Directed differentiation of human embryonic stem cells 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 ES cell line pancreas 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 hes Recapitulate embryonic development in vitro to derive lineage precursors 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 T25-T75 'Cut and paste' for stock maintenance and transfer to bulk culture Enzymatic passage to establish bulk cultures Enzymatic passage for expansion for use in FACS analysis, differentiation, genetic manipulation Elefanty/Stanley Laboratory Enzymatic passage for short term maintenance (15-25 passages) T75-T150
Enzymatically expanded HESCs express stem cell markers HES 3 d0 Tra 1 60 SSEA 4 E Cadherin control 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 animal product free, containing only recombinant human proteins reproducible, quantifiable outcomes HESCs 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 differentiation with different d0 d1 d2 d3 d4 media batches #2 #3 #4 Elizabeth Ng
Directed differentiation of ES cells to hematopoietic mesoderm hes Mesendoderm Ectoderm Hematopoietic Mesoderm Endoderm
BMP4 directs differentiation of ES cells to hematopoietic mesoderm hes BMP4 induces MIXL1 BMP4 induces hematopoietic mesoderm genes Mesendoderm Hematopoietic Mesoderm Marjorie Pick
BMP4 and VEGF are required for efficient formation of CD34+ cells and hematopoietic CFCs Hematopoietic Mesoderm Hematopoietic 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 CD34 CD45 RFP RFP + cells are GFP dim 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
RFP expression is confined to erythroid cells in ErythRED hematopoietic colonies 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 Inner Cell Mass Primitive endoderm Epiblast Embryonic ectoderm Primitive Streak Extraembryonic and Embryonic mesoderm 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 MIXL1 MIXL1 GFP/wt cells Richard Davis
Correlation between GFP and MIXL1 protein expression in differentiating MIXL1 GFP/w HESCs GFP/w HESCs Richard Davis & Elizabeth Ng
Hematopoietic CFCs are enriched in the GFP + PDGFRa + fraction of differentiating d4 MIXL1 GFP/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 maintenance Spin EB set up 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 Marjorie Karin Anna Claire Richard Amanda Aude Lisa Elizabeth Xueling Sue Steve Robyn Kathy
Research supported by: The Australian Stem Cell Centre Juvenile Diabetes Research Foundation National Health and Medical Research Council