Human embryonic and fetal mesenchymal stem cells differentiate toward three different cardiac lineages in contrast to their adult counterparts

Abstract

Mesenchymal stem cells (MSCs) show unexplained differences in differentiation potential. In this study, differentiation of human (h) MSCs derived from embryonic, fetal and adult sources toward cardiomyocytes, endothelial and smooth muscle cells was investigated. Labeled hMSCs derived from embryonic stem cells (hESC-MSCs), fetal umbilical cord, bone marrow, amniotic membrane and adult bone marrow and adipose tissue were co-cultured with neonatal rat cardiomyocytes (nrCMCs) or cardiac fibroblasts (nrCFBs) for 10 days, and also cultured under angiogenic conditions. Cardiomyogenesis was assessed by human-specific immunocytological analysis, whole-cell current-clamp recordings, human-specific qRT-PCR and optical mapping. After co-culture with nrCMCs, significantly more hESC-MSCs than fetal hMSCs stained positive for α-actinin, whereas adult hMSCs stained negative. Furthermore, functional cardiomyogenic differentiation, based on action potential recordings, was shown to occur, but not in adult hMSCs. Of all sources, hESC-MSCs expressed most cardiac-specific genes. hESC-MSCs and fetal hMSCs contained significantly higher basal levels of connexin43 than adult hMSCs and co-culture with nrCMCs increased expression. After co-culture with nrCFBs, hESC-MSCs and fetal hMSCs did not express α-actinin and connexin43 expression was decreased. Conduction velocity (CV) in co-cultures of nrCMCs and hESC-MSCs was significantly higher than in co-cultures with fetal or adult hMSCs. In angiogenesis bioassays, only hESC-MSCs and fetal hMSCs were able to form capillary-like structures, which stained for smooth muscle and endothelial cell markers.Human embryonic and fetal MSCs differentiate toward three different cardiac lineages, in contrast to adult MSCs. Cardiomyogenesis is determined by stimuli from the cellular microenvironment, where connexin43 may play an important role.

Figure 1. Characterization of hESC-MSCs.

(A1) Bright field image of a hESC colony in which the cells at the periphery are differentiating toward spindle-shaped fibroblast-like cells and (A2–A3) pure cultures of hESC-MSC. (B1–B2) Confirmation of the human origin of the hMSCs derived from hESC colonies with the aid of a human-specific lamin A/C antibody. Incubation of murine MSCs (mMSCs; negative control cells) with this antibody (B1) did not produce signal corroborating its species specificity. (C1–E2) Immunostaining of hESC colonies and hMSCs derived from these colonies for the embryonic stem cell marker SSEA-4 and the pluripotency markers Oct-4 and Nanog. Nuclei were detected with Hoechst.

Figure 2. Immunocytological assessment of cardiomyogenic differentiation of different types of hMSCs after 10 days of co-culture with nrCMCs.

(A1–B3) A fraction of eGFP-labeled, human-specific lamin A/C positive hESC-MSCs and fetal amniotic, BM and UC hMSCs expressed α-actinin (indicated as α-act), while (C1–C2) adult BM and adipose hMSCs did not. (D) eGFP-labeled human fetal skin fibroblasts (hSFBs; negative control cells) in co-culture with nrCMCs did not stain positive for α-actinin. (E) Quantitative analysis of the cardiomyogenic differentiation of different types of hMSCs. The graph is based on a minimum of 1,200 cells analyzed from 4 separate isolations per hMSC type. ^# P<0.001 vs all fetal and adult hMSC types; ^* P<0.05 vs adult hMSCs; ND is not detected. (F) Intracellular electrophysiological measurements in fetal (amniotic) and adult (adipose) hMSCs at day 10 of co-culture with nrCMCs and after pharmacological uncoupling of gap junctions. Intrinsic action potentials could be recorded from eGFP-labeled fetal cells, while adult hMSCs showed only steady membrane potentials.

Figure 3. Study of cardiomyogenic differentiation in hESC-MSCs and fetal hMSCs after co-culture with nrCFBs for 10 days assessed by immunocytological analysis.

(A1–A2) No α-actinin expression, indicated as α-act, was detected in eGFP-labeled, human-specific lamin A/C positive hESC-MSCs or fetal hMSCs after co-incubation with nrCFBs. Nuclei were detected with Hoechst. (B) Quantitative analysis of cardiomyogenic differentiation of hESC-MSCs and all of the fetal hMSC types co-cultured with nrCFBs or nrCMCs. The graph is based on a minimum of 1,200 cells analyzed from 3 separate isolations per hMSC type. ^# P<0.001 vs hESC-MSCs and fetal hMSCs co-cultured with nrCFBs and fetal hMSCs in co-culture with nrCMCs; ^* P<0.01 vs fetal hESC-MSCs and fetal hMSCs co-cultured with nrCFBs; ND is not detected.

