Induced Pluripotent Stem Cell Differentiation Protocols

What are Induced Pluripotent Stem Cells?

Adult somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs) with the overexpression of key reprogramming genes (OCT4, KLF4, SOX2, cMYC, NANOG and LIN28). Human iPSCs have the unique ability to differentiate into any cell type of the body including:

• Ectodermal: Neuron, Astrocyte, Oligodendrocyte, Retinal Epithelial Cell (RPE), Epidermal, Hair and Keratinocytes.
• Endodermal: Hepatocyte, Pancreatic β-islet Cell, Intestinal Epithelial Cell, Lung Alveolar Cells.
• Mesodermal: Hematopoietic, Endothelial Cell, Cardiomyocyte, Smooth Muscle Cell, Skeletal Muscle Cell, Renal cell, Adipocyte, Chondrocyte and Osteocytes.

Figure 1. IPSC Pathway

iPSC Collection

We offer a large collection of cell culture media, supplements, bioactive small molecules, and growth factors used to control the cell fate of human iPSCs. The table below highlights the most widely used protocols, media and characterization antibodies used to differentiate human iPSC’s into different cell lineages.

Differentiated Cell Type	Media and Supplements	Characterization Antibodies	Reference
Neural Stem Cell	DMEM/F12, Glutamine, Neurobasal Media, N2, B27, SB431542, CHIR99021, Compound E, Human LIF, Noggin, bFGF, EGF Order Complete Media	Nestin, PAX6, OTX2	Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling1
Cortical Neuron	DMEM/F12, Glutamine, Neurobasal Media, N2, B27, SB431542, CHIR99021, Compound E, Noggin, bFGF, EGF Order Complete Media	MAP2, TBR1, CTIP2, VGLUT1	Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks2
Dopaminergic Neuron	DMEM/F12, Glutamine, Neurobasal Media, N2, B27, SB431542, CHIR99021, Compound E, Human LIF, SHH, FGF8, BDNF, GDNF, TGFβ3, cAMP Order Complete Media	TH, TUJ-1, LMX1A, FOXA2, NURR1	Directed differentiation of dopamine neurons from human pluripotent stem cells3
Motor Neurons	DMEM/F12, Glutamine, Neurobasal Media, N2, B27, SB431542, CHIR99021, Compound E, all-trans retinoic acid (ATRA), SHH, BDNF, CNTF, GDNF	Nestin, TUJ-1, ChAT, Islet1, Hb9, Hoxa2	Directed differentiation of human-induced pluripotent stem cells generates active motor neurons4
Astrocyte	DMEM/F12, Glutamine, B27, nonessential amino acids, Penicillin-Streptomycin, FBS, bFGF, CNTF, BMP4, Activin A, Heregulin 1β, IGF-1	GFAP, TUJ-1, CD44	Efficient Generation of Astrocytes from Human Pluripotent Stem Cells in Defined Conditions5
Oligodendrocyte	DMEM/F12, Glutamine, Neurobasal Media, N2, B27, SB431542, CHIR99021, Compound E, Human LIF, all-trans retinoic acid (ATRA), SHH, SAG, PDGF, IGF-I, NT3, Insulin, T3, cAMP	PAX6, OLIG2, NKX2.2, O4, SOX10, MBP, MAP2	Efficient Generation of Myelinating Oligodendrocytes from Primary Progressive Multiple Sclerosis Patients by Induced Pluripotent Stem Cells6
Cardiomyocyte	RPMI 1640, B27, rH-albumin, L-ascorbic acid 2-phosphate, Lactate, CHIR99021, Wnt-C59	TNNT2, α-SMA, α-Actinin, vWF, MLC2A, MLC2V	Chemically defined generation of human cardiomyocytes7
Skeletal Muscle	IMDM, FBS, Glutamine, Penicillin-Streptomycin, nonessential amino acids, 2-mercaptoethanol, oleic acid, linoleic acid, iron sulfate, sodium ferric gluconate, Horse Serum, Y-27632, bFGF	CD13, CD31, CD56, CD44, CD45, CD49b, CD146, MyHC	Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells8
Vascular Endothelial/ Smooth Muscle	DMEM/F12, Glutamine, Neurobasal Media, N2, B27, CHIR99021, BIO, Y-27632, BMP4, VEGF, Forskolin, PDGF-BB, Activin A, Heparin	CD31, CD144, vWF, I-CAM1, SMA, MyHC, VECAD, PECAM	Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells9
Hepatocyte	RPMI 1640, B27, Activin A, BMP4, bFGF, HGF, Oncostatin M	FOXA2, SOX17, GATA4, HNF4-α, AFP, ALB	Highly Efficient Generation of Human Hepatocyte–like Cells from Induced Pluripotent Stem Cells10
Pancreatic β-cell	DMEM/F12, IMDM, bovine serum albumin, N2, B27, ITS, BMP4, Activin A, FGF7, Noggin, bFGF, EGF, nicotinamide, Exendin-4, all-trans retinoic acid (ATRA)	PDX1, FOXA2, SOX9, HNF1β, c-peptide, Insulin, Somatostatin	Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells11
Lung	DMEM/F12, Glutamine, B27, N2, ascorbic acid, monothioglycerol, bovine serum albumin, Penicillin-Streptomycin, Y-27632, Dorsomorphin, SB431542, IWP2, CHIR99021, BMP4, bFGF, Activin A, FGF-10, FGF7,  EGF, all-all-trans retinoic acid (ATRA)	FOXA2, SOX2, NKX2.1, p63	Efficient generation of lung and airway epithelial cells from human pluripotent stem cells12

