Delayed enrichment of mesenchymal cells promotes cardiac lineage and calcium transient development

Abstract

Bone marrow-derived mesenchymal stem cells (BM-MSCs) can be induced to differentiate into myogenic cells. Despite their potential, previous studies have not been successful in producing a high percentage of cardiac-like cells with a muscle phenotype. We hypothesized that cardiac lineage development in BM-MSC is related to cell passage, culture milieu, and enrichment for specific cell subtypes before and during differentiation. Our study demonstrated that Lin(-) BM-MSC at an intermediate passage (IP; P8-P12) expressed cardiac troponin T (cTnT) after 21 days in culture. Cardiac TnT expression was similar whether IP cells were differentiated in media containing 5-azacytidine+2% FBS (AZA; 14%) or 2% FBS alone (LS; 12%) and both were significantly higher than AZA+5% FBS. This expression was potentiated by first enriching for CD117/Sca-1 cells followed by differentiation (AZA, 39% and LS, 28%). A second sequential enrichment for the dihydropyridine receptor subunit alpha2delta1 (DHPR-alpha2) resulted in cardiac TnT expressed in 54% of cultured cells compared to 28% of cells after CD117/Sca-1(+) enrichment. Cells enriched for CD117/Sca-1 and subjected to differentiation displayed spontaneous intracellular Ca(2+) transients with an increase in transient frequency and a 60% decrease in the transient duration amplitude between days 14 and 29. In conclusion, IP CD117/Sca-1(+) murine BM-MSCs display robust cardiac muscle lineage development that can be induced independent of AZA but is diminished under higher serum concentrations. Furthermore, temporal changes in calcium kinetics commensurate with increased cTnT expression suggest progressive maturation of a cardiac muscle lineage. Enrichment with CD117/Sca-1 to establish lineage commitment followed by DHPR-alpha2 in lineage developing cells may enhance the therapeutic potential of these cells for transplantation.

Figure 1

Description of first and second series of differentiation experiments: In the first experiment (EXP1) only IP cells expressed cTnT. In the second experiment (EXP2) following CD117/Sca-1 enrichment, EP cells did not adhere and LP cells contained negligible CD117/Sca-1⁺ cells. Only IP cells were adherent and contained CD117/Sca-1⁺ cells and thus were used for the remaining experiments.

Figure 2

Immunofluorescence from cTnT stained IP CD117/Sca-1 enriched cells compared to IP unenriched (control) cells cultured for up to 21 days: A. cTnT staining of control cells treated with LS. B. cTnT staining of CD117/Sca-1⁺ cells treated with LS. C. cTnT staining of control cells treated with AZA. D. cTnT staining of CD117/Sca-1⁺ cells treated with AZA. E. Mean cTnT positive cells for enriched and control fractions cultured in AZA or LS. The number of sampled fields, n, from left to right was 31, 53, 33, and 46 respectively. Values represent mean ± SEM, and significant differences (*) are P<0.05. There was no significant difference between the two enriched groups treated with AZA or LS.

Figure 3

Temporal expression of cTnT and changes in BM-MSC Morphology: Representative immunofluorescent images of cTnT staining observed in IP CD117/Sca-1 enriched cells following differentiation treatment in LS. A. Day 5, B. Day 9, C. Day 15, D. Day 21. E. cTnT⁺ cells area relative to Day 5. The average number of fields and the number of cells imaged per field was (10, 17), (6, 20), (3, 33), (4, 41) for Day 5, 9, 15, and 21 respectively.

Figure 4

Confocal line scan images of IP CD117/Sca-1⁺ cells loaded with Fluo-4/AM showing spontaneous intracellular Ca²⁺ transients: A. Representative Ca²⁺ cycling by Day 14 of culture. B. Representative Ca²⁺ cycling by Day 29. See also supplemental Video-1. C-E. Intracellular Ca²⁺ transients of spontaneously cycling cells at Day 14, 19, and 29 in LS differentiating media: (C) Transient duration at 50% amplitude (TD₅₀). (D) Transient duration at 90% amplitude (TD₉₀). (E) Mean Ca²⁺ transient frequency. The number of cells and the corresponding cycles analyzed were (10, 42), (7, 27), and (9, 76) for days 14, 19, and 29 respectively. Values represent mean + SEM and significant differences (*) are p<0.05.

Figure 5

IP CD117/Sca-1⁺ cell differentiated with LS for 26 days: A. Fluo-4 AM loaded cell. B. Line scan of intracellular Ca²⁺ transient of cell on (A) showing spontaneous changes in [Ca]_i. C. After fixation and staining, cell on (A) was indentified on the plate and verified to be cTnT⁺.

Figure 6

DHPR-α2 enrichment increased the percent of cTnT⁺ cells in LS differentiating media: A. RT-PCR analysis for gene expression evaluation of DHPR-α2, cTnT, cTnI, GATA-4, and GAPDH of IP cells differentiated after day 5, day 15, day 20 and day 26 respectively. An adult heart was used for positive control. B. DHPR-α2 staining of CD117/Sca-1⁺ enriched cells after 20 days in differentiating media. C. DHPR-α2 flow cytometry analysis of CD117/Sca-1⁺ enriched cells after 26 days in differentiating media. D. Representative immunofluorescent image of cTnT staining of IP CD117/Sca-1 and DHPR-α2 enriched cells. E. Mean cTnT positive cells 30 days after DHPR-α2 enrichment (n=45) compared to control (n=46) and CD117/Sca-1⁺ (n=33) cells. Values represent Mean + SEM, and significant differences (*) are P<0.05.

Figure 7

Intracellular Ca²⁺ transients of spontaneously cycling and stimulated cells after CD117/Sca-1 and DHPR-α2 enrichment in LS differentiating media. A. Transient duration at 50% amplitude (TD₅₀). B. Transient duration at 90% amplitude (TD₉₀). C. Mean transient amplitude (TA, ΔF/F₀). Values represent Mean ± SEM and significant differences (*) are p<0.05. The number of cells and the corresponding cycles analyzed were (6, 32), and (5, 26). Refer to supplemental data for representative Ca²⁺ transient examples.

Figure 8

Nifedipine inhibition of Ca²⁺ transients. A. Representative line scan of one cell from CD117/Sca-1 and DHPR-α2 enriched cells in differentiating media after application of 40 μM nifedipine. Cells were continuously stimulated with 0.7 Hz, 80V, 5ms electrical pulse. B. Mean transient amplitude (TA, ΔF/F₀) at sequential time points following application of nifedipine. Values represent Mean + SEM, n=3 cells. Supplemental Video-3 demonstrates that not all calcium transients were inhibited by the L-type calcium channel blocker.