Therapeutic and Research Potential of Human Stem Cells – Prospects and Challenges Dr Stephen L Minger Dr Stephen L Minger Stem Cell Biology Laboratory Stem Cell Biology Laboratory Wolfson Centre for Age- -Related Diseases Related Diseases Wolfson Centre for Age King’ ’s College London s College London King Focal Point, Biomedicine in China Focal Point, Biomedicine in China Stem Cell Expert – – UK Gene Therapy UK Gene Therapy Stem Cell Expert Advisory Committee (GTAC) Advisory Committee (GTAC)
Manipulation of Stem Cells for Manipulation of Stem Cells for Research &/or Therapeutic Use Research &/or Therapeutic Use � Expansion & Differentiation of Pluripotent Expansion & Differentiation of Pluripotent � Cells for Transplantation Cells for Transplantation � Stimulation of Endogenous Tissue Stimulation of Endogenous Tissue- -Specific Specific � Stem/Progenitor Cells for Tissue Repair Stem/Progenitor Cells for Tissue Repair � Expansion & Differentiation of Pluripotent Expansion & Differentiation of Pluripotent � Cells for Drug Discovery/Toxicology Cells for Drug Discovery/Toxicology � Generation of Disease Generation of Disease- -Specific Pluripotent Specific Pluripotent � Cell Line for “ “Disease in a dish Disease in a dish” ” Modelling Modelling Cell Line for
Parkinson’ ’s Disease and the Brain s Disease and the Brain Parkinson
Progressive and sustained improvement in transplant function over ten years in human graft recipient Piccini et al, 1999, Nature Neurosci, 2
Stem Cells – Types, Flavours and Sources Pluripotent Multipotent Multipotent Multipotent
Cell transplantation and stem cells: and stem cells: Cell transplantation minimal requirements for success minimal requirements for success � Stem/progenitor cells must proliferate for extended Stem/progenitor cells must proliferate for extended � periods and generate 100,000,000,000+ s of cells periods and generate 100,000,000,000+ s of cells � Stem/progenitor cell phenotype must be stable over time Stem/progenitor cell phenotype must be stable over time � with no loss of cellular potency with no loss of cellular potency � Stem/progenitor cells must be capable of generating Stem/progenitor cells must be capable of generating � required cell types upon differentiation required cell types upon differentiation � Stem/progenitor cells or their differentiated progeny Stem/progenitor cells or their differentiated progeny � must survive implantation, functionally integrate into must survive implantation, functionally integrate into host adult tissue, evade immune rejection, and provide host adult tissue, evade immune rejection, and provide long- -term therapeutic benefit. term therapeutic benefit. long
Austin Smith, 2003
A B With Good Karma, Embryos, and Culture Conditions, Human ES Cells Can Be Established At Frequency of ~ 10% A, B, Cystic Fibrosis Line (CF-1) C HES Cell Line (WT-4) C C
Oct-4 β -Tubulin(ectoderm) Albumin (endoderm) Smooth Muscle Actin (mesoderm)
Generation of multipotent somatic stem cells from pluripotent embryonic stem cells Neurones Oligos Astrocytes Neural Stem Cells Insulin Glucagon Pancreatic Stem Cells ES cells Tissue-specific cell types Other Stem Cells Multipotent Bi or Tri- potential Pluripotent Tissue-specific Progenitor cells stem cells
Generation of Differentiated Somatic Cell Generation of Differentiated Somatic Cell Populations from Human ES Cells Populations from Human ES Cells Embryoid Body (EB) Formation Embryoid Body (EB) Formation � � Efficient process – – gives rise to many diff cell types gives rise to many diff cell types Efficient process � � Very mixed populations ( endo endo- -, , meso meso- - ectoderm) ectoderm) Very mixed populations ( � � EB Formation with Defined Mitogenic EB Formation with Defined Mitogenic Factors Factors � � Uncertainty as to which factors support cell types of interest Uncertainty as to which factors support cell types of interest � � Direct Selection by Mitogenic Mitogenic Selection Selection Direct Selection by � � Only cells capable of responding to individual factors should proliferate oliferate Only cells capable of responding to individual factors should pr � � Co- -Culture of Culture of EBs EBs/ES cells with Somatic Cell