stem%cell% biology%
TRANSCRIPT
Stem Cell Biology
Jim Hue0ner 11/18/2014
suggested readings • Solter D. (2006) From teratocarcinomas to embryonic stem cells and beyond:
a history of embryonic stem cell research. Nat Rev Genet. 7:319-‐27. • Buganim Y, Faddah DA, Jaenisch R. Mechanisms and models of somaTc cell
reprogramming. Nat Rev Genet. 2013 Jun;14(6):427-‐39. • Buganim Y, Markoulaki S, van Wietmarschen N, et al. (2014) The
developmental potenTal of iPSCs is greatly influenced by reprogramming factor selecTon. Cell Stem Cell. 15:295-‐309..
• Cahan P, Li H, Morris SA, Lummertz da Rocha E, Daley GQ, Collins JJ. (2014) CellNet: network biology applied to stem cell engineering. Cell. 158:903-‐15.
• Fox IJ, Daley GQ, Goldman SA, Huard J, Kamp TJ, Trucco M. (2014) Stem cell therapy. Use of differenTated pluripotent stem cells as replacement therapy for treaTng disease. Science 345(6199):1247391.
• Schwartz SD, Regillo CD, Lam BL et al., (2014) Human embryonic stem cell-‐derived reTnal pigment epithelium in paTents with age-‐related macular degeneraTon and Stargardt’s macular dystrophy: follow-‐up of two open-‐label phase 1/2 studies. Lancet. e-‐pub October 15.
Stem Cells: definiTon • Self Renewal -‐ undifferenTated cells that can divide repeatedly while maintaining their undifferenTated state.
• Pluripotency – ability to differenTate into a variety of different cell types
Donovan and Gearhart, 2001
In vitro differenTaTon: • Cell/Tssue replacement therapies • Human model systems of disease and development
Types of Stem Cells Embryonic – from the inner cell mass of pre-‐implantaTon embryos, prior to formaTon of the 3 germ layers (ectoderm, mesoderm, endoderm)
SomaTc – undifferenTated cells found in specific locaTons in “mature” Tssues
iPS cells – induced pluripotent stem cells generated by reprogramming differenTated cells (or cell nuclei, i.e. therapeuTc cloning)
Potency
• ToTpotent – able to generate every cell type including extraembryonic Tssues
• Pluripotent – able to generate cells from all three embryonic germ layers
• MulTpotent – able to generate a variety of cells from a parTcular somaTc structure
• Unipotent – only generate one cell type
Time Line
ferTlizaTon gastrulaTon
toTpotent pluripotent mulTpotent
somaTc differenTaTon
zygote morula blastocyst
implantaTon
h0p://stemcells.nih.gov/info/scireport/pages/chapter1.aspx
Inner cell mass
Epiblast: embryo Hypoblast: yolk sac
Early Embryology
h0p://stemcells.nih.gov/info/scireport/pages/appendixa.aspx
Human Mouse
making a knockout mouse
h0p://en.wikipedia.org/wiki/Knockout_mouse
First IsolaTon of ES cells Mouse:
Evans MJ, Kaufman MH. (1981) Establishment in culture of pluripotenTal cells from mouse embryos. Nature. 292:154-‐6. MarTn GR. (1981) IsolaTon of a pluripotent cell line from early mouse embryos cultured in medium condiToned by teratocarcinoma stem cells. P.N.A.S. U S A. 78:7634-‐8.
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. 282:1145-‐7.
GeneTc and Developmental Normality (140 cycles): Suda Y, Suzuki M, Ikawa Y, Aizawa S. (1987) Mouse embryonic stem cells exhibit indefinite proliferaTve potenTal. J Cell Physiol. 133:197-‐201.
Pluripotency markers • Stage-‐specific anTgens: AnT-‐SSEA 3 and 4 recognize globo-‐series gangliosides
• Tra1-‐60 and Tra1-‐81: keraTn sulfate surface anTgens
• Oct3/4, Sox2, Nanog – transcripTon factors involved with maintaining pluripotency
• Normal karyotype, and pre-‐X-‐inacTvaTon?
Two types of ES cells?