Figure 4. Analysis by qRT-PCR of expression of pluripotency and cardiac genes in hMSCs alone or after co-incubation with nrCMCs.

(A) hESC-MSCs expressed most cardiac-specific genes, which were significantly upregulated after co-incubation with nrCMCs. Expression of the cardiac progenitor genes, Islet-1 and c-kit, was downregulated in the presence of nrCMCs. (B–D) The fetal hMSC types expressed a variety of cardiac-specific genes, which were upregulated after co-culture with nrCMCs. Islet-1 and c-kit mRNA levels decreased in the presence of nrCMCs with the exception of the upregulation of c-kit gene expression in fetal BM MSCs following their co-culture with nrCMCs. (E–F) ANP, Cx43, VEGF and c-kit gene expression was detected in adult hMSCs before and after co-incubation with nrCMCs. (A–F) hMSCs did not express the pluripotency genes Oct-4 and Nanog. ^# P<0.05 vs specific hMSC monoculture; ^* P<0.01 vs specific hMSC monoculture; ^† P<0.001 vs specific hMSC monoculture.

Figure 5. Assessment of CV by optical mapping in co-cultures of nrCMCs and different types of hMSCs.

(A) The presence of hMSCs and nrCFBs after optical mapping was confirmed by immunostaining for human-specific lamin A/C and collagen type I, respectively. Nuclei were detected with Hoechst. (B) Activation maps of the different (co-)cultures reveal significantly higher CVs in nrCMCs monocultures and in hESC-MSCs/nrCMC and fetal amniotic hMSCs/nrCMC co-cultures than in co-cultures of nrCMCs with adult adipose hMSCs or with nrCFBs. CVs were also significantly higher in nrCMC monocultures and in hESC-MSCs/nrCMC co-cultures than in co-cultures between nrCMCs and fetal amniotic hMSCs. Spacing of isochronal lines in activation maps is 4 ms, and colors indicate temporal sequence of activation, starting from the red area. (C) Bar graph of the CVs in nrCMC monocultures and in co-cultures (+) between nrCMCs and nrCFBs or different types of hMSCs as indicated. ^# P<0.001 vs fetal amniotic hMSCs, adult adipose hMSCs and nrCFBs co-incubated with nrCMCs; ^* P<0.01 vs adult adipose hMSCs and nrCFBs co-incubated with nrCMCs.

Figure 6. Angiogenic differentiation capacity of different types of hMSCs assessed by formation of capillary-like structures and expression of angiogenic markers following their culture on Matrigel.

(A1–D1) Bright field images show that hESC-MSCs and fetal hMSCs were able to form stable cellular networks on a basement membrane matrix, while (E1–F1) adult hMSCs could not. (A2–D2) hESC-MSCs and all fetal hMSC types stained positive for the endothelial cell protein, PECAM-1, and the smooth muscle cell protein, smMHC, while (E2–F2) the adult hMSCs were negative for these markers. Nuclei were stained with Hoechst.

Figure 7. Analysis by immunofluorescence microscopy, qRT-PCR and Western blotting of Cx43 expression in hMSC monocultures and in co-cultures of nrCMCs or nrCFBs with different types of hMSCs.

(A1–A2) Immunocytological analysis shows high Cx43 levels in monocultures of hESC-MSCs and fetal amniotic hMSCs. Cx43 was also detected at the interfaces of these young hMSCs with nrCMCs but not with nrCFBs. nrCMCs were visualized by staining with α-actinin (indicated as α-act), while an antibody against human-specific lamin A/C was used to detect hMSCs. Nuclei were detected with Hoechst. (A3–A4) Adult adipose hMSCs contain very low amounts of Cx43 in both monocultures and co-cultures with nrCMCs or nrCFBs. Also in nrCFB monocultures and nrCFB/nrCMC co-cultures Cx43 is barely detectable. (B1–B3) Bar graphs of the assessment by qRT-PCR of Cx43 mRNA levels in hESC-MSC, fetal amniotic hMSC and adult adipose hMSC monocultures and co-cultures (+) of these cells with either nrCMCs or nrCFBs as indicated. (C) Picture of representative part of a Western blot showing that hESC-MSCs and fetal amniotic hMSCs contain large amounts of Cx43 in contrast to adult adipose hMSCs and nrCFBs. The housekeeping protein glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used to check for equal protein loading. ^# P<0.05 vs hMSCs in co-culture with nrCFBs; ^* P<0.01 vs hMSC monocultures and hMSCs in co-culture with nrCFBs.