Stem Cell Biology. Jim Huettner 11/21/2017

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1 Stem Cell Biology Jim Huettner 11/21/2017

2 suggested readings Solter D. (2006) From teratocarcinomas to embryonic stem cells and beyond: a history of embryonic stem cell research. Nat Rev Genet. 7: Buganim Y, Faddah DA, Jaenisch R. Mechanisms and models of somatic cell reprogramming. Nat Rev Genet Jun;14(6): Buganim Y, Markoulaki S, van Wietmarschen N, et al. (2014) The developmental potential of ipscs is greatly influenced by reprogramming factor selection. Cell Stem Cell. 15: De Los Angeles et al. (Daley GQ). (2015) Hallmarks of pluripotency. Nature 525: Fox IJ, Daley GQ, Goldman SA, Huard J, Kamp TJ, Trucco M. (2014) Stem cell therapy. Use of differentiated pluripotent stem cells as replacement therapy for treating disease. Science 345(6199): Schwartz SD, Regillo CD, Lam BL et al., (2014) Human embryonic stem cellderived retinal pigment epithelium in patients with age-related macular degeneration and Stargardt s macular dystrophy: follow-up of two open-label phase 1/2 studies. Lancet. e-pub October 15.

3 Stem Cells: definition Self Renewal - undifferentiated cells that can divide repeatedly while maintaining their undifferentiated state. Pluripotency ability to differentiate into a variety of different cell types

4 In vitro differentiation: Cell/tissue replacement therapies Human model systems of disease and development Donovan and Gearhart, 2001

5 Types of Stem Cells Embryonic from the inner cell mass of preimplantation embryos, prior to formation of the 3 germ layers (ectoderm, mesoderm, endoderm) Somatic undifferentiated cells found in specific locations in mature tissues ips cells induced pluripotent stem cells generated by reprogramming differentiated cells (or cell nuclei, i.e. therapeutic cloning)

6 Potency Totipotent able to generate every cell type including extraembryonic tissues Pluripotent able to generate cells from all three embryonic germ layers Multipotent able to generate a variety of cells from a particular somatic structure Unipotent only generate one cell type

7 Time Line totipotent pluripotent multipotent fertilization implantation gastrulation somatic differentiation zygote morula blastocyst

8 Inner cell mass Epiblast: embryo Hypoblast: yolk sac

9 Early Embryology Human Mouse

10 making a knockout mouse

11 First Isolation of ES cells Mouse: Evans MJ, Kaufman MH. (1981) Establishment in culture of pluripotential cells from mouse embryos. Nature. 292: Martin GR. (1981) Isolation of a pluripotent cell line from early mouse embryos cultured in medium conditioned by teratocarcinoma stem cells. P.N.A.S. U S A. 78: Human: Thomson JA, Itskovitz-Eldor J, Shapiro SS, Waknitz MA, Swiergiel JJ, Marshall VS, Jones JM. (1998) Embryonic stem cell lines derived from human blastocysts. Science. 282: Genetic and Developmental Normality (140 cycles): Suda Y, Suzuki M, Ikawa Y, Aizawa S. (1987) Mouse embryonic stem cells exhibit indefinite proliferative potential. J Cell Physiol. 133:

12 Pluripotency markers Stage-specific antigens: Anti-SSEA 3 and 4 recognize globo-series gangliosides Tra1-60 and Tra1-81: keratin sulfate surface antigens Oct3/4, Sox2, Nanog transcription factors involved with maintaining pluripotency Normal karyotype, and pre-x-inactivation?

13 Two types of ES cells? Naïve (ICM-like) Blastocyst chimera (+) High cloning efficiency Short doubling time Xa Xa Distal Oct4 enhancer High Nanog, Klf2/4, Rex1 Primed (Epi-SC) Blastocyst chimera (-) Low cloning efficiency Long doubling time Xa Xi Proximal Oct4 enhancer Low Nanog, Klf2/4, Rex1 Both types can self renew and give rise to cells from all 3 germ layers in teratomas or following in vitro differentiation

