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Stem Cells, Lineage Development, Plasticity, & Regeneration

Stem Cells, Lineage Development, Plasticity, & Regeneration. Functional Histology Dean Tang, SPRD, MDACC Aug. 31, 2009. Flatworm (planarian). Newt. MRL mice. Stem cell development. Terminal differentiation. Progenitors/ Precursor cells. Senescence. Death (PCD). Stem cells.

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Stem Cells, Lineage Development, Plasticity, & Regeneration

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  1. Stem Cells, Lineage Development, Plasticity, & Regeneration Functional Histology Dean Tang, SPRD, MDACC Aug. 31, 2009

  2. Flatworm (planarian) Newt MRL mice

  3. Stem cell development Terminal differentiation Progenitors/ Precursor cells Senescence Death (PCD) Stem cells

  4. Stem Cell Biology 1. Characteristics & Definition 2. Types: germ stem cells (GSC), embryonic SC (ESC), and somatic (adult) SC (SSC). Adult SCs in many cases are called adult progenitor or precursor cells. 3. (Adult) SC Identification: multiple approaches 4. SC Niche: location, location, location! 5. Plasticity, Reprogramming, & iPS Cells 6. SCs & Tissue Regeneration 7. SCs & Cancer

  5. Stem Cells • Rare • Are generally small • - Normally localized in a ‘protected’ environment called • NICHE, where they only occasionally divide • - But they possess HIGH PROLIFERATIVE POTENTIAL • and can give rise to large clones of progeny in vitro or in • vivo following injury or appropriate stimulation • - Possess the ability to SELF-RENEW (i.e, asymmetric • or symmetric cell division) • - Can generate (i.e., DIFFERENTIATE into) one or • multiple or all cell types (uni-, oligo-, multi-, pluri-, or • toti-potent)

  6. SC Self-renewal, Proliferation, and Differentiation SC Committed cells

  7. SC Development: Self-renewal, proliferation, differentiation Differentiation Transformation targets Self-renewal ? LT-SC ST-SC Late progenitors Differen- tiating cells Differen- tiated cells Early progenitors Proliferation Niche Commitment Differentiation

  8. Stem Cell Biology 1. Characteristics & Definition 2. Types: germ stem cells (GSC), embryonic SC (ESC), and somatic (adult) SC (SSC). Adult SCs in many cases are called adult progenitor or precursor cells. 3. (Adult) SC Identification: multiple approaches 4. SC Niche: location, location, location! 5. Plasticity, Reprogramming, & iPS Cells 6. SCs & Tissue Regeneration 7. SCs & Cancer

  9. Embryonic Stem Cells (ESCs) • Mouse ESCs were generated early 1980s by Evans and • Martin • mES cells are cultured on mouse fibroblast feeders • (irradiated or mitomycin C-treated) together with LIF • .mES cells are widely used in gene targeting • Human ES (hES) cells were first created by Jim Thomson • (Uni. Wisconsin) in 1998 • hES cells were initially cultured also on mouse fibroblast • feeders but without LIF. Now they can be maintained in • defined medium with high bFGF (100 ng/ml), activin, • and some other factors

  10. How can hES cells be derived? • Leftover or Dead-end IVF embryos (PGD)

  11. 16-cell morula

  12. Primitive ectoderm Trophectoderm Primitive Endoderm A. Nagy

  13. ES cells A. Nagy

  14. TS cells A. Nagy

  15. A. Nagy

  16. A. Nagy

  17. lacZ hAP GFP wildtype A. Nagy

  18. A. Nagy

  19. heart pancreas testis liver brain kidney A. Nagy

  20. GFP positive ES cell contribution to the brain A. Nagy

  21. ES cells Tetraploid A. Nagy

  22. ES cell <-> tetraploid embryo aggregation A. Nagy

  23. Other ‘embryonic’ SCs Germline Stem Cells (GSC) Cord Blood Stem Cells (CB-SC) • Derived from umbilical cord • Primarily blood stem cells • Also contain mesenchymal stem cells that can differentiate • into bone, cartilage, heart muscle, brain, liver tissue etc. • *CB-SC could be stimulated to differentiate into neuron, • endothelial cell, and insulin-producing cells

  24. Stem Cell Biology 1. Characteristics & Definition 2. Types: germ stem cells (GSC), embryonic SC (ESC), and somatic (adult) SC (SSC). Adult SCs in many cases are called adult progenitor or precursor cells. 3. (Adult) SC Identification: multiple approaches 4. SC Niche: location, location, location! 5. Plasticity, Reprogramming, & iPS Cells 6. SCs & Tissue Regeneration 7. SCs & Cancer

  25. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

  26. Hematopoietic and progenitor cell lineages (~1:5,000 or 0.02%; lifetime self-renewal) (~1:1,000 or 0.1%; self-renewal for 8 wks) (No self-renewal) Passegué, Emmanuelle et al. (2003) Proc. Natl. Acad. Sci. USA 100, 11842-11849

