Diabetic Mesenchymal Stem Cells Research Articles

Photobiomodulation therapy compensate the impairments of diabetic bone marrow mesenchymal stem cells.

Pathophysiologic conditions associated with diabetes mellitus affect mesenchymal stem cells (MSCs), and this phenomenon may lead to some diabetic secondary complications. The present study was conducted to evaluate the impact of photobiomodulation (PBM) on rat diabetic MSC (DMSC) behavior in vitro. For the purpose of PBM, we used helium-neon laser with a wavelength of 632.8nm at three different energy densities (0.5, 1, 2J/cm2) and radiation periodicity of once, twice, and thrice. The survival, proliferation, and apoptosis in the normal MSCs (NMSCs), DMSCs, and diabetic MSCs, which were laser irradiated (DMSCs+L), were assessed using MTT assay, Ki67 immunofluorescence staining, and TUNEL assay, respectively. Our results demonstrated that DMSCs have significantly lower survival (P < 0.05) and proliferation rates (P < 0.001), and dramatically higher population doubling time (PDT, P < 0.001) and apoptosis rates (P < 0.001) as compared to NMSCs. Moreover, PBM with energy density of 1J/cm2 and the periodicity of 1 or 2 times could improve diabetic MSC capabilities in the term of survival, proliferation, and apoptosis. Considering these findings, it is suggested that PBM could improve the ability of diabetic MSCs in vitro prior to transplantation or may rise their capabilities in their native niche in vivo.

Invited commentary

Preventing the advancement of or, better yet, reversing peripheral vascular occlusive disease could save many limbs and provide a better quality of life for our patients. The “magic bullet” of an individual agent placed locally to change the milieu sufficiently to improve collateral vascular development or to stimulate angiogenesis has not been successful to date. However, providing cells that can change the local milieu by providing a slurry of beneficial agents is more promising. Mesenchymal stem cells (MSCs) can provide stromal support and paracrine effects of potential benefit. Unfortunately, one group of patients who could benefit most are those with diabetes, and yet their MSCs (dMSCs) have a deficient expansion capacity. This paper tests the hypothesis that dMSCs have a reversible defect in the epidermal growth factor (EGF) receptor pathway that may blunt cell growth and survival. Human platelet lysate (PL), known to be rich in several growth factors including EGF, might reverse this deficiency, as might EGF alone. It tests whether dMSCs can provide angiogenic stimulus of human umbilical vein endothelial cells (ECs) and, therefore, be a viable regenerative treatment. The study demonstrates that MSCs from the bone marrow and adipose tissue of limbs of well-controlled, nonsmoking diabetic patients have similar population doublings and entered senescence after passage 11, confirming deficient expansion capacity. In addition, dMSCs do not express EGF in their secretome while demonstrating robust expression of other angiogens. Supplementing the dMSCs with PL increased phosphorylation of AKT to healthy MSC levels, whereas providing EGF alone increased only phosphorylated extracellular signal-regulated protein kinases 1 and 2 (ERK1/2) to similar levels as that of PL supplementation. MSC invasion increased over time, but there were inconsistent differences of invasion length and chemokine concentration across MSC types and patients. Both cell types were able to stimulate EC proliferation like standard EC growth media and in co-culture demonstrated longer EC sprouts. In contrast to past literature, this study confirms a lack of EGF expression by dMSCs, providing a deficiency that is cell culture modifiable. Adding EGF increased phosphorylated ERK1/2 levels but not pAKT levels, whereas PL increased both, which may be important because both AKT and ERK1/2 signaling pathways promote cell survival, proliferation, and sustained paracrine activity. The lack of EGF may explain the early senescence noted in the study, but exogenous EGF certainly does not fully correct deficient signaling pathways, whereas PL does better but likely not totally in considering prior studies by the same authors. Both types of dMSCs promote EC proliferation and sprouting, which supports the regenerative benefits of these cells if sufficient numbers for local injection are available. This study has limitations regarding sample size, potential lack of generalization (the population was nonsmoking, well-controlled diabetic patients), and potential bias because the control MSCs were from the iliac crest whereas the experimental MSCs were from the tibia. Provided is some detailed explanation as to how these limitations can be interpreted. Overall, this study is another step forward in the basic science wheel of evidence that will eventually lead to a clinical advancement in regenerative medicine aimed at limb salvage. The opinions or views expressed in this commentary are those of the author and do not necessarily reflect the opinions or recommendations of the Journal of Vascular Surgery the Society for Vascular Surgery. Reversible secretome and signaling defects in diabetic mesenchymal stem cells from peripheral arterial disease patientsJournal of Vascular SurgeryVol. 68Issue 6PreviewRegenerative medicine seeks to stall or to reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral arterial disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). Full-Text PDF Open Archive

Reversible secretome and signaling defects in diabetic mesenchymal stem cells from peripheral arterial disease patients