14 maintenance of pluripotency - 1 Initial work done on mouse embryonic fibroblast (MEF) feeder cells in medium supplemented with animal serum One factor produced by feeder cells that helps maintain mouse ES cells in their undifferentiated state is leukemia inhibitory factor (LIF) which activates the Stat3 pathway. Good Manufacturing Process (GMP) guidelines for isolation and propagation of cells that would be used for replacement therapy. Ideally they would be xeno-free. The push for xeno-free conditions, combined with work to optimize reprogramming, has driven screening of factors that can enable serum-free maintenance of pluripotency

15 maintenance of pluripotency - 2 naïve Positive Regulators primed LIF - Stat3 BMP4 - Smad1/5 Wnt (GSK-3 inhibitors) IGF TGFβ/activin Smad2/3 FGF2 ERK1/2 Wnt (GSK-3 inhibitors) IGF Negative Regulators TGFβ/activin-Smad2/3 FGF2 ERK1/2 BMP4 Smad1/5

16 maintenance of pluripotency - 3 Mouse (2008) Human (2013) LIF - Stat3 GSK-3 inhibitors (Wnt) ERK1/2 inhibitors (2i/LIF) For serum free growth also need: Insulin, transferrin, progesterone, putrescine, selenium Current Standard Conditions LIF Stat3 GSK-3 inhibitors (Wnt) ERK1/2 inhibitors PKC inhibitor p38 inhibitor JNK inhibitor ROCK inhibitor FGF2 TGF-β1 but see: Takashima et al., Cell 158: (2014)

17 Attempts to define Stemness Early microarray profiles showed surprising lack of agreement (limitations in microarray technology or platform/lab/primary cell or cell line differences) (Science 302:393, 2003) Relatively weak overlap between mouse and human ES cells (~25%) compared to >90% typical for differentiated tissues. (Stem Cell Reviews 1: , 2005) but this may reflect confusion between naïve and primed ES cells Recent evidence for totipotent mouse stem cells: Blockade of pro-differentiation signaling pathways can maintain cells at approximately the 8-cell morula stage or drive naïve mes cells to a pre-naïve 8-cell like state See Yang et al. (2017) Establishment of mouse expanded potential stem cells. Nature 550:

18 In vitro differentiation Different culture conditions alter the fate of ES cells in vitro Protocols exist for all three germ layers Many, but not all, protocols involve aggregation of ES cells in embryoid bodies Most protocols do not yield a single type of cell Selection steps can help to remove undesired cell types Need to ask: How far? & How faithful? Pancreatic β cells: Pagliuca FW, et al. (2014) Generation of Functional Human Pancreatic β Cells In Vitro. Cell. Oct, 159: Rezania A, et al. (2014) Reversal of diabetes with insulin-producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol. Nov, 32:

19 ES cells neurons pluripotent functionally immortal genetically & developmentally normal postmitotic polarized excitable heterogeneous

20 Serum Free Medium SFD 4 d 4 d 4 d 6 d ESC + RA SF+RA Kim et al., Developmental Biology 328: , 2009

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22 β-tubulin nestin Hoechst

23 GAP43 MAP2 Hoechst GABA β-tubulin Hoechst

24 voltage-gated Na + and K + currents Developmental Biology 168: , 1995

25 Journal of Neuroscience 16: , 1996

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27 Hierarchical clustering by frequency of Gene Ontology terms Developmental Biology 328: , 2009

28 Reprogramming SCNT somatic cell nuclear transfer (reproductive and therapeutic cloning) deterministic and fairly rapid ips induced pluripotent stem cells slow and stochastic (until recently) Transdifferentiation conversion of one terminally differentiated cell type into another without dedifferentiation to an immature phenotype. Must rule out cell fusion or other explanations.

29 Reprogramming: somatic cell nuclear transfer

30 Reprogramming Firsts: SCNT Frog: Gurdon JB. (1962) Adult frogs derived from the nuclei of single somatic cells. Dev Biol. 4: Sheep: Campbell KH, McWhir J, Ritchie WA, Wilmut I. (1996) Sheep cloned by nuclear transfer from a cultured cell line. Nature. 380:64-6. Human: (2004) Claim of human SCNT that proved to be unfounded! Tachibana M, et al. (2013) Human embryonic stem cells derived by somatic cell nuclear transfer. Cell. 153:

