Generation and characterization of human cardiac resident and non-resident mesenchymal stem cell

Abstract

Despite the surgical and other insertional interventions, the complete recuperation of myocardial disorders is still elusive due to the insufficiency of functioning myocardiocytes. Thus, the use of stem cells to regenerate the affected region of heart becomes a prime important. In line with this human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) have gained considerable interest due to their potential use for mesodermal cell based replacement therapy and tissue engineering. Since MSCs are harvested from various organs and anatomical locations of same organism, thus the cardiac regenerative potential of human cardiac-derived MSCs (hC-MSCs) and human umbilical cord Wharton's Jelly derived MSC (hUC-MSCs) were tested concurrently. At in vitro culture, both hUC-MSCs and hC-MSCs assumed spindle shape morphology with expression of typical MSC markers namely CD105, CD73, CD90 and CD44. Although, hUC-MSCs and hC-MSCs are identical in term of morphology and immunophenotype, yet hUC-MSCs harbored a higher cell growth as compared to the hC-MSCs. The inherent cardiac regenerative potential of both cells were further investigated with mRNA expression of ion channels. The RT-PCR results demonstrated that both MSCs were expressing a notable level of delayed rectifier-like K(+) current (I KDR ) ion channel, yet the relative expression level was considerably varied between hUC-MSCs and hC-MSCs that Kv1.1(39 ± 0.6 vs 31 ± 0.8), Kv2.1 (6 ± 0.2 vs 21 ± 0.12), Kv1.5 (7.4 ± 0.1 vs 6.8 ± 0.06) and Kv7.3 (27 ± 0.8 vs 13.8 ± 0.6). Similarly, the Ca2(+)-activated K(+) current (I KCa ) channel encoding gene, transient outward K(+) current (I to ) and TTX-sensitive transient inward sodium current (I Na.TTX ) encoding gene (Kv4.2, Kv4.3 and hNE-Na) expressions were detected in both groups as well. Despite the morphological and phenotypical similarity, the present study also confirms the existence of multiple functional ion channel currents IKDR, IKCa, Ito, and INa.TTX in undifferentiated hUC-MSCs as of hC-MSCs. Thus, the hUC-MSCs can be exploited as a potential candidate for future cardiac regeneration.

Keywords: Cardiac resident stem cell and umbilical cord stem cell; Electrophysiology; Mesenchymal stem cell.

Fig. 1

Characterization of mesenchymal stem cells (MSC) derived from human umbilical cord and heart biopsy. a, b Images are showing morphology of adherent hUC-MSC and hC-MSC. c Flow Cytometry analysis showing the immunophenotype of hUC-MSC and hC-MSC. Upper panel histogram shows the expression of positive markers for hUC-MSC. d Lower panel histogram shows the expression of positive markers for hC-MSC. e The table shows the mean value of percentage of positive cells (±) standard deviation to the total number of sample analyzed (n = 3). Cells used in this analysis were obtained from the homogenous confluent monolayer at the end of third/fourth passage. The picture was taken using phase contrast microscope at 100× magnification. Scale bar = 200 μm

Fig. 2

Proliferation rate and fold change of hUC-MSCs and hC-MSCs. A 1 × 10³ cells/well of hUC-MSCs and hC-MSCs were seeded into 96 well plates and harvested by Trypan blue dye exclusion test after 24, 48, 72, 96 and 120 h incubation by trypsinization for Trypan blue dye exclusion test after 24, 48, 72, 96 and 120 h incubation. B The fold change was checked over a period of 120 h. C 1 × 10³ cells/well hUC-MSC and hC-MSCs were seeded into 96 well plates and incubated over 24, 48, 72, 96 and 120 h. The MTT reagent was added post incubation at each time points and measured with ELISA plate reader. Cell proliferation was plotted using O.D absorbance values. D The fold change was calculated over a period of 120 h. The variation within each set of triplicates is shown with mean ± SD:^*# P < 0.05 (n = 3)

Fig. 3

Characterization of the two-lineage potential of mesenchymal stem cells (MSCs) from human umbilical cord (hUC) and heart biopsy at Passage 4. a Morphological appearance of undifferentiated and adipogenic differentiated MSC’s. Red color stained cells indicating the accumulation of fat droplets in adipogeneic lineage cells, were not seen in undifferentiated MSC’s. b Morphological images of undifferentiated and osteogenic differentiated MSC’s. Red color stained cells indicate the presence of calcium mineralized droplets in osteogeneic lineage MSC’s. The picture was taken using phase contrast microscope at 100× magnification. Scale bar = 200 μm. (Color figure online)

Fig. 4

Characterization of multiple functional ion channel currents subtypes in different sources of MSCs. A Reverse transcription-polymerase chain reaction data compared between hUC-MSCs, hC-MSCs and heart biopsy. The gel image on the left is showing the mRNAs expression of ion channel subunits. Primer and heart biopsy mRNA were used as a negative and positive control, respectively. GAPDH and ß-Actin were used as an internal control gene. The experiment was conducted in replicate of technical triplicates. B Bar graphs comparing the relative mRNA expression of functional ion channel currents (K⁺ and Na⁺) between experimental groups. The expression of K⁺ channel current was analyzed by quantification of Kv1.1, Kv2.1, Kv1.5, Kv7.3, KCNN3, KCNN4, Kv4.2, Kv4.3 gene expressions and Na⁺ channel current was hNE-Na gene expression. The sources of mRNAs of these cells were obtained from the homogenous confluent monolayer at fourth passage. The variation within each set of triplicates is shown with mean of SD ± :^*#@ P < 0.05 (n = 3)