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Generation of multipotent induced cardiac progenitor cells from mouse fibroblasts and potency testing in ex vivo mouse embryos

Nature Protocols volume 12, pages 1029–1054 (2017)Cite this article

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Abstract

Here we describe a protocol to generate expandable and multipotent induced cardiac progenitor cells (iCPCs) from mouse adult fibroblasts using forced expression of Mesp1, Tbx5, Gata4, Nkx2.5 and Baf60c (MTGNB) along with activation of Wnt and JAK/STAT signaling. This method does not use iPS cell factors and thus differs from cell activation and signaling-directed (CASD) reprogramming to cardiac progenitors. Our method is specific to direct CPC reprogramming, whereas CASD reprogramming can generate various cell types depending on culture conditions and raises the possibility of transitioning through a pluripotent cell state. The protocol describes how to isolate and infect primary fibroblasts; induce reprogramming and observe iCPC colonies; expand and characterize reprogrammed iCPCs by immunostaining, flow cytometry and gene expression; differentiate iCPCs in vitro into cardiac-lineage cells; and test the embryonic potency of iCPCs via injection into the cardiac crescent of mouse embryos. A scientist experienced in molecular cell biology and embryology can reproduce this protocol in 12–16 weeks. iCPCs can be used for studying cardiac biology, drug discovery and regenerative medicine.

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Figure 1: Experimental design.
Figure 2: Transduction of adult fibroblasts with cardiac factors and identification of cells undergoing reprogramming based on morphological changes and characterization of iCPCs.
Figure 3: Differentiation of iCPCs into cardiac-lineage cells.
Figure 4: Generation of tools for embryo dissection and cell injection.
Figure 5: Identification of resorbing embryos.
Figure 6: Injection of iCPCs into cardiac crescent for embryonic potency test.

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Acknowledgements

This research was supported by the US National Institutes of Health grants U01HL099773, U01HL134764, R01 HL129798 and S10RR025644 (to T.J.K.); R01 HD042706 and R01 HD079481 (to K.M.D.); T32 HD041921 (to A.M.R.); a predoctoral fellowship R25GM083252 (to A.M.R.); and an AHA predoctoral fellowship 12PRE9520035 (to P.A.L.)

Author information

Authors and Affiliations

  1. Department of Medicine, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Pratik A Lalit & Timothy J Kamp

  2. Stem Cell and Regenerative Medicine Center, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Pratik A Lalit, Adriana M Rodriguez, Karen M Downs & Timothy J Kamp

  3. Department of Cell and Regenerative Biology, University of Wisconsin–Madison, Madison, Wisconsin, USA

    Adriana M Rodriguez, Karen M Downs & Timothy J Kamp

Authors
  1. Pratik A Lalit
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  2. Adriana M Rodriguez
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  3. Karen M Downs
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  4. Timothy J Kamp
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Contributions

P.A.L. conceived project, designed and performed experiments, analyzed data and wrote the final manuscript. K.M.D. and A.M.R. carried out animal husbandry. K.M.D. made all the embryo reagents/tools, designed the potency experiments, performed all the embryo dissections/manipulations/injections/potency testing, analyzed data and wrote the embryonic potency testing section of the manuscript. A.M.R. recorded and edited the supplemental videos. T.J.K. designed experiments, analyzed data, contributed to writing the manuscript, provided funding and approved the final manuscript.

Corresponding author

Correspondence to Timothy J Kamp.

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Competing interests

T.J.K. is a consultant for Cellular Dynamics International, a division of FujiFilm, which is a stem cell biotechnology company.

Supplementary information

Reflexion of uterine muscle and liberation of decidua. (MOV 6976 kb)

Grasping of decidual swellings for transfer to a fresh dish of embryo dissection medium. (MOV 1613 kb)

41596_2017_BFnprot2017021_MOESM412_ESM.mov

Splitting of decidual swellings to expose conceptuses. The first deciduum splits nicely into halves, whereas the second requires an additional snip with the forceps to split it into halves. (MOV 8407 kb)

41596_2017_BFnprot2017021_MOESM413_ESM.mov

Shelling out of conceptuses from the decidual halves. The first conceptus is removed directly from the decidual half; its Reichert's membrane will be removed in a separate step (Supplementary Video 5). The second conceptus demonstrates the option of reflecting Reichert's membrane in situ before removing it from the decidual half. (MOV 5603 kb)

Reflecting of Reichert's membrane. (MOV 2189 kb)

Trimming of Reichert's membrane. (MOV 1984 kb)

Aspiration of cells into the injection pipette for injection into cardiac crescent. (MOV 3251 kb)

41596_2017_BFnprot2017021_MOESM417_ESM.mov

Injection of cells into cardiac crescent. Successful injection into the right cardiac crescent is followed by a deep penetration of the injection pipette into the left cardiac crescent, causing cells to enter the amniotic cavity. (MOV 5648 kb)

41596_2017_BFnprot2017021_MOESM418_ESM.mov

After trimming the ectoplacental cone with the hypodermic needles, an untrimmed conceptus is brought in for a side-by-side comparison. (MOV 1491 kb)

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Lalit, P., Rodriguez, A., Downs, K. et al. Generation of multipotent induced cardiac progenitor cells from mouse fibroblasts and potency testing in ex vivo mouse embryos. Nat Protoc 12, 1029–1054 (2017). https://doi.org/10.1038/nprot.2017.021

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