Differentiated PDGFRα-Positive Cells: A Novel In-Vitro Model for Functional Studies of Neuronal Nitric Oxide Synthase

Abstract

It is evident that depletion of interstitial cells and dysfunction of nitric oxide (NO) pathways are key players in development of several gastrointestinal (GI) motility disorders such as diabetic gastroparesis (DGP). One of the main limitations of DGP research is the lack of isolation methods that are specific to interstitial cells, and therefore conducting functional studies is not feasible. The present study aims (i) to differentiate telomerase transformed mesenchymal stromal cells (iMSCs) into platelet-derived growth factor receptor-α-positive cells (PDGFRα-positive cells) using connective tissue growth factor (CTGF) and L-ascorbic acids; (ii) to investigate the effects of NO donor and inhibitor on the survival rate of differentiated PDGFRα-positive cells; and (iii) to evaluate the impact of increased glucose concentrations, mimicking diabetic hyperglycemia, on the gene expression of neuronal nitric oxide synthase (nNOS). A fibroblastic differentiation-induction medium supplemented with connective tissue growth factor was used to differentiate iMSCs into PDGFRα-positive cells. The medium was changed every day for 21 days to maintain the biological activity of the growth factors. Gene and protein expression, scanning electron and confocal microscopy, and flow cytometry analysis of several markers were conducted to confirm the differentiation process. Methyl tetrazolium cell viability, nitrite measurement assays, and immunostaining were used to investigate the effects of NO on PDGFRα-positive cells. The present study, for the first time, demonstrated the differentiation of iMSCs into PDGFRα-positive cells. The outcomes of the functional studies showed that SNAP (NO donor) increased the survival rate of differentiated PDGFRα-positive cells whereas LNNA (NO inhibitor) attenuated these effects. Further experimentations revealed that hyperglycemia produced a significant increase in expression of nNOS in PDGFRα-positive cells. Differentiation of iMSCs into PDGFRα-positive cells is a novel model to conduct functional studies and to investigate the involvement of NO pathways. This will help in identifying new therapeutic targets for treatment of DGP.

Cryopreservation and validation of differentiated PDGFRα-positive cells for long term usage in experimentation

“…The expression of cell surface markers CD140α/PDGFRα, CD44, and CD34 were measured and analysed in iMSCs, fibroblasts, and PDGFRα-positive cells using flowcytometry. The details of the results can be obtained from published data [ 1 ] [10.3390/ijms22073514].…”

“…The validation of the cryopreserved differentiated PDGFRα-positive cells was done by measuring the Gene expression of Extracellular Matrix (ECM) Proteins (COL I, DEC, ELA, HAS3, and TIMP1) and Stem Cell Differentiation (SCD) markers (ALP, AGG, CD44, and FSP-1) in fibroblasts, iMSCs, and PDGFRα-positive cells. The details of the results from qRT PCR can be obtained from previously published data [ 1 ] [10.3390/ijms22073514] of our group.…”

“…A study published by our research group [ 1 ] has established that telomerase transformed immortalized Mesenchymal Stromal Cells (iMSCs) can be successfully differentiated into PDGFRα-positive cells using connective tissue growth factor (CTGF) and l -ascorbic acid (LAA) [ 1 , 2 ]. The iMSC-derived PDGFRα-positive cells thus obtained have been successfully used as an in-vitro model to conduct functional studies associated with Diabetic Gastroparesis [ 1 , 3 ]. We and others have used this protocol to differentiate iMSCs into PDGFRα-positive cells; however, one of the limitations of the protocol was the waiting time of 21 days for each round of differentiation.…”

Nitric Oxide: From Gastric Motility to Gastric Dysmotility

“…In this view, the observation that in mice expressing a heme-deficient NO-GC (apo-sGC mouse) gastric emptying was delayed, suggests apo-sGC mice as a valid model to investigate motility diseases such as gastroparesis [ 104 ]. Interestingly, it has been recently reported that differentiated PDGFRα+ cells may represent a novel in vitro model to conduct functional studies of NOS and to identify new therapeutic targets for treatment of diabetic gastroparesis [ 105 ].…”

Electroacupuncture Ameliorates Gastrointestinal Motility in Rats with Functional Dyspepsia Via PLC/IP3 Signaling

“…As a classical pathway, PLC hydrolyzes phosphatidylinositol 4,5-bisphosphate (PIP 2 ) at the plasma membrane to generate IP 3 , which is then released into the cytoplasm to trigger intracellular Ca 2+ release and activate related protein kinases, resulting in SMC contraction/relaxation 19 . The PDGFRα + cellmediated pathway is critical for controlling GI smooth muscle tone 20 . Considering the potentially important but poorly clari ed role of PLC in the etiology of GI diseases, we blocked the PLC pathway to explore the effects of this pathway on FD.…”