Human amniotic mesenchymal stem cell-derived induced pluripotent stem cells may generate a universal source of cardiac cells

Abstract

Human amniotic mesenchymal stem cells (hAMSCs) demonstrated partially pluripotent characteristics with a strong expression of Oct4 and Nanog genes and immunomodulatory properties characterized by the absence of HLA-DR and the presence of HLA-G and CD59. The hAMSCs were reprogrammed into induced pluripotent stem cells (iPSCs) that generate a promising source of universal cardiac cells. The hAMSC-derived iPSCs (MiPSCs) successfully underwent robust cardiac differentiation to generate cardiomyocytes. This study investigated 3 key properties of the hAMSCs and MiPSCs: (1) the reprogramming efficiency of the partially pluripotent hAMSCs to generate MiPSCs; (2) immunomodulatory properties of the hAMSCs and MiPSCs; and (3) the cardiac differentiation potential of the MiPSCs. The characteristic iPSC colony formation was observed within 10 days after the transduction of the hAMSCs with a single integration polycistronic vector containing 4 Yamanaka factors. Immunohistology and reverse transcription-polymerase chain reaction assays revealed that the MiPSCs expressed stem cell surface markers and pluripotency-specific genes. Furthermore, the hAMSCs and MiPSCs demonstrated immunomodulatory properties enabling successful engraftment in the SVJ mice. Finally, the cardiac differentiation of MiPSCs exhibited robust spontaneous contractility, characteristic calcium transience across the membrane, a high expression of cardiac genes and mature cardiac phenotypes, and a contractile force comparable to cardiomyocytes. Our results demonstrated that the hAMSCs are reprogrammed with a high efficiency into MiPSCs, which possess pluripotent, immunomodulatory, and precardiac properties. The MiPSC-derived cardiac cells express a c-kit cell surface marker, which may be employed to purify the cardiac cell population and enable allogeneic cardiac stem cell therapy.

FIG. 1.

Immunostain and reverse RT-PCR of the immunomodulatory properties of hAMSCs. (A) Immunostain demonstrates the specific immune characteristics of the hAMSCs: (+)CD59, (+)HLA-G, and (−)HLA-DR. Hoechst 33258 was used for nuclei staining. (B) Reverse RT-PCR analysis similarly confirms the (+)CD59, (+)HLA-G, and (−)HLA-DR profile of hAMSCs. hAMSCs, human amniotic mesenchymal stem cells; RT-PCR, reverse transcription–polymerase chain reaction.

FIG. 2.

Generation of the MiPSCs from hAMSCs. (A) Timeline of the MiPSC generation from the hAMSCs demonstrates the first ESC-like colony found within the first 11 days after transduction. At day 20, the characteristic iPSC colonies were observed, which displayed a cobblestone appearance with prominent nucleoli and a distinct individual cell border. (B) The MiPSCs demonstrated high alkaline phosphatases activity similar to the H7 hESCs but that was not found in the hAMSCs. (C) Teratoma derived from the MiPSCs after transplantation into the hind limbs of SCID mouse was found 5 weeks after the injection. Hematoxylin and eosin stain confirmed that the tumor contained the tissue from 3 germ lines, including gut-like epithelial tissues (endoderm), muscle (mesoderm), and epidermis tissues (ectoderm). iPSC, induced pluripotent stem cells; MiPSC, hAMSC-derived iPSCs; hESCs, human embryonic stem cells.

FIG. 3.

Stemness properties of the hAMSCs and MiPSCs. (A) Immunostaining of hAMSCs demonstrated(+)Tra-1-81, (+)SSEA3, (+)SSEA4, and (−)Tra-1-60 demonstrating partial pluripotency of the hAMSCs. (B) RT-PCR was performed to compare a total of 23 ESC marker expressions between the H7 hESCs and MiPSCs. (C) Immunostain of the MiPSCs was positive for SSEA3, SSEA4, Tra-1-60, and Tra-1-80. Note Tra-1-60 was negative for the hAMSCs. (D) Comparison of the stemness properties between the hAMSCs and MiPSCs demonstrated the absence of Sox2 and Rex1 in the hAMSCs.

FIG. 4.

Immune characteristics of the MiPSCs. (A) Immunostain confirms that both CD 59 and HLA-G were positive and HLA-DR was negative in the MiPSCs (left). However, this profile was not seen with the H7 hESCs (right). (B) RT-PCR analysis was performed to confirm the absence of HLA-DR and HLA-C in the MiPSCs. (C) Splenocytes cocultured with MEF, hESCs, MiPSCs, mouse iPSCs, and hAMSCs for 24 h. Significantly decreased cytotoxicity (P<0.05, n=5) was observed in the cocultures containing the MiPSCs, hAMSCs, and mouse iPSCs (syngeneic). (D) Mouse ESCs, MiPSCs, and hAMSCs were injected into the hind limbs of immunocompetent SVJ mice. The mESCs and hAMSCs demonstrated robust BLI survival signals, while the MiPSCs did not do so at week 1. MEF, mouse embryonic fibroblasts.

FIG. 5.

Cardiac differentiation profile of the MiPSCs. (A) C-kit (CD 117) surface marker was positive in MiPSCs. (B) C-kit (CD 117) surface marker was negative in H7 hESCs. (C) Flow cytometry for MiPSCs demonstrated significant c-kit (+) MiPSC sub-population (arrow). The proportion of c-kit (+) MiPSCs was ∼55%. The control group, IgM isotype stain, exhibited negligible c-kit expression. (D) The contractile areas of the differentiated MiPSCs were characterized by the colocalization of cTNT, Connexin 43, and alpha-sarcomeric actin. Hoechst 33258 was used for nuclei staining (blue). (E) Flow cytometry analysis revealed ∼40.3% cTNT+ cells derived from c-kit (+) MiPSCs, 19.9%* cTnT+ cells derived from unselected MiPSCs, and 4.5%* cTNT+ cells derived from H7 hESCs (*P<0.05, n=3). The cells derived from both MiPSCs and H7 cells were stained with mouse IgM isotype antibodies to be used as the control group, respectively. cTNT, cardiac troponin T.

FIG. 6.

Electrophysiological characterization of the contractile MiPSC-derived cardiac cells. (A) Live-cell calcium imaging of the contractile MiPSC-derived cardiac cells exhibited a characteristic calcium transient with each depolarization. The left image represents a time-lapsed fluorescence image of a line scan of confocal stain, and the right image indicates the calcium peaks. The MiPSC-derived cardiac cells demonstrated mature cardiomyocyte characteristics. (B) The histogram of atomic force microscopy that evaluates the force exerted by each contraction of a representative MiPSC-derived cardiac cell (left image). The maximal contractile force measured for 4 representative beating cells was comparable to that of native cardiomyocytes (right image, 10-0.4∼10-0.6 nN).