• Blastocyst chimera (+) • High cloning efficiency • Short doubling Tme • Xa Xa • Distal Oct4 enhancer • High Nanog, Klf2/4, Rex1
• Blastocyst chimera (-‐) • Low cloning efficiency • Long doubling Tme • Xa Xi • Proximal Oct4 enhancer • Low Nanog, Klf2/4, Rex1
“Naïve” (ICM-‐like)
“Primed” (Epi-‐SC)
Both types can self renew and give rise to cells from all 3 germ layers in teratomas or following in vitro differenTaTon
maintenance of pluripotency -‐ 1 • IniTal 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 undifferenTated state is leukemia inhibitory factor (LIF) which acTvates the Stat3 pathway.
• Good Manufacturing Process (GMP) – guidelines for isolaTon and propagaTon of cells that would be used for replacement therapy. Ideally they would be xeno-‐free.
• The push for xeno-‐free condiTons, combined with work to opTmize reprogramming, has driven screening of factors that can enable serum-‐free maintenance of pluripotency
maintenance of pluripotency -‐ 2
• LIF -‐ Stat3 • BMP4 -‐ Smad1/5 • Wnt (GSK-‐3 inhibitors) • IGF
• TGFβ/acTvin-‐Smad2/3 • FGF2 • ERK1/2
• TGFβ/acTvin – Smad2/3 • FGF2 • ERK1/2 • Wnt (GSK-‐3 inhibitors) • IGF
• BMP4 – Smad1/5
“naïve” “primed” Posi%ve Regulators
Nega%ve Regulators
maintenance of pluripotency -‐ 3
• LIF -‐ Stat3 • GSK-‐3 inhibitors (Wnt) • ERK1/2 inhibitors
(2i/LIF)
• LIF – Stat3 • GSK-‐3 inhibitors (Wnt) • ERK1/2 inhibitors • PKC inhibitor • p38 inhibitor • JNK inhibitor • ROCK inhibitor • FGF2 • TGF-‐β1
Mouse (2008) Human (2013)
“Current Standard” Condi%ons
For serum free growth also need: Insulin, transferrin, progesterone, putrescine, selenium
but see: Takashima et al., Cell 158:1254-‐1269 (2014)
A0empts to define “Stemness”
• Early microarray profiles showed surprising lack of agreement (limitaTons in microarray technology or platorm/lab/primary cell or cell line differences) (Science 302:393, 2003)
• RelaTvely weak overlap between mouse and human ES cells (~25%) compared to >90% typical for differenTated Tssues. (Stem Cell Reviews 1:111-‐118, 2005) but this may reflect confusion between naïve and primed ES cells
In vitro differenTaTon • Different culture condiTons alter the fate of ES cells in vitro • Protocols exist for all three germ layers
• Many, but not all, protocols involve aggregaTon of ES cells in “embryoid bodies”
• Most protocols do not yield a single type of cell
• SelecTon steps can help to remove undesired cell types
• Need to ask: How far? & How faithful?
PancreaTc β cells: • Pagliuca FW, et al. (2014) GeneraTon of FuncTonal Human PancreaTc β Cells In Vitro. Cell. Oct, 159:428-‐39.
• Rezania A, et al. (2014) Reversal of diabetes with insulin-‐producing cells derived in vitro from human pluripotent stem cells. Nat Biotechnol. Nov, 32:1121-‐33.
ES cells → neurons • pluripotent • funcTonally immortal • geneTcally & developmentally normal
• postmitoTc • polarized • excitable • heterogeneous
4 d 4 d 4 d 6 d
Serum Free Medium
+ RA
ESC
SFD
SF+RA
Kim et al., Developmental Biology 328:456-‐471, 2009
β-tubulin nestin Hoechst
GABA β-tubulin Hoechst GAP43 MAP2 Hoechst
voltage-‐gated Na+ and K+ currents
Developmental Biology 168:342-‐357, 1995
Journal of Neuroscience 16:1056-‐65, 1996
Developmental Biology 328:456-‐471, 2009
Hierarchical clustering by frequency of Gene Ontology terms
Reprogramming
• SCNT – somaTc cell nuclear transfer (reproducTve and therapeuTc cloning) – determinisTc and fairly rapid
• iPS – induced pluripotent stem cells – slow and stochasTc (unTl recently)
• TransdifferenTaTon – conversion of one terminally differenTated cell type into another without de-‐differenTaTon to an immature phenotype. Must rule out cell fusion or other explanaTons.