31 Reprogramming Firsts: ips cells Mouse: Takahashi K, Yamanaka S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126: Human: Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. (2007) Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131: Yu J, Vodyanik MA, Smuga-Otto K, et al., (2007) Induced pluripotent stem cell lines derived from human somatic cells. Science. 318:

32 Generating ips cells Express transcription factors: Oct3/4, Sox2, Klf4 and c-myc OR Oct3/4, Sox2, Nanog and Lin28 Initial de-differentiation and proliferation (day 1-3, enhanced by Myc); histone modification and chromatin reorganization 2 nd wave of gene expression - stem cell and development related genes (day 9-12); DNA demethylation and X reactivation

33 Graf T. Cell Stem Cell 9: , 2011

34 Nature Reviews Genetics 14: , 2013

35 Removing the bottle neck? Rais et al., Nature 502:65-70, 2013 implicate Mbd3, a component in the NuRD complex that mediates gene repression via histone deacetylation and chromatin remodeling. Argue that the reprogramming factors recruit both repressive (Mbd3/NuRD) and de-repressive (Wdr5 and Utx) complexes, and reprogramming only occurs when the Mbd3/NuRd repression loses. Achieve nearly 100% reprogramming within 7 days in cells with Mbd3 reduced or eliminated.

36 Skipping the bottle neck? Jaenisch lab (Cell Stem Cell 15: , 2014) used SNEL factors from the deterministic phase (Sall4, Nanog, Esrrb and Lin28). Obtained fewer but higher quality mouse ipsc colonies as judged by production of all-ipsc mice from 4n blastocyst injections, and lack of trisomy 8. Has not worked yet in humans Is this de-differentiation or transdifferentiation?

37 Transdifferentiation Conversion from one differentiated cell type to another without evident de-differentiation and re-differentiation Must not be confused by cell fusion or selection for rare pluripotent cells in the source material. Induced by expression of transcription factors and micrornas

38 Graf T. Cell Stem Cell 9: , 2011

39 Fibroblasts to neurons Wernig and colleagues screened 19 transcription factors via lentiviral expression Found 5 were most critical Asc1, Brn2, Olig2, Zic1 and Myt1l, and 3 were sufficient 20% conversion within 2 weeks For human fibroblast conversion also require NeuroD1 and it is less efficient (2-4%) and slower (5-6 weeks for functional synapses)

40 Yang et al., Cell Stem Cell 9: , 2011

41 Conversion process Asc1 bhlh transcription factor binds to many of the same genomic loci when expressed in fibroblasts, myoblasts or neural progenitors. These sites are marked by specific histone modifications (H3K4me1, H3K27acetyl, H3K9me3) these sites are not accessible in keratinocytes or osteoblasts, which resist transdifferentiation into neurons. Brn2 Pou-Homeodomain transcription factor is recruited by Asc1 to a subset of locations

42 Evaluation SCNT vs ipscs from isogenic cells: Ma H, Morey R, O'Neil RC, et al., (2014) Abnormalities in human pluripotent cells due to reprogramming mechanisms. Nature 511: Johannesson B, Sagi I, Gore A, et al., (2014) Comparable frequencies of coding mutations and loss of imprinting in human pluripotent cells derived by nuclear transfer and defined factors. Cell Stem Cell 15: Origin-dependence after ipsc differentiation: Hargus G, Ehrlich M, Araúzo-Bravo MJ, et al., (2014) Origin-dependent neural cell identities in differentiated human ipscs in vitro and after transplantation into the mouse brain. Cell Reports 8: An optimization strategy? Morris SA, Cahan P, Li H, et al., (2014) Dissecting engineered cell types and enhancing cell fate conversion via CellNet. Cell 158: Retention of cellular age? Huh CJ, Zhang B, Victor MB, Dahiya S, Batista LF, Horvath S, Yoo AS. Maintenance of age in human neurons generated by micrornabased neuronal conversion of fibroblasts. Elife Sep 20;5.

43 Goals of Reprogramming: Models of human disease Isogenic cells for replacement therapy Muffat et al. (2016) CNS disease models with human pluripotent stem cells in the CRISPR age. Curr Opin Cell Biol. 43: Wu et al. (2016) Stem cells and interspecies chimaeras. Nature 540: Aldhous, 2001

44 Proof of Concept Hanna et al., Science 318: , 2007