembryonic stemstem cellscells TECAN Symposium 2008 Biologics - From Benchtop to Production Wednesday 17 September 2008 Andrew Elefanty Embryonic Stem Cell Differentiation Laboratory, MISCL ES cells as a system to dissect development hES ES cells derived from inner cell mass of preimplantation blastocyst Functionally similar, patient specific cells generated by reprogramming adult or fetal cells: • Somatic cell nuclear transfer (SCNT) - oocyte cytoplasm reprograms somatic cell • Induced pluripotential stem cell (iPS) – reprogramming is initiated by genes and/or growth factors introduced into adult cells ES cells can differentiate into all the cell types in the body blood cells differentiation liver heart muscle pancreas ES cell line nerve In vitro differentiation of embryonic stem cells provides an avenue to • study early development • generate tissue specific stem cells and mature end cells to study disease to screen drugs/therapeutic agents for cell therapies Directed differentiation of ES cells Recapitulate embryonic development in vitro to derive lineage precursors hES Mesendoderm Ectoderm Mesoderm Endoderm Factors that play key roles in embryogenesis also direct differentiation of ES cells hES BMP/Activin/Wnt/FGF Mesendoderm FGF/noggin BMP/Wnt Activin Ectoderm Mesoderm Endoderm Elements that facilitate directed differentiation of HESCs in vitro •large, uniform populations of undifferentiated HESCs •robust, reproducible differentiation protocol incorporating a serum free defined medium permissive for evaluation of effects of growth factors •availability of genetically modified ES cell lines to monitor differentiation or transplantation Maintenance and expansion of HESCs 'Cut and paste' for stock maintenance and transfer to bulk culture T25-T75 Enzymatic passage Enzymatic for expansion for passage to use in FACS establish bulk analysis, cultures differentiation, genetic manipulation Enzymatic passage for short term Elefanty/Stanley Laboratory maintenance (15-25 passages) T75-T150 Enzymatically expanded HESCs express stem cell markers HES 3 d0 control E Cadherin SSEA 4 Tra 1 60 CD 9 Elizabeth Ng, Robyn Mayberry & Amanda Bruce Elements that facilitate directed differentiation of HESCs in vitro •large, uniform populations of undifferentiated HESCs •robust, reproducible differentiation protocol incorporating a serum free defined medium permissive for evaluation of effects of growth factors •availability of genetically modified ES cell lines to monitor differentiation or transplantation Spin EB differentiation of HESCs in APEL, a serum free culture medium APEL Medium • a 'neutral' medium that permits evaluation of effects of GFs HESCs •animal product free, containing only recombinant human proteins • reproducible, quantifiable outcomes Ng et al Curr Protoc Stem Cell Biol, 2008; Ng et al Nat Protocols 2008 Reproducible EB formation in spin EBs generated in APEL medium EB size relates to cell input number 500 1000 2000 3000 4000 Reproducible d0 d1 d2 d3 d4 differentiation with different media batches #2 #3 #4 Elizabeth Ng Directed differentiation of ES cells to hematopoietic mesoderm hES Mesendoderm Hematopoietic Ectoderm Endoderm Mesoderm BMP4 directs differentiation of ES cells to hematopoietic mesoderm BMP4 induces MIXL1 BMP4 induces hematopoietic mesoderm genes hES Mesendoderm Hematopoietic Mesoderm Marjorie Pick BMP4 and VEGF are required for efficient formation of CD34+ cells and hematopoietic CFCs Hematopoietic Hematopoietic Mesoderm Progenitors Hematopoietic CFCs Marjorie Pick Hematopoietic blast colony CFC are present transiently during differentiation BMP/VEGF/SCF SF methyl cellulose + VEGF/SCF/IL3/IL6/Epo Elizabeth Ng Elements that facilitate directed differentiation of HESCs in vitro •large, uniform populations of undifferentiated HESCs •robust, reproducible differentiation protocol incorporating a serum free defined medium