Calcium Dependent CAMTA1 in Adult Stem Cell Commitment to a Myocardial Lineage

The phenotype of somatic cells has recently been found to be reversible. Direct reprogramming of one cell type into another has been achieved with transduction and over expression of exogenous defined transcription factors emphasizing their role in specifying cell fate. To discover early and novel endogenous transcription factors that may have a role in adult-derived stem cell acquisition of a cardiomyocyte phenotype, mesenchymal stem cells from human and mouse bone marrow and rat liver were co-cultured with neonatal cardiomyocytes as an in vitro cardiogenic microenvironment. Cell-cell communications develop between the two cell types as early as 24 hrs in co-culture and are required for elaboration of a myocardial phenotype in the stem cells 8-16 days later. These intercellular communications are associated with novel Ca(2+) oscillations in the stem cells that are synchronous with the Ca(2+) transients in adjacent cardiomyocytes and are detected in the stem cells as early as 24-48 hrs in co-culture. Early and significant up-regulation of Ca(2+)-dependent effectors, CAMTA1 and RCAN1 ensues before a myocardial program is activated. CAMTA1 loss-of-function minimizes the activation of the cardiac gene program in the stem cells. While the expression of RCAN1 suggests involvement of the well-characterized calcineurin-NFAT pathway as a response to a Ca(2+) signal, the CAMTA1 up-regulated expression as a response to such a signal in the stem cells was unknown. Cell-cell communications between the stem cells and adjacent cardiomyocytes induce Ca(2+) signals that activate a myocardial gene program in the stem cells via a novel and early Ca(2+)-dependent intermediate, up-regulation of CAMTA1

Microscale Flow Control and Droplet Generation Using Arduino-Based Pneumatically-Controlled Microfluidic Device

Microfluidics are crucial for managing small-volume analytical solutions for various applications, such as disease diagnostics, drug efficacy testing, chemical analysis, and water quality monitoring. The precise control of flow control devices can generate diverse flow patterns using pneumatic control with solenoid valves and a microcontroller. This system enables the active modulation of the pneumatic pressure through Arduino programming of the solenoid valves connected to the pressure source. Additionally, the incorporation of solenoid valve sets allows for multichannel control, enabling simultaneous creation and manipulation of various microflows at a low cost. The proposed microfluidic flow controller facilitates accurate flow regulation, especially through periodic flow modulation beneficial for droplet generation and continuous production of microdroplets of different sizes. Overall, we expect the proposed microfluidic flow controller to drive innovative advancements in technology and medicine owing to its engineering precision and versatility

Intracellular Ca²⁺ signals acquired from a dsRed hMSC and neonatal cardiac myocyte, after 48 hrs in co-culture.

<p>(A) Illustrates the three dimensional distribution of hMSCs and cardiac myocytes in co-culture. The image shows a 1.0 µm confocal optical section from a 14 µm thick co-culture. The section shown corresponds to the regions indicated by the blue arrows, confirming that the cells were not overlapping (right upper quadrant). A line scan was acquired along the green horizontal line. Scale bar = 20 µm. (B) Using confocal line scan microscopy Ca²⁺ oscillations corresponding to the hMSCs (red arrow in A) and a cardiac myocyte (white arrow in A) were recorded. Top panel shows Ca²⁺ oscillations recorded in the cytosol of a cardiac myocyte, noted by the white arrow in A. Bottom panel shows perinuclear Ca²⁺ oscillation recorded from a young dsRed hMSC adjacent to a cardiac myocyte (red arrow in A). Note the difference in Ca²⁺ signal amplitude at this early time point. Ca²⁺ signals were recorded on a Zeiss laser scanning confocal microscope at room temperature. All cells were labeled with Fluo-4 AM. Cardiac myocytes were beating spontaneously. (C) Human MSCs response to ionomycin. Fluo-4 fluorescence measured in naive hMSCs in monoculture (left panel), 10 sec after the application of 1 µMol/L ionomycin (middle panel), and 60 sec after the application of 2 mMol/L EGTA (right panel). (D) Transcriptional response to ionomycin-induced intracellular calcium in hMSCs in monocultures. Human MSCs in monocultures were stimulated for 6 hrs with 0.6 µMol/L ionomycin. RT-qPCR from RNA harvested from A: Untreated, control hMSCs. B: hMSCs after 6 hr ionomycin stimulation, C: hMSCs stimulated for 6 hrs with ionomycin, washed, cultured in fresh medium and harvested after 24 hrs of ‘recovery’. Expression of CAMTA1 (left panel); RCAN (right panel). The bars show mean ± SEM, *p<0.05.</p

Regulation of cardiac transcription factors in liver stem cells co-cultured with cardiomyocytes after minimizing CAMTA1 expression.

<p>Except for the control conditions (A and B) the stem cells were pre-transfected with specific siRNA 24 hrs before they were added to the co-culture with cardiomyocytes. A: control, naive stem cells in monoculture for 72 hrs. B: naive stem cells cultured for 24 hrs, then co-cultured with cardiomyocytes for 48 hrs. C: Stem cells pre-transfected with a scrambled siRNA 24 hrs before they were co-cultured for 48 hrs with cardiomyocytes. D: Stem cells were pre-transfected with a human pool of CAMTA1 siRNAs for 24 hrs before they were co-cultured for 48 hrs with cardiomyocytes. The graphs illustrate expression of the transcription factors CAMTA1, CAMTA2, Nk×2.5, Myocardin, cTnT and BetaMHC. A significant decrease in the expression of transcription factors Nkx2.5, Myocardin, cTnT and BetaMHC were measured when CAMTA1 expression was minimized. Note that no significant change in CAMTA2 expression was observed. The bars show mean ± SEM, *p<0.05.</p

Bone marrow-derived MSCs in co-culture with cardiomyocytes.

<p>(A) A nascent cardiomyocyte derived from mMSCs of a βMHC-YFP genotype mouse at 8 days in co-culture with cardiomyocytes. The βMHC-YFP cell is pseudo colored green for visualization of the striations. Left panel shows novel endogenous expression of βMHC-YFP fluorescence along stress fibers and striations (magnified in insert). Middle panel shows surrounding rat cardiomyocyte. Merged images in right panel. (B) GFP-hMSCs co-cultured with cardiomyocytes for 16 days and immunostained for α-actinin or troponin T (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038454#pone.0038454-MullerBorer1" target="_blank">[23]</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038454#pone.0038454.s003" target="_blank">Supporting Information S1</a>). Acquisition of a differentiated cardiac phenotype is demonstrated in the inserts where cardiomyocyte striations are visible. Nuclear DAPI in blue. Scale bar = 20 µm.</p