Functional properties of cells obtained from human cord blood CD34⁺ stem cells and mouse cardiac myocytes in coculture

Abstract

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34+ cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34+ cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP+ cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca2+] ([Ca2+]i) homeostasis by [Ca2+]i imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP+ cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca2+]i transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca2+]i oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34+ cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.

Knockdown of Cyclin-dependent Kinase Inhibitors Induces Cardiomyocyte Re-entry in the Cell Cycle

“…Neonatal cardiomyocytes were isolated from 1-2-day-old SWISS CD1 mice as described previously (40) and specified in the supplemental "Experimental Procedures." To minimize the number of residual proliferating cardiomyocytes and of contaminating fibroblasts, cell cultures were cultured in minimal essential medium containing 10% FBS in the presence 20 M cytosine ␤-D-arabinofuranoside (AraC, C1768 Sigma) for 48 h. After a 3-day recovery time in AraC-free medium, neonatal cardiomyocytes were transfected.…”

Human cord blood CD34+ progenitor cells acquire functional cardiac properties through a cell fusion process

“…In a previous work (36), we showed that human cord blood CD34 ϩ cells, when cocultured on neonatal mouse cardiomyocytes, exhibit excitation-contraction coupling features similar to those of cardiomyocytes, even though no human genes were upregulated. The aims of the present work are to investigate whether human CD34 ϩ cells, isolated after 1 wk of coculture with neonatal ventricular myocytes, possess molecular and functional properties of cardiomyocytes and to discriminate, using a reporter gene system, whether cardiac differentiation derives from a (trans)differentiation or a cell fusion process.…”

“…ϩ UCB cells in coculture with mouse neonatal cardiomyocytes functionally integrate within the substrate, no data about the time course of this integration were previously described (36) (Fig. 1A), while the EGFP ϩ / CD34 ϩ cells did not contract and were electrically inactive and depolarized ( Fig.…”

“…Depending on the experiment, a number of cells ranging from 3 ϫ 10 5 to 5 ϫ 10 5 CD34 ϩ were transduced with Lenti-GFP vector at multiplicity of infection of 50 or 100 in the cytokine-containing EM (see above), as previously reported (36), in the presence of polybrene (8 g/ml, Sigma-Aldrich) and incubated at 37°C and 5% CO 2 atmosphere for 24 h. Next, cells were harvested and extensively washed three times in 10 ml of PBS supplemented with 5% FCS. Lenti-RFP vector supernatants, at a ratio of 1:1 between viral supernatant and rat cardiomyocyte maintaining medium, were used to obtain RFP-labeled neonatal rat cardiomyocytes.…”

“…Recently, the physiological properties of mouse bone marrow (BM)-derived c-kit ϩ cells and human cord blood CD34 ϩ cells, cocultured with rodent neonatal cardiac myocytes, have been investigated (29,36) by us. However, in those two reports, the electrophysiological properties of cells reisolated from the coculture and the role of cell fusion in the acquisition of a molecular repertoire and a typical excitable/contractile phenotype were not investigated.…”