permissive for evaluation of effects of growth factors •availability of genetically modified ES cell lines to monitor differentiation or transplantation Genetic tags • Provide reagents for quantifying the effects of specific growth factors. • Provide simply visual assay during the course of ES cell differentiation- allowing the association between different cell types to be observed in real time. • Provide a means to isolate and analyse viable cells at specific differentiation stages in vitro and in transplantation assays in vivo Generation of ErythRED HESC line Time course of RFP expression in differentiating ErythRED HESCs RFP GFP CD45 CD34 RFP RFP+ cells are GFPdim Most RFP+ cells are CD34- CD45- Tanya Hatzistavrou Characterisation of d14 sorted EB populations from ErythRED cells GFP dims during erythroid differentiation Tanya Hatzistavrou Expression of erythroid genes is upregulated in RFP+ ErythRED cells * * * * Tanya Hatzistavrou RFPRFP expressionexpression isis confinedconfined toto erythroiderythroid cellscells inin ErythREDErythRED hematopoietichematopoietic coloniescolonies Tanya Hatzistavrou Transplantation of d7 ErythRED EBs under the kidney capsule of immunodeficient mice d7 EBs Grafts harvested d7 – d28 BMP/VEGF/SCF No teratomas RFP+ cells in 11/12 grafts at d28 Tanya Hatzistavrou Donor RFP+ ErythRED cells distinguished from host erythroid cells in kidney capsule grafts Tanya Hatzistavrou & Sue Micallef Precursors of mesoderm and endoderm pass through the primitive streak during gastrulation BLOOD Primitive Embryonic endoderm ectoderm Extraembryonic and Embryonic mesoderm Inner Cell Epiblast Primitive Mass Streak Definitive endoderm ES cells Primitive Streak MIXL1 MIXL1 is expressed in the Primitive Streak Targeting GFP to MIXL1 locus in HESC lines Clone Validation • Excision of selectable marker • Single cell cloning • Karyotypic analysis • Expression of ES cell markers • Formation of teratomas • Sequencing of genomic junctions • Southern Blotting (exclude multiple integration) Richard Davis • Gene expression in differentiating MIXL1GFP/wt cells Richard Davis CorrelationCorrelation betweenbetween GFPGFP andand MIXL1MIXL1 proteinprotein expressionexpression inin differentiatingdifferentiating MIXL1MIXL1GFP/w HESCsHESCs Richard Davis & Elizabeth Ng Hematopoietic CFCs are enriched in the GFP+PDGFRa+ fraction of differentiating d4 MIXL1GFP/w EBs Richard Davis & Elizabeth Ng Application of robotics to HESC differentiation Stage 2: Spin EB differentiation Enzymatic passage for expansion for use in FACS analysis, differentiation, genetic manipulation Stage 1: HESC Spin EB set up maintenance Growth factor addition and removal Harvesting EBs for further culture or analysis Stage 3: High content image analysis Reporter targeted HESC lines for high content screening analysis Reporter targeted HESC lines for high content screening analysis Reporter targeted HESC lines for high content screening analysis – fluorescent signal specificity Summary Enzymatic HESC expansion and passaging • enables generation of sufficient cell numbers for differentiation, analysis and genetic manipulation Differentiation in serum free animal product free medium (APEL) using a robust multiwell format (spin EB) •enables unbiased assessment of growth factors influencing differentiation Lineage specific fluorescent reporter cells •enable objective assessment of differentiation, in vitro and in vivo Protocols lend themselves to application of robotics and high content screening assays Embryonic Stem Cell Differentiation Laboratory, MISCL Maggie Tanya Michael Lloyd Andrew Sue Mei Ed Alex Karin Anna Claire Marjorie Richard Amanda Aude Elizabeth Lisa Steve Xueling Robyn Sue Kathy Research supported by: The Australian Stem Cell Centre Juvenile Diabetes Research Foundation National Health and Medical Research Council.
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