Pluripotent stem cell application guide

Learn how to reprogram induced pluripotent stem cells (iPSCs), maintain PSCs, differentiate them and which markers to use for different cell types

Learn how to reprogram induced pluripotent stem cells (iPSCs), maintain PSCs, differentiate them and which markers to use for germ layers, neural cells et


Research using pluripotent stem cells (PSCs) has revolutionized regenerative medicine and the way we model human diseases. These cells are usually grouped into two types; embryonic stem cells (ESCs) derived from a blastocyst stage embryo and induced pluripotent stem cells (iPSCs) induced to become pluripotent from somatic adult cells. Both PSC types are commonly generated and cultured for disease research, drug discovery, and the study of normal human development.

Here we cover the most important steps in your PSC workflow including the following. 

Contents


1. iPSC reprogramming

iPSCs can be induced by forcing somatic cells to express transcription factors associated with pluripotency eg Oct4, Klf4, Sox2, cMyc, etc (Takahashi et al., 2006). Many different methods have been successfully used to express different combinations of these pluripotency factors resulting in effectively reprogrammed iPSCs. Reprogramming of iPSCs is usually carried out by either chemical methods (Huangfu et al., 2008) or lentiviral and retroviral transfection to incorporate DNA encoding the pluripotency genes into the host genome (Yu et al., 2007).

We provide many different biochemicals you can use to reprogram somatic adult cells into iPSCs. Some key examples of this include the following:

Human breast cancer cells (MDA-MB-468) can be reprogrammed using a combination of rapamycin (mTOR inhibitor) and Y27632 (ROCK inhibitor) with 90% efficacy after seven days of induction (Yuan et al., 2018). This could be achieved using 2 μM Y27632 and rapamycin.

Glycogen synthase kinase 3 (GSK-3) inhibitor, CHIR99021, can be used to reprogram mouse embryonic fibroblasts (MEFs) already transduced to express Oct4 and Klf4 (Li et al., 2009). This was the first example of an iPSC generated without the expression of Sox2. The MEF cells we treated with 10 μM CHIR99021 for four weeks.

BIX 01294, histone methyltransferase inhibitor can be used to reprogram mouse embryonic fibroblasts (MEFs) into PSCs when combined with Oct4 and Klf4 expression (Shi et al., 2008)

2. PSC maintenance

To keep your PSCs in a pluripotent state it is important to handle them with care. PSCs require strict culture media and passaging to keep them healthy and undifferentiated. The two culture systems commonly used for PSCs are either feeder-dependent or feeder-free culture systems.

Feeder-dependent culture

Feeder-dependent cultures use a layer of feeder cells which will provide the PSCs with the growth factors and extracellular matrix proteins they need to keep them healthy and able to expand. MEFs are commonly used as feeder cells.

Feeder-free culture

Feeder-free culture systems use replacement media which provide all the growth factors the PSCs need. This may make things much easier as the cells can grow on simple matrix-coated plates, so you won’t need to worry about removing the feeder-cells for downstream experiments. You can also use biochemicals to help maintain pluripotency in feeder-free culture such as SC-1 (Pluripotin), Dual kinase and GTPase inhibitor.

It is also important to check that your PSCs are still in a pluripotent state. A good way to check for this is to use undifferentiated cell markers in applications such as western blot or ICC. Presence of undifferentiated cell markers such as Oct4 and Sox2 are a good first step to check if your cells are pluripotent.

We recommend that you use these undifferentiated cell markers:

Recombinant Anti-Oct4 antibody [EPR17929] - ChIP Grade (ab181557)

Recombinant Anti-SOX2 antibody [EPR3131] (ab92494)

Recombinant Anti-KLF4 antibody [EPR20753-25] (ab214666)

Recombinant Anti-c-Myc antibody [Y69] (ab32072)

Recombinant Anti-Nanog antibody [EPR20694] - ChIP Grade (ab214549)

You could also try our embryonic stem cell marker panel, to find the right marker for your research.

Embryonic Stem Cell Marker Panel (Human: Oct4, Nanog, Tra-1-60, SOX2, SSEA4) (ab109884)

Undifferentiated cell markers like those listed here are expressed by undifferentiated PSC however using these markers alone does not guarantee that the cells you are working with are pluripotent. To be entirely certain of pluripotency it is essential to also check your PSCs using a functional assay.

3. Differentiation

Once you have your PSCs growing in culture and you have checked that they are expressing undifferentiated cell markers, you can start to differentiate your PSCs into your new cell type of interest. Due to the pluripotent nature of PSCs they have the potential to differentiate into any cell type from neural cells to cardiomyocytes. This differentiation can be done simply using a cocktail of different biochemicals and growth factors. Here we outline some of the biochemicals you can purchase on our catalog and the different cell types you can make using these.