  27. (Nestin) (PDGFR) (Mash-1) (NeuM) (Pax6) (A2B5) (GFAP) (MBP) (NG2)

  28. Sue Fischer

  29. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

  30. Anchorage- independ. survival Plating efficiency Clonogenic ‘In-gel’ assays (plate cells at low density) Prolif. potential Prolif. Clonal *Plate cells at clonal density (50-100 cells/well in 6-well plate or 10-cm dish or T25 flask) ‘On-gel’ assays (plate at low density) *Plate single cells into 96-well plates (or using flow sorting) - limiting dilution Gels: Agar Agarose Methylcellulose Matrigel Poly-HEMA fibroblasts Spheres (sphere-formation assays) Colonies (colony-formation assays) Holoclone Mero- or paraclone A colony/sphere: a 3-D structure Efficiency (%) Colony/sphere size (cell number) Colony/sphere development (tracking) Immunostaining/tumor exp. CLONAL vs CLONOGENIC ASSAYS A clone: a two-dimensional structure Cloning efficiency (CE; %) Clonal size (cell number/clone) Clonal development (tracking) Clone types

  31. Mixing Experiments to Demonstrate the Clonality of Clones/Spheres DU145:DU145 GFP (1:1) Clonal Assay phase DU145:DU145 GFP (1:1) MC GFP DU145 RFP:DU145 GFP (1:1) MC

  32. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

  33. Identification of HSC by SP Zhou et al., Nature Med 7, 1028, 2001

  34. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

  35. LRCs in the Bulge ARE Stem Cells Tumbar et al., Science 303, 359-363, 2004; Fuchs et al., Cell 116, 769, 2004 Fuchs E: The tortoise and the hair: Slow-cycling cells in the stem cell race. Cell 137, 811-819, 2009.

  36. How to identify and characterize (adult) stem cells? Marker analysis Clonal/clonogenic assays Side population (SP): BCRP or ABCG2 Label-retaining cells (LRC) Aldefluor assay (Aldh1 expression) Cell size-based enrichment Genetic marking

  37. Stem Cell Biology 1. Characteristics & Definition 2. Types: germ stem cells (GSC), embryonic SC (ESC), and somatic (adult) SC (SSC). Adult SCs in many cases are called adult progenitor or precursor cells. 3. (Adult) SC Identification: multiple approaches 4. SC Niche: location, location, location! 5. Plasticity, Reprogramming, & iPS Cells 6. SCs & Tissue Regeneration 7. SCs & Cancer

  38. Stem Cell Niche The most important function of a stem cell niche is to keep the stem cells quiescent and from differentiating and simultaneously maintain their “stemness” (i.e., the repertoire of gene expression profiles characteristic of stem cells).

  39. Stem Cell Niche in Hair Follicles: The Bulge Moore KA & Lemischka IR. Science 311, 1880-1885, 2006

  40. Bulge Stem Cells Tumbar et al., Science 303, 359-363, 2004; Fuchs et al., Cell 116, 769, 2004

  41. Stem Cell Niche in Small Intestine: The Crypt Moore KA & Lemischka IR. Science 311, 1880-1885, 2006

  42. Stem Cell Niche in BM: The Osteoblast Niche Moore KA & Lemischka IR. Science 311, 1880-1885, 2006

  43. Stem Cell Niche In Drosophila, the GSC niches can be experimentally “emptied” - but the niches still persist and can even “recruit” SSC into the niches and maintain their SC properties for some time, although the SSCs never become GSC (Kai and Spradling, PNAS 100, 4633, 2003). Sato T et al., Single Lgr5 stem cells build crypt-villus structures in vitro without a mesenchymal niche. Nature 459, 262-265, 2009.

  44. Stem Cell Biology 1. Characteristics & Definition 2. Types: germ stem cells (GSC), embryonic SC (ESC), and somatic (adult) SC (SSC). Adult SCs in many cases are called adult progenitor or precursor cells. 3. (Adult) SC Identification: multiple approaches 4. SC Niche: location, location, location! 5. Plasticity, Reprogramming, & iPS Cells 6. SCs & Tissue Regeneration 7. SCs & Cancer

  45. Stem cell lineage Differentiated cells Progenitors/ Precursor cells Other cell(s) Senescence Death (PCD) Stem cells

  46. Adult Stem Cell Plasticity Plasticity: the ability of SCs to regenerate and trans-differentiate into (many) other cell types (the cell type- specific programming of apparently committed primary progenitors is not irrevocably fixed, but may be radically re-specified in response to a single transcriptional regulator. Heyworth C et al., EMBO J. 21, 3770-3781, 2002). **Transdifferentiation vs Dedifferenitation: Transdifferentiation refers to adult stem cells directly differentiating into other cell lineages of cells; de-differentiation refers to somatic stem/progenitor cells first reverting back to a more primitive state then differentiating into a specific cell type.

  47. Blelloch R. Nature 455, 604-605, 2008

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