Generation and purification of iPSC-derived cardiomyocytes for clinical applications

Abstract

Background: Over the past decade, the field of cell therapy has rapidly expanded with the aim to replace and repair damaged cells and/or tissue. Depending on the disease many different cell types can be used as part of such a therapy. Here we focused on the potential treatment of myocardial infarction, where currently available treatment options are not able to regenerate the loss of healthy heart tissue.

Method: We generated good manufacturing practice (GMP)-compatible cardiomyocytes (iCMs) from transgene- and xenofree induced pluripotent stem cells (iPSCs) that can be seamless adapted for clinical applications. Further protocols were established for replating and freezing/thawing iCMs under xenofree conditions.

Results: iCMs showed a cardiac phenotype, with the expression of specific cardiac markers and absence of pluripotency markers at RNA and protein level. To ensure a pure iCMs population for in vivo applications, we minimized risks of iPSC contamination using RNA-switch technology to ensure safety.

Conclusion: We describe the generation and further processing of xeno- and transgene-free iCMs. The use of GMP-compliant differentiation protocols ab initio facilitates the clinical translation of this project in later stages.

Keywords: Cardiomyocytes; Cell therapy; Clinical translation; Induced pluripotent stem cells.

Fig. 1

Schematic overview of the workflow to produce purified transgene- and xenofree human iPSC-derived cardiomyocytes (iCMs). First somatic cells from a patient are reprogrammed into induced pluripotent stem cells (iPSCs). These iPSCs are differentiated into cardiomyocytes, followed by purification. Human iCMs are either replated or frozen for further applications, such as tissue engineering or disease modelling

Fig. 2

Human blood derived-iPSCs show the pluripotent nature of generated iPSCs. (A) Schematic representation of the timeline and experimental setup. (B) Morphology of an iPSC colony in culture. Immunostaining for pluripotency markers OCT4, SOX2, TRA-1-60, TRA-1-81 (all Alexa 594) and Alkaline phosphatase staining of cultured iPSCs. DAPI is shown in blue. (C) Reprogrammed cells show a normal karyotype. (D) Quantitative qRT-PCR assay for expression analyses of pluripotency markers OCT4, SOX2, NANOG, REX1, and DNTM3 in iPSCs. Data are normalized with GAPDH and relative to the original PBMC. (E) Differentiation of iPSCs into three germ layers. Differentiated cells were analyzed by qRT-PCR for the expression of endodermal (FOXA2 and CXCR4), mesodermal (MSX1 and Hand1) and ectodermal (PAX6 and OTX2) lineage markers. Data are normalized with GAPDH and relative to undifferentiated iPSCs. Quantitative data are presented as mean ± standard deviation (n = 3). One representative clone DW4 is shown in Fig. 2. (Scale bars: 200 μm)

Fig. 3

Human iPSC-derived cardiomyocytes show a cardiac phenotype. (A) Schematic representation of the timeline and experimental setup. (B) After differentiation of iPSC into cardiomyocytes immunostaining was performed for following markers: alpha smooth muscle actin (αSMA), cTNT, Myl2, Oct4, Ki67, Nanog and Dapi. (C) Flow cytometry dot plot of Oct4, cTnT and SIRPA expression for iCMs DW4 and undifferentiated iPSCs. (D) Quantitative RT-PCR assay for expression analyses of mesodermal, cardiac and pluripotent markers at day10 and day17 after differentiation. Data were normalized with GAPDH and relative to the undifferentiated iPSCs. Quantitative data are presented as mean ± standard deviation. One representative clone DW4 is shown. MIM: Mesoderm Induction Media, CMM: Cardiac Maintenance Media, CIM: Cardiac Induction Media, CIM II: Cardiac Induction Media II. (Scale bars: 100 μm)

Fig. 4

Purified human iPSC-derived cardiomyocytes are a safe cell source for in vivo application. (A) Schematic representation of the experimental setup. B + C) Flow cytrometry dot plot of iCMs and iPSC before (upper row) and after (lower row) purification. The proportion of artificially mixed iPS cells in the iCMs is 10% (B) and 50% (C) respectively. Pie charts show percentage of iCMs (red, cTNT positive) and iPSC (blue, Oct4 positive) for the respective marker. (D) Measurement of the volume (mm³) over time after injecting subcutaneously matrigel containing either iCMs (1,000,000 cells) or iPSCs (200,000 cells) using an immunodeficient NSG mouse model. (n = 5) (E) Summary of the tumor formation after subcutaneous injection of iCMs and iPSCs. One representative clone is shown in Fig. 4 (DW4)

Fig. 5

Human iPSC-derived cardiomyocytes can be replated and frozen/thawed for later applications without phenotype change. (A) Quantitative RT-PCR assay for expression analyses of mesodermal, cardiac and pluripotent markers at day10 after differentiation, after replating (P1) and after freeze/thawing (F/T). Data were normalized with GAPDH and relative to the undifferentiated iPSCs. Quantitative data are presented as mean ± standard deviation. (B + C) Immunostaining for different markers (alpha smooth muscle actin (αSMA), Nkx2.5, cTNT, Myl2, Nanog, Ki67, Actinin, Oct4, and Dapi) of cultured iCMs after replating or freeze/thawing. (D) Pie charts of analyzed flow cytometry show percentage of iCM (red, cTNT positive) and iPSC (blue, Oct4 positive) for the respective marker after replating or freeze/thawing. One representative clone DW4 is shown