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
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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 MIXL1MIXL1GFP/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 MIXL1MIXL1GFP/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 and genetic manipulation
Differentiation in serum free animal product free medium (APEL) using a robust multiwell format (spin EB) numbers for differentiation, analysis •enables unbiased assessment of growth
Lineage specific fluorescent reporter cells •enable objective assessment of differentiation, in vitro and in vivo
factors influencing differentiation 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