Biochemicals

Neural cell types

SB431542, ALK inhibitor can be used in combination with Noggin to differentiate PSCs into neural progenitor cells (Chambers et al., 2009).

AICAR (Acadesine/AICA riboside), AMPK activator induces astroglial differentiation of neural stem cells (Zhang et al., 2008).

Cardiac cell types

5-Azacytidine has been shown to promote the differentiation of mesenchymal stem cells into cardiomyocytes (Makino et al., 1999).

Glycogen synthase kinase 3 (GSK-3) inhibitor, CHIR99021 can be used to differentiate PSCs into cardiomyocytes (Lian et al., 2013).

Hepatic cell types

Sodium butyrate can be used to generate hepatocytes from ESCs (Rambhatla et al., 2003).

Pancreatic cell types

RepSox, TGF-beta type 1 receptor inhibitor will induce PSCs to become pancreatic β-cells (Hosoya et al., 2012).

Growth factors and cytokines

Members of the fibroblast growth factor (FGF) family play key roles in the differentiation of stem cells into the 3 germ layers. Applying different FGF proteins in combination with other growth factors eg bone morphogenetic proteins (BMPs), Wnts, and epidermal growth factor (EGF) will lead to the differentiation of these germ layers into specific cell types. Here are some growth factor combinations for different cell types.

Neural cell types

FGF-2 and EGF can be combined to make neural progenitor cells from PSCs (Ostenfeld et al.,2004).

Cardiac cell types

BMP-2, BMP-4, Activin A, FGF, and Wnt5a all used in the differentiation of PSCs to cardiac progenitors (Rajala et al., 2011).

Pancreatic cell types

FGF-10 and FGF-7 are used to differentiate PCSs into pancreatic β cell progenitors (Shahjalal et al., 2018)

Hepatic cell types

Wnt3a, Activin A, FGF4, BMP-4, hepatocyte growth factor (HGF) are all used to generate liver progenitor cells (Du et al., 2018).

Lung cell types

BMP-4, Wnt3a, FGF-10, EGF, and retinoic acid (RA) are all used in the production of alveolar type II-like epithelial cells from PSCs (Ghaedi et al., 2015).

4. Cell type characterization

Once you have differentiated your PSCs into your desired cell type eg neural, cardiac, lung, etc, it is important to check that the cells are no longer expressing the undifferentiated cell markers described above (Oct4, KLF-4, c-Myc, Sox2, etc). You can do this as described in section 2: PSC maintenance using the undifferentiated cell markers described there.

It is also now important to check that the cell types you have generated are the cell types you expect them to be. Here we will list some of the antibodies we recommend that you can use to check the presence of markers on different cell types.

Germ layers

Mesoderm

Brachyury: Recombinant Anti-Brachyury / Bry antibody [EPR18113] (ab209665)

CD31: Recombinant Anti-CD31 antibody [EP3095] (ab134168)

CD34: Recombinant Anti-CD34 antibody [EP373Y] (ab81289)

BMP2: Recombinant Anti-BMP2 antibody [EPR20807] (ab214821)

BMP4: Recombinant Anti-BMP4 antibody [EPR6211] (ab124715)

N-cadherin: Anti-N Cadherin antibody (ab18203)

Nodal: Recombinant Anti-Nodal antibody [EPR2057] (ab109317)

Snail: Recombinant Anti-SNAIL antibody [EPR21043] (ab216347)

 

Endoderm

Sox17: Recombinant Anti-SOX17 antibody [EPR20684] (ab224637)

FOXA2: Recombinant Anti-FOXA2 antibody [EPR4466] (ab108422)

GATA4: Recombinant Anti-GATA4 antibody [EPR4768] (ab134057)

 

Ectoderm

Nestin: Recombinant Anti-Nestin antibody [SP103] (ab105389)

Notch1: Recombinant Anti-Notch1 antibody [EP1238Y] (ab52627)

TP63: Recombinant Anti-p63 antibody [EPR5701] (ab124762)

 

Neural cell types

Neural stem cells

Fas: Recombinant Anti-Fas antibody [EPR5700] (ab133619)

Frizzled-9: Recombinant Anti-Frizzled 9 antibody [EPR4011] (ab108628)

MSI1 (Musashi-1): Recombinant Anti-Musashi 1 / Msi1 antibody [EP1302] (ab52865)

MSl2 (Musashi-2): Recombinant Anti-MSI2 antibody [EP1305Y] (ab76148)

Nestin: Recombinant Anti-Nestin antibody [SP103] (ab105389)

Noggin: Recombinant Anti-Noggin antibody [EPR1561] (ab124977)

Pax-6: Recombinant Anti-PAX6 antibody [EPR15858] (ab195045)