References

1. Chambers SM, Fasano CA, Papapetrou EP, Tomishima M, Sadelain M, Studer L. 2009. Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nat Biotechnol. 27(3):275-280. https://doi.org/10.1038/nbt.1529

2. Shi Y, Kirwan P, Livesey FJ. 2012. Directed differentiation of human pluripotent stem cells to cerebral cortex neurons and neural networks. Nat Protoc. 7(10):1836-1846. https://doi.org/10.1038/nprot.2012.116

3. Ma L, Liu Y, Zhang S. 2011. Directed Differentiation of Dopamine Neurons from Human Pluripotent Stem Cells.411-418. https://doi.org/10.1007/978-1-61779-201-4_30

4. Karumbayaram S, Novitch BG, Patterson M, Umbach JA, Richter L, Lindgren A, Conway AE, Clark AT, Goldman SA, Plath K, et al. 2009. Directed Differentiation of Human-Induced Pluripotent Stem Cells Generates Active Motor Neurons. 27(4):806-811. https://doi.org/10.1002/stem.31

5. Shaltouki A, Peng J, Liu Q, Rao MS, Zeng X. 2013. Efficient Generation of Astrocytes from Human Pluripotent Stem Cells in Defined Conditions. 31(5):941-952. https://doi.org/10.1002/stem.1334

6. Douvaras P, Wang J, Zimmer M, Hanchuk S, O’Bara M, Sadiq S, Sim F, Goldman J, Fossati V. 2014. Efficient Generation of Myelinating Oligodendrocytes from Primary Progressive Multiple Sclerosis Patients by Induced Pluripotent Stem Cells. Stem Cell Reports. 3(2):250-259. https://doi.org/10.1016/j.stemcr.2014.06.012

7. Burridge PW, Matsa E, Shukla P, Lin ZC, Churko JM, Ebert AD, Lan F, Diecke S, Huber B, Mordwinkin NM, et al. 2014. Chemically defined generation of human cardiomyocytes. Nat Methods. 11(8):855-860. https://doi.org/10.1038/nmeth.2999

8. Maffioletti SM, Gerli MFM, Ragazzi M, Dastidar S, Benedetti S, Loperfido M, VandenDriessche T, Chuah MK, Tedesco FS. 2015. Efficient derivation and inducible differentiation of expandable skeletal myogenic cells from human ES and patient-specific iPS cells. Nat Protoc. 10(7):941-958. https://doi.org/10.1038/nprot.2015.057

9. Patsch C, Challet-Meylan L, Thoma EC, Urich E, Heckel T, O’Sullivan JF, Grainger SJ, Kapp FG, Sun L, Christensen K, et al. 2015. Generation of vascular endothelial and smooth muscle cells from human pluripotent stem cells. Nat Cell Biol. 17(8):994-1003. https://doi.org/10.1038/ncb3205

10. Si-Tayeb K, Noto FK, Nagaoka M, Li J, Battle MA, Duris C, North PE, Dalton S, Duncan SA. 2010. Highly efficient generation of human hepatocyte-like cells from induced pluripotent stem cells. Hepatology. 51(1):297-305. https://doi.org/10.1002/hep.23354

11. Zhang D, Jiang W, Liu M, Sui X, Yin X, Chen S, Shi Y, Deng H. 2009. Highly efficient differentiation of human ES cells and iPS cells into mature pancreatic insulin-producing cells. Cell Res. 19(4):429-438. https://doi.org/10.1038/cr.2009.28

12. Huang SXL, Islam MN, O'Neill J, Hu Z, Yang Y, Chen Y, Mumau M, Green MD, Vunjak-Novakovic G, Bhattacharya J, et al. 2014. Efficient generation of lung and airway epithelial cells from human pluripotent stem cells. Nat Biotechnol. 32(1):84-91. https://doi.org/10.1038/nbt.2754