Lines /ES cells with Somatic Cell Lines Co � � Often requires physical contact/close proximity to target cells Often requires physical contact/close proximity to target cells � � Cells often of animal origin and require serum for growth Cells often of animal origin and require serum for growth � � Lineage Specification and Selection Lineage Specification and Selection � � Requires cell surface antigen unique to cell population of interest Requires cell surface antigen unique to cell population of inter est � � Requires knowledge of transcription factors expressed early in development of evelopment of Requires knowledge of transcription factors expressed early in d � � target tissue – – genetic modification and selection genetic modification and selection target tissue Requires Selection of Target Population - Requires Selection of Target Population -> Gene Transfer, > Gene Transfer, FACs FACs, , � � Immunoselection Immunoselection
Human Embryoid Bodies – 6 days post-differentiation
In vitro expression of germ layer markers during differentiation. Mean fold change in target WT3 (Transcript copy number) 200 gene expression NCAM-1 - - 150 AFP 100 VEGFR2 50 40 20 0 0 5 10 15 20 Differentiation time (Days) 400 150 CF1 WT4 250 125 100 100 100 75 75 50 50 25 25 0 0 0 5 10 15 20 0 5 10 15 20 All data are expressed relative to an undifferentiated hES cell and normalised for the GAPDH house-keeping gene. n=5. Bars represent S.E.M.
Human Neural Stem/Progenitor Cells Derived from Human Embryonic Stem Cells 120 Mean percentage of immuno- 100 reactive cells (%) 80 60 40 20 0 PSA-NCAM Nucleostemin Sox-2 Musashi-1 Neural stem cell marker Taylor and Minger, unpublished
Generation of Differentiated Somatic Cell Generation of Differentiated Somatic Cell Populations from Human ES Cells Populations from Human ES Cells Embryoid Body (EB) Formation Embryoid Body (EB) Formation � � Efficient process – Efficient process – gives rise to many diff cell types gives rise to many diff cell types � � Very mixed populations (endo endo- -, , meso meso- - ectoderm) ectoderm) Very mixed populations ( � � EB Formation with Defined Mitogenic EB Formation with Defined Mitogenic Factors Factors � � Uncertainty as to which factors support cell types of interest Uncertainty as to which factors support cell types of interest � � Direct Selection by Mitogenic Mitogenic Selection Selection Direct Selection by � � Only cells capable of responding to individual factors should proliferate oliferate Only cells capable of responding to individual factors should pr � � Co- -Culture of Culture of EBs EBs/ES cells with Somatic Cell Lines /ES cells with Somatic Cell Lines Co � � Often requires physical contact/close proximity to target cells Often requires physical contact/close proximity to target cells � � Cells often of animal origin and require serum for growth Cells often of animal origin and require serum for growth � � Lineage Specification and Selection Lineage Specification and Selection � � Requires cell surface antigen unique to cell population of interest est Requires cell surface antigen unique to cell population of inter � � Requires knowledge of transcription factors expressed early in Requires knowledge of transcription factors expressed early in � � development of target tissue – development of target tissue – genetic modification and selection genetic modification and selection Requires Selection of Target Population - -> Gene Transfer, > Gene Transfer, FACs FACs, , Requires Selection of Target Population � � I mmunoselection I mmunoselection
Onset Of DA Neuron Markers During Embryonic Onset Of DA Neuron Markers During Embryonic Development Of Human Midbrain Development Of Human Midbrain Gestational 4 5 6.5 week Post-mitotic Neuroepithelial Dopaminergic Differentiated differentiating stem cell Progenitor Cell neuron DA neuron En1, Nurr-1, TH, Ptx3 Lmx1a Aldh1, Nurr-1 Sox1 En2, Ptx3, RET Msx1 Lmx1b Wnt1, Lmx1b, Ngn2 Pax2, En1, Pax5 En2, Aldh1, TH Proposed induction and development of mesencephalic dopaminergic neurons in the human midbrain. Taylor and Minger, 2005
Timeline for Clinical Translation of PD Cell Therapy
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