Sox1: Recombinant Anti-SOX1 antibody [EPR4766] (ab109290)

Sox2: Recombinant Anti-SOX2 antibody [SP76] (ab93689)

 

Neuronal cells

α-Synuclein: Recombinant Anti-Alpha-synuclein antibody [MJFR1] (ab138501)

NCAM: Recombinant Anti-NCAM1 antibody [EP2567Y] (ab75813)

Doublecortin: Recombinant Anti-Doublecortin antibody [EPR10935(B)] (ab167400)

GABA B Receptor: Recombinant Anti-GABA B Receptor 1 antibody [EPR22954-47] (ab238130)

GAP-43: Recombinant Anti-GAP43 antibody [EP890Y] (ab75810)

Glutamine Synthetase: Recombinant Anti-Glutamine Synthetase antibody [EPR13022(B)] (ab176562)

MASH1: Recombinant Anti-MASH1/Achaete-scute homolog 1 antibody [EPR19840] (ab211327)

mGluR1α: Recombinant Anti-mGluR1a antibody [EPR13540] (ab183712)

Nestin: Recombinant Anti-Nestin antibody [SP103] (ab105389)

Nicastrin: Recombinant Anti-Nicastrin antibody [EPR2575Y] (ab68145)

Pax-5: Recombinant Anti-PAX5 antibody [EPR3730(2)] (ab109443)

P-glycoprotein: Recombinant Anti-P Glycoprotein antibody [EPR10364-57] - BSA and Azide free (ab216656)

Synapsin I: Recombinant Anti-Synapsin I (phospho S553) antibody [E377] (ab32532)

Synaptophysin: Recombinant Anti-Synaptophysin antibody [YE269] (ab32127)

 

Astrocytes

GFAP: Recombinant Anti-GFAP antibody [EPR1034Y] (ab68428)

EAAT1: Recombinant Anti-EAAT1 antibody [EPR12686] (ab181036)

EAAT2: Recombinant Anti-EAAT2 antibody [EPR19794] (ab205247)

Glutamine synthetase: Recombinant Anti-Glutamine Synthetase antibody [EPR13022(B)] (ab176562)

S100 beta: Recombinant Anti-S100 beta antibody [EP1576Y] (ab52642)

ALDH1L1: Recombinant Anti-ALDH1L1 antibody [EPR12743(B)] (ab177463)

 

Oligodendrocytes

NG2: Recombinant Anti-NG2 antibody [EPR9195] (ab139406)

Olig1: Anti-Olig1 antibody [EPR6790] (ab124908)

Oligodendrocyte specific protein (OSP): Recombinant Anti-Oligodendrocyte Specific Protein antibody [EPR12726] (ab175236)

Myelin basic protein (MBP): Recombinant Anti-Myelin Basic Protein antibody [IGX3421] (ab209328)

Myelin oligodendrocyte glycoprotein (MOG): Recombinant Anti-Myelin oligodendrocyte glycoprotein antibody [EPR4282] (ab108505)

Sox10: Recombinant Anti-SOX10 antibody [EPR4007-104] (ab180862)

 

Pancreatic cell types

Glucagon: Recombinant Anti-Glucagon antibody [EP3070] (ab92517)

Insulin: Recombinant Anti-Insulin antibody [EPR17359] (ab181547)

Islet-1: Recombinant Anti-Islet 1 antibody [EP4182] (ab109517)

Pancreatic polypeptide: Recombinant Anti-Pancreatic Polypeptide antibody [EPR22853-61] (ab255827)

PDX-1: Recombinant Anti-PDX1 antibody [EPR3358(2)] (ab134150)

 

Hepatic cell types

α-Fetoprotein: Recombinant Anti-alpha 1 Fetoprotein antibody [EPR9309] (ab169552)

Albumin: Recombinant Anti-Albumin antibody [EPSISR1] (ab137885)

HNF-1α: Recombinant Anti-HNF1 alpha antibody [EPR23054-142] (ab242140)

Tat-SF1: Recombinant Anti-Tat-SF1 antibody [EPR9105(B)] (ab134921)

 

Cardiac cell types

α-Actinin: Recombinant Anti-Sarcomeric Alpha Actinin antibody [EP2529Y] (ab68167)

Cardiac Troponin I: Recombinant Anti-Cardiac Troponin I antibody [EP1106Y] (ab52862)

Cardiac Troponin T: Recombinant Anti-Cardiac Troponin T antibody [EPR3695] (ab91605)

GATA4: Recombinant Anti-GATA4 antibody [EPR4768] (ab134057)

GATA6: Recombinant Anti-Gata6 antibody [EPR3990(N)] (ab175927)

Myogenin: Recombinant Anti-Myogenin antibody [EPR4789] (ab124800)

Nkx2.5: Recombinant Anti-Nkx2.5 antibody [EPR20168] (ab205263)

 

If you’re having trouble finding the right antibody to characterize your differentiated cell type, try our antibody and reagent identification program and let us find the antibody for you.