Reprogramming: somaTc cell nuclear transfer
h0p://www.biotechnologyonline.gov.au/images/contentpages/scnt.gif
Reprogramming Firsts: SCNT Frog:
Gurdon JB. (1962) Adult frogs derived from the nuclei of single somaTc cells. Dev Biol. 4:256-‐73.
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 somaTc cell nuclear transfer. Cell. 153:1228-‐38.
Reprogramming Firsts: iPS cells Mouse:
Takahashi K, Yamanaka S. (2006) InducTon of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell. 126:663-‐76
Human: Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. (2007) InducTon of pluripotent stem cells from adult human fibroblasts by defined factors. Cell. 131:861-‐72. Yu J, Vodyanik MA, Smuga-‐O0o K, et al., (2007) Induced pluripotent stem cell lines derived from human somaTc cells. Science. 318:1917-‐20.
GeneraTng iPS cells
• Express transcripTon factors: Oct3/4, Sox2, Klf4 and c-‐Myc OR Oct3/4, Sox2, Nanog and Lin28
• IniTal de-‐differenTaTon and proliferaTon (day 1-‐3, enhanced by Myc); histone modificaTon and chromaTn reorganizaTon
• 2nd wave of gene expression -‐ stem cell and development related genes (day 9-‐12); DNA demethylaTon and X reacTvaTon
Graf T. Cell Stem Cell 9:504-‐516, 2011
Nature Reviews GeneTcs 14:427-‐439, 2013
Removing the bo0le neck?
• Rais et al., Nature 502:65-‐70, 2013 implicate Mbd3, a component in the NuRD complex that mediates gene repression via histone deacetylaTon and chromaTn 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.
Skipping the bo0le neck?
• Jaenisch lab (Cell Stem Cell 15:295-‐309, 2014) used SNEL factors from the determinisTc phase (Sall4, Nanog, Esrrb and Lin28).
• Obtained fewer but “higher quality” mouse iPSC colonies as judged by producTon of all-‐iPSC mice from 4n blastocyst injecTons, and lack of trisomy 8.
• Has not worked yet in humans
• Is this de-‐differenTaTon or transdifferenTaTon?
TransdifferenTaTon
• Conversion from one differenTated cell type to another without evident de-‐differenTaTon and re-‐differenTaTon
• Must not be confused by cell fusion or selecTon for rare pluripotent cells in the source material.
• Induced by expression of transcripTon factors and microRNAs
Graf T. Cell Stem Cell 9:504-‐516, 2011
Fibroblasts to neurons
• Wernig and colleagues screened 19 transcripTon factors via lenTviral expression
• Found 5 were most criTcal 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 funcTonal synapses)
Yang et al., Cell Stem Cell 9:517-‐525, 2011
Conversion process • Asc1 bHLH transcripTon factor binds to many of the same genomic loci when expressed in fibroblasts, myoblasts or neural progenitors.
• These sites are marked by specific histone modificaTons (H3K4me1, H3K27acetyl, H3K9me3)
• these sites are not accessible in keraTnocytes or osteoblasts, which resist transdifferenTaTon into neurons.
• Brn2 Pou-‐Homeodomain transcripTon factor is recruited by Asc1 to a subset of locaTons
EvaluaTon • SCNT vs iPSCs from isogenic cells:
Ma H, Morey R, O'Neil RC, et al., (2014) AbnormaliTes in human pluripotent cells due to reprogramming mechanisms. Nature 511:177-‐83. Johannesson B, Sagi I, Gore A, et al., (2014) Comparable frequencies of coding mutaTons and loss of imprinTng in human pluripotent cells derived by nuclear transfer and defined factors. Cell Stem Cell 15:634-‐642.
• Origin-‐dependence a�er iPSC differenTaTon: Hargus G, Ehrlich M, Araúzo-‐Bravo MJ, et al., (2014) Origin-‐dependent neural cell idenTTes in differenTated human iPSCs in vitro and a�er transplantaTon into the mouse brain. Cell Reports 8:1697-‐703.
• An opTmizaTon strategy? Morris SA, Cahan P, Li H, et al., (2014) DissecTng engineered cell types and enhancing cell fate conversion via CellNet. Cell 158:889-‐902.
Aldhous, 2001
Goals of Reprogramming: • Models of human disease
• Isogenic cells for replacement therapy
Proof of Concept
Hanna et al., Science 318:1920-‐1923, 2007