Viability Of Marrow Stromal Cells Research Articles

Influence of bone marrow stromal microenvironment on forodesine-induced responses in CLL primary cells

AbstractForodesine, a purine nucleoside phosphorylase inhibitor, displays in vitro activity in chronic lymphocytic leukemia (CLL) cells in presence of dGuo, which is the basis for an ongoing clinical trial in patients with fludarabine-refractory CLL. Initial clinical data indicate forodesine has significant activity on circulating CLL cells, but less activity in clearing CLL cells from tissues such as marrow. In tissue microenvironments, lymphocytes interact with accessory stromal cells that provide survival and drug-resistance signals, which may account for residual disease. Therefore, we investigated the impact of marrow stromal cells (MSCs) on forodesine-induced response in CLL lymphocytes. We demonstrate that spontaneous and forodesine-induced apoptosis of CLL cells was significantly inhibited by human and murine MSCs. Forodesine-promoted dGuo triphosphate (dGTP) accumulation and GTP and ATP depletion in CLL cells was inhibited by MSCs, providing a mechanism for resistance. Also, MSCs rescued CLL cells from forodesine-induced RNA- and protein-synthesis inhibition and stabilized and increased Mcl-1 transcript and protein levels. Conversely, MSC viability was not affected by forodesine and dGuo. Collectively, MSC-induced biochemical changes antagonized forodesine-induced CLL cell apoptosis. This provides a biochemical mechanism for MSC-derived resistance to forodesine and emphasizes the need to move toward combinations with agents that interfere with the microenvironment's protective role for improving current therapeutic efforts.

Liquid storage of marrow stromal cells

Marrow stromal cells (MSCs) for clinical trials are inevitably stored before administration, but little is known about the effects of storage on MSCs. The effects of short-term liquid storage on the in vitro function of MSCs intended for a clinical trial were studied. Early-passage human MSCs were suspended in 0.9% saline or culture medium and stored at 4 degrees C or room temperature for up to 72 hours followed by assessment of cell loss, viability, and growth in culture. When stored in saline at 4 degrees C, MSC counts decreased by 5% to 20%, MSC viability decreased 17% to 37%, and MSC growth decreased 65% to 100% after 24-hour storage. Similar results were obtained by MSC storage at room temperature or in Dulbecco's modified Eagle's medium or by addition of 1% human serum albumin (HSA) from two manufacturers. Storage of MSCs in saline with HSA from a third manufacturer maintained MSC viability at prestorage levels and improved poststorage MSC growth versus saline (32 +/- 9% vs. 9 +/- 9%; p < 0.05) or saline with two other HSA preparations (4 +/- 4 and 8 +/- 11%; p < 0.05). MSCs stored at 4 degrees C or room temperature, in saline or culture medium, rapidly become nonviable. HSA preparations vary significantly in their ability to maintain poststorage MSC viability and growth. These results provide insight regarding the effects of storage on MSCs and indicate the need to screen HSA preparations before their use as additives to preserve MSCs during short-term liquid storage.

Erythropoietin Enhances the Angiogenic Potency of Autologous Bone Marrow Stromal Cells in a Rat Model of Myocardial Infarction

Background: Transplantation of marrow stromal cells (MSC) has been shown to improve heart perfusion and cardiac function after ischemia. Erythropoietin (EPO) is capable of inducing angiogenesis and inhibiting cell apoptosis. The aim of this study was to investigate the effect of EPO on the therapeutic potency of MSC transplantation in a rat model of myocardial infarction. Methods: MSC viability was detected by MTT andflow cytometry following culture in serum-free medium for 24 h with or without EPO. Release of vascular endothelial growth factor (VEGF) by MSC incubated with different doses of EPO was assayed using ELISA. Immediately after coronary ligation, autologous MSC (3 × 10⁶ cells) were injected into the ischemic myocardium (MSC and MSC-EPO groups). EPO (3,000 U/kg body weight) was injected daily for 3 consecutive days starting 1 day prior to ligation. The same EPO dose was also injected for consecutive 3 days starting 15 days after surgery (EPO and MSC-EPO groups). Control animals were injected saline solution for the same time period. Cardiac function was assessed by echocardiography 2 and 21 days after surgery, respectively. Western blot and immunohistological assessments were performed to examine the effects of treatments. Results: In vitro, EPO inhibited MSC apoptosis induced by serum-free medium and increased vascular endothelial growth factor (VEGF) release by MSC. In vivo, cardiac infarct size was significantly smaller, cardiac function significantly improved, and capillary density obviously higher in the MSC and EPO groups than in the control group. Combined treatment with EPO infusion and MSC transplantation demonstrated a further decrease in infarct size, a further improvement in cardiac function, and a further increase in capillary density compared with MSC or EPO alone. Furthermore, a higher ratio of phosphorylated Akt to total Akt was measured by Western blot; Bcl-2 was upregulated and Bax was downregulated by immunohistochemistry in the MSC-EPO group compared to the other three groups. Conclusion: Transplantation of MSC combined with EPO infusion is superior to MSC monotherapy for angiogenesis and cardiac function recovery.