 

References

Chambers, S. M., Fasano, C. A., Papapetrou, E. P., Tomishima, M., Sadelain, M., & Studer, L. (2009). Highly efficient neural conversion of human ES and iPS cells by dual inhibition of SMAD signaling. Nature Biotechnology, 27(3), 275–280.

Du, C., Feng, Y., Qiu, D., Xu, Y., Pang, M., Cai, N., … Zhang, Q. (2018). Highly efficient and expedited hepatic differentiation from human pluripotent stem cells by pure small-molecule cocktails. Stem Cell Research and Therapy, 9(1), 1–15.

Ghaedi, M. Niklason L.E., and Williams J (2015) Development of Lung Epithelium from Induced Pluripotent Stem Cells. Curr Transplant Rep. 2(1): 81–89.

Hosoya, M. (2012). Preparation of pancreatic β-cells from human iPS cells with small molecules. Islets, 4(3), 249–252.

Huangfu, D., Maehr, R., Guo, W., Eijkelenboom, A., Snitow, M., Chen, A. E., & Melton, D. A. (2008). Induction of pluripotent stem cells by defined factors is greatly improved by small-molecule compounds. Nature Biotechnology, 26(7), 795–797.

Li, W., Zhou, H., Abujarour, R., Zhu, S., Joo, J. Y., Lin, T., … Ding, S. (2009). Generation of human-induced pluripotent stem cells in the absence of exogenous Sox2. Stem Cells, 27(12), 2992–3000.

Lian, X., Zhang, J., Azarin, S. M., Zhu, K., Hazeltine, L. B., Bao, X., … Palecek, S. P. (2013). Directed cardiomyocyte differentiation from human pluripotent stem cells by modulating Wnt/β-catenin signaling under fully defined conditions. Nature Protocols, 8(1), 162–175.

Makino S, K Fukuda, S Miyoshi, F Konishi, H Kodama, J Pan, M Sano, T Takahashi, S Hori, H Abe, J Hata, A Umezawa and S Ogawa. (1999). Cardiomyocytes can be generated from marrow stromal cells in vitro. J Clin Invest 103:697–705.

Ostenfeld T, Svendsen CN. (2004) Requirement for neurogenesis to proceed through the division of neuronal progenitors following differentiation of epidermal growth factor and fibroblast growth factor-2-responsive human neural stem cells. Stem Cells. 22(5):798-811.

Rambhatla, L., Chiu, C. P., Kundu, P., Peng, Y., & Carpenter, M. K. (2003). Generation of hepatocyte-like cells from human embryonic stem cells. Cell Transplantation, 12(1), 1–11.

Rajala, K., Pekkanen-mattila, M., & Aalto-set, K. (2011). Cardiac Differentiation of Pluripotent Stem Cells. 2011(1).

Shahjalal, H. M., Abdal Dayem, A., Lim, K. M., Jeon, T. Il, & Cho, S. G. (2018). Generation of pancreatic β cells for treatment of diabetes: Advances and challenges. Stem Cell Research and Therapy, 9(1).

Shi, Y., Desponts, C., Do, J. T., Hahm, H. S., Schöler, H. R., & Ding, S. (2008). Induction of Pluripotent Stem Cells from Mouse Embryonic Fibroblasts by Oct4 and Klf4 with Small-Molecule Compounds. Cell Stem Cell, 3(5), 568–574.

Takahashi, K., & Yamanaka, S. (2006). Induction of Pluripotent Stem Cells from Mouse Embryonic and Adult Fibroblast Cultures by Defined Factors. Cell, 126(4), 663–676.

Usselman, C. W. N. S. S. J. R. B. (2017). HHS Public Access. Physiology & Behavior, 176(3), 139–148.

Yu, J., Vodyanik, M. A., Smuga-Otto, K., Antosiewicz-Bourget, J., Frane, J. L., Tian, S., … Thomson, J. A. (2007). Induced pluripotent stem cell lines derived from human somatic cells. Science, 318(5858), 1917–1920.

Yuan, J., Zhang, F., You, M., & Yang, Q. (2018). Identification of protein kinase inhibitors to reprogram breast cancer cells. Cell Death and Disease, 9(9).

Zang, Y., Yu, L. F., Pang, T., Fang, L. P., Feng, X., Wen, T. Q., … Li, J. (2008). AICAR induces astroglial differentiation of neural stem cells via activating the JAK/STAT3 pathway independently of AMP-activated protein kinase. Journal of Biological Chemistry, 283(10), 6201–6208. 





Sign up