Snapshot: Directed Differentiation of Pluripotent Stem Cells Luis A

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a 5 02©02Esve n. DOI 10.1016/j.cell.2012.05.015 Cell 149 , May 25,2012 ©2012 Elsevier Inc. 1174 SnapShot: Directed Differentiation of Pluripotent Stem Cells Luis A. Williams, Brandi N. Davis-Dusenbery, and Kevin C. Eggan HHMI, Harvard University, Cambridge, MA 02138, USA

GERM CELLS ECTODERM DERIVATIVES

BMP4 BMP7 BMP8B Primordial germ Meiotic Haploid + + + transduction of cell-like cells cells cells DAZ genes Retinal pigment Epidermis epithelium Photoreceptors Otic hair cells Astrocytes Oligodendrocytes Cortical neurons

Insulin RA Utricle FGF2 Egf Taurine stroma SHH Neural crest hCG Culture on coculture PDGF Retinoids arti cial CNTF p75+, HNK1+ stem cells ACTIVIN A Adherent Sort Spontaneous matrix Fgf2 FGF8 differentiation culture Igf1 Serum Primordial germ FGF2 SHH Oocyte-like Follicle-like cell-like cells EGF cells + structures FGF SHH Ectoderm BMP4 Bmp4 Keratinocytes Neural Scf GABA neurons DA neurons Sort NOGGIN progenitors RA Transplantation Integrin-β3+ Lif Activin a SHH into testes Ssea1+ Egf Fgf2 DKK LDN/ Ascorbic Dorsomorphin WNT acid Sort

BMP4 Nicotinamide Spermatozoa Primordial germ Epiblast-like cells NODAL LEFTY Spinal motor neurons cell-like cells ACTIVIN CD73+ KSR or adherent culture SB431542 Cortical layers BMP4 PA6/MS-5 coculture Optic cup FGF2 Neural retina (NR) EGF SFEBq culture 3D matrigel Retinal pigment VEGF Sort CXCR+,CD117high ACTIVIN A Neuroectoderm culture epithelium (RPE)

Multipotent Deﬁnitive Pluripotent endodermal endoderm Stem Cells INSULIN, TRANSFERIN Serum Sort CD73+ FGF10 progenitor cells RA A-83-01 (ESCs/iPSCs) Selenium OP-9 coculture FGF2, PDGF-BB OP-9 coculture Mesenchymal stem/ TGFβ precursor cells ACTIVIN/ BMP4 Semisolid NODAL VEGF culture BMP4 Mesenchymo- Dexa, IBXT β-GP SCF angioblast Dexa HGF NOGGIN TPO FGF2 Insulin SB431542 Ascorbic Sort FGF4 FLT-3 ACTIVIN A Adipogenic cells EGF Hepatic WNT3A BMP4 Primitive streak acid NCAM+ FGF4 mesoderm progenitors Ascorbic acid VEGF, NT4 FGF10RA CSF, IL-3, IL-6

See online version for legend and references. and legend for version online See TGF 3 SCF, VEGF, BMP4 IL-3, IL-6, BMP4, FGF2 NT4, GDF5 β FOLLISTATIN BMP4, FGF2 Hepatocytes FLT-3, EPO, TPO IL-11, SCF SHH Osteogenic Semisolid BMP4 Chondrogenic cells N2 DAPT culture OP-9 -Serum Cyclopamine VEGF cells TPO coculture DKK DKK NOTCH Pancreatic progenitors EPO Anterior Hemangioblast Hematopoietic IL-3 WNT WNT progenitors IL-6 endoderm VEGF VEGF Skeletal IGF1 IL-11 myoblasts HGF EGF SCF Hindgut BMP4 SCF NOGGIN BMP4 Monocyte/ Cardiovascular Nicotinamide endoderm FGF2 TPO OP-9 or MS-5 macrophage WNTs IL-3 coculture colony-forming cells 3D matrigel FP6 progenitors culture EPO M-CSF Smooth β cells RANKL VEGF SB431542 FGF2 muscle cells

TGFβ Multipotent Erythropoietic Lymphoid Intestinal lung progenitors cells progenitors Osteoclasts Fibronectin, EGM2 tissue Endothelial cells Cardiomyocytes ENDODERM DERIVATIVES MESODERM DERIVATIVES SnapShot: Directed Differentiation of Pluripotent Stem Cells Luis A. Williams, Brandi N. Davis-Dusenbery, and Kevin C. Eggan HHMI, Harvard University, Cambridge, MA 02138, USA

Embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) posses great potential for applications in regenerative medicine, disease modeling, and developmental biology studies. This potential relies on the ability of these cells to differentiate into the hundreds of cell types within the body. Here, we highlight some of strategies for directing the differentiation of ESCs and iPSCs into defined cell types. Most cell types and pathways depicted correspond to published work on human cells, except for the production of spermatozoa, oocyte-like cells, otic hair cells, cortical layers, and optic cup, which were generated with mouse ESCs or iPSCs. In order to uncover these differentiation strategies, stem cell biologists have relied heavily on previous research in model organisms, including Drosophila, Xenopus, chick, and mouse. Early in embryonic development, one of the first cellular differentiation events occurs during gastrulation, which results in the formation of the three germ layers: ectoderm, mesoderm, and endoderm. Similarly, many of the directed differentiation methods rely on the initial specification of ESCs or iPSCs into one of these multipotent lineages, followed by the generation of the particular cell type of interest. The relevant signaling pathways to manipulate in vitro can be gleaned from developmental studies; however, directing the differentiation to defined cell types requires considerable optimization of the precise concentrations, timing, and combinations of factors and small molecules. For this SnapShot, we have indicated key growth and differentiation factors for each pathway in blue and small molecules in red; specific culturing conditions such as coculture with stromal cells are also indicated. Successful differentiation toward a particular cell type is typically determined by the expression of specific markers identified through in vivo studies, but in many cases, additional phenotypic analyses of cells derived from pluripotent stem cells are performed. Although the field of directed differentiation has made rapid progress, many issues remain, including the validation of the maturity and functionality of many of the human cell types derived in vitro (e.g., pancreatic β cells, hepatocytes) and the generation of more complex structures with tissue- or organ-like organization and function. Nevertheless, these challenges represent exciting opportuni- ties for future studies and discoveries.

Abbreviations β-GP, β-glycerol phosphate; BMP, bone morphogenetic protein; CNTF, ciliary neurotrophic factor; CSF, colony-stimulating factor; Dexa, dexamethasone; DKK, Dickkopf; EGF, epidermal growth factor; EPO, erythropoietin; FGF, fibroblast growth factor; FLT, fms-like tyrosine kinase ligand; FP6, IL-6 + IL-6 soluble receptor; GDF, growth differentiation factor; hCG, human chorionic gonadotropin; HGF, hepatocyte growth factor; IBXT, isobutylxanthine; IGF, insulin-like growth factor; IL, interleukin; KSR, knockout serum replace- ment; Lif, leukemia inhibitory factor; M-CSF, macrophage colony-stimulating factor; NT4, neurotrophin; PDGF, platelet-derived growth factor; RA, retinoic acid; RANKL, receptor activator of nuclear factor kappa b ligand; SCF, stem cell factor; SFEBq, Serum-free of embryoid body-like aggregates; SHH, Sonic hedgehog; TGF-β, transforming growth factor b; TPO, thrombopoietin; VEGF, vascular endothelial growth factor.

ACKNOWLEDGEMENTS

We apologize to our many colleagues whose excellent work could not be included due to space constraints.

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