Engineering Ex Vivo–Expanded Marrow Stromal Cells to Secrete Calcitonin Gene–Related Peptide Using Adenoviral Vector

Calcitonin gene-related peptide (CGRP) is a target for cardiovascular gene therapy. Marrow stromal cells (MSCs) hold promise for use in adult stem cell-based cell and gene therapy. To determine the feasibility of adenoviral-mediated CGRP gene transfer into ex vivo-expanded MSCs, rat MSCs were isolated, ex vivo expanded, and transduced with adenoviruses. Adprepro-CGRP and AdntlacZ, adenoviral vectors containing prepro-CGRP or nuclear-targeted beta-galactosidase reporter gene ntlacZ under the control of Rous sarcoma virus promoter, were used. In this study, it can be shown that transduction efficiency of adenoviral-mediated gene transfer into ex vivo-expanded MSCs is dose dependent, transgene expression persists for more than 21 days in culture, and adenoviral transduction does not alter the proliferation or viability of MSCs. Transduced MSCs retain multipotentiality and transgene expression after cell differentiation. The expression and secretion of CGRP by Adprepro- CGRP-transduced MSCs was confirmed by Western blot analysis and enzyme immunoassay. The secretion of CGRP by Adprepro-CGRP-transduced MSCs is dose dependent, and the transduced cells release as much as 9.5 +/- 0.4 pmol CGRP/1 x 10(6) cells/48 hours (mean +/- standard error of mean, n = 3) into culture medium at a multiplicity of infection of 300. Furthermore, culture supernatant from Adprepro-CGRP-transduced MSCs increases intracellular cyclic AMP levels in pulmonary artery smooth muscle cells in culture. These findings suggest that replication-deficient recombinant adenovirus can be used to gene engineer ex vivo-expanded MSCs and that high-level secretion of biologically active CGRP can be achieved, underscoring the clinical potential of using this novel adult stem cell-based cell and gene therapy strategy for the treatment of cardiovascular diseases.

In vitro cytotoxicity of redox radical initiators for cross-linking of oligo(poly(ethylene glycol) fumarate) macromers.

A novel hydrogel system based on oligo(poly(ethylene glycol) fumarate) (OPF) is currently being investigated as an injectable carrier for marrow stromal cells (MSCs) for orthopedic tissue engineering applications. This hydrogel is cross-linked using the redox radical initiators ammonium persulfate (APS) and ascorbic acid (AA). In this study, two different persulfate oxidizing agents (APS and sodium persulfate (NaPS)) with three reducing agents derived from ascorbic acid (AA, sodium ascorbate (Asc), and magnesium ascorbate-2-phosphate (Asc-2)) and their combinations were examined to determine the relationship between pH, exposure time, and cytotoxicity for rat MSCs. In addition, gelation times for specific combinations were determined using rheometry. pH and cell viability data after 2 h for combinations ranging from 10 to 500 mM in each reagent showed that there was a smaller pH change and a corresponding higher viability at lower concentrations, regardless of the reagents used. At 10 mM, there was less than a 1.5 unit drop in pH and greater than 90% viability for all initiator combinations examined. However, MSC viability was significantly reduced with concentrations of 100 mM and higher of the initiator combinations. At 100 mM, exposure to NaPS/Asc-2 resulted in significantly more live cells than exposure to APS/AA or NaPS/Asc, but at this concentration, NaPS/Asc-2 exhibited significantly longer OPF gelation onset times than APS/AA. At all combination concentrations, exposure time (10 min vs 2 h) did not significantly affect MSC viability. These data indicate that final pH and/or radical formation have a large impact on MSC viability and that multiple, intertwined testing procedures are required for identification of appropriate initiators for cell encapsulation applications.

In vitro cytotoxicity of unsaturated oligo[poly(ethylene glycol) fumarate] macromers and their cross-linked hydrogels.

Currently, oligo[poly(ethylene glycol) fumarate] (OPF) hydrogels are being investigated as an injectable and biodegradable system for tissue engineering applications. In this study, cytotoxicity of each component of the OPF hydrogel formulation and the resulting cross-linked network was examined. Specifically, OPF synthesized with poly(ethylene glycol) (PEG) of different molecular weights (MW), the cross-linking agent [PEG-diacrylate (PEG-DA)], and the redox initiator pair [ammonium persulfate (APS) and ascorbic acid (AA)] were evaluated for cytotoxicity at 2 and 24 h using marrow stromal cells (MSCs) as model cells. The effect of leachable byproducts of OPF hydrogels on cytotoxicity was also investigated. Upon exposure to various concentrations of OPF for 2 h, greater than 50% of the MSCs were viable, regardless of OPF molecular weight or concentration in the media. After 24 h, the MSCs maintained more than 75% viability except for OPF concentrations higher than 25% (w/v). When examining the cross-linking agent, PEG-DA of higher MW (3400) demonstrated significantly higher viability compared to PEG-DA with MW 575 at all concentrations tested. Considering initiators, when MSCs were exposed to AA and APS, as well as the combination of AA and APS, higher viability was observed at lower concentrations. Once cross-linked, the leachable products from the OPF hydrogels had minimal adverse effects on the viability of MSCs (percentage of live cells was higher than 90% regardless of hydrogel types). The results suggest that, after optimization of cross-linking parameters, OPF-based hydrogels hold promise as novel injectable scaffolds or cell carriers in tissue engineering.