ObjectiveRegenerative medicine seeks to stall or to reverse the pathologic consequences of chronic diseases. Many people with diabetes have peripheral arterial disease (PAD), which increases their already high risk of major amputation. Cellular therapies are a promising regenerative medicine approach to PAD that can be used to focally inject regenerative cells to endangered tissue beds. Mesenchymal stem cells (MSCs) are known to promote tissue regeneration through stromal support and paracrine stimulation of new blood vessels (angiogenesis). Whereas little is known about human diabetic MSCs (dMSCs), particularly those from patients with PAD, dMSCs have a limited expansion capacity but can be improved with human platelet lysate (PL) supplementation. PL is rich in many growth factors, including epidermal growth factor (EGF), which is known to be important to cell proliferation and survival signaling pathways. We hypothesize that dMSCs have a reversible defect in EGF receptor pathways. The objective of this work was to test this hypothesis using dMSCs from PAD patients. MethodsThe secretome expression of EGF and prominent angiogens was characterized from bone marrow (BM)-derived and adipose tissue-derived (ATD) dMSCs from five patients (six limbs) undergoing major amputation. Western blot was used to characterize the AKT and extracellular signal-regulated protein kinases 1 and 2 expression in dMSCs under standard culture (5% fetal bovine serum plus fibroblast growth factor 2 [FGF2]), 5% human PL, or 5% fetal bovine serum plus EGF. Healthy donor MSCs were control cells. The angiogenic activity of BM- and ATD-dMSCs was tested on human umbilical vein endothelial cells (ECs). Paired t-test, analysis of variance, and Kruskal-Wallis tests were used as appropriate. ResultsBoth BM- and ATD-dMSCs had typical MSC surface marker expression and similar expansion profiles, and they did not express EGF in their secretome. PL supplementation of dMSCs improved AKT signaling, but they were resistant to FGF2 activation of extracellular signal-regulated protein kinases 1 and 2. EGF supplementation led to similar AKT expression as with PL, but PL had greater phosphorylation of AKT at 30 and 60 minutes. The conditioned media from both BM- and ATD-dMSCs had robust levels of prominent angiogens (vascular endothelial growth factor, monocyte chemoattractant protein 1, hepatocyte growth factor), which stimulated EC proliferation and migration, and the co-culture of dMSCs with ECs led to significantly longer EC sprouts in three-dimensional gel than EC-alone pellets. ConclusionsPL and EGF supplementation improves AKT expression in dMSCs over that of FGF2, but PL improved pAKT over that of EGF. Thus, PL supplementation strategies may improve AKT signaling, which could be important to MSC survival in cellular therapies. Furthermore, BM- and ATD-dMSCs have similar secretomes and robust in vitro angiogenic activity, which supports pursuing dMSCs from both reservoirs in regenerative medicine strategies.

Human diabetic mesenchymal stem cells from peripheral arterial disease patients promote angiogenesis through unique secretome signatures

BackgroundDiabetic patients are at increased risk of complications from severe peripheral arterial disease. Mesenchymal stem cells (MSC) may be useful in limiting these complications. Our objective is to test the angiogenic potential of diabetic versus healthy MSCs. MethodsMSCs' angiogenic potential was tested by endothelial cell (EC) proliferation, migration, and 3-dimensional sprouting. Diabetic conditions were simulated with 5.5, 20, or 40 mM glucose. MSC secretome was quantified by enzyme-linked immunosorbent assay. ResultsHuman aortic ECs were most sensitive to glucose conditions and were used for all MSC experiments. Diabetic MSCs had greater 3-dimensional invasion than healthy MSCs (P < .05), but EC sprouting was decreased in high glucose conditions in both diabetic and healthy MSCs. Secretome analysis demonstrated that 20mM glucose stimulated epidermal growth factor (EGF) expression in diabetic and healthy MSCs, but that diabetic MSCs had a unique secretome with increased levels of chemokine (C-X-C motif) ligand 1 (CXCL-1), interleukin six (IL-6), and monocyte chemoattractant protein 1 (MCP-1) (P < .05). ConclusionDespite having similar in vitro angiogenic activity, diabetic MSCs secrete a unique and inflammatory angiogenic signature that may influence MSC survival and function after transplantation in cell therapy applications. Strategies that normalize secretome in diabetic patients may improve the utility of autologous MSCs in this population of patients.

The Compromised Anti-microbial Function of Mesenchymal Stem Cells in Diabetic Patients

Category: Basic Sciences/Biologics Introduction/Purpose: Diabetic foot infection (DFI), including skin infection and osteomyelitis, is a severe complication of late- stage diabetes. Mesenchymal stem cells (MSCs) facilitate bacterial clearance. In bacterial infection, MSCs, via paracrine mediators, regulate the host cell metabolism and inflammatory response. Particularly, MSCs augment the antibacterial function of neutrophils. It is generally believed that hyperglycemia in diabetes is toxic to MSCs/progenitors and detrimental to their regenerative function. It is unknown, however, whether the antibacterial function of MSCs is compromised in diabetes. Methods: Bone marrow samples from 6 diabetic and 4 non-diabetic patients (approved by IRB) were used for MSC isolation. 1. MSCs from both diabetic and non-diabetic patients were treated with lipopolysaccharides (LPS), a bacterial wall component, for 6 hours. The tissue culture media were collected as conditioned medium. E. coli from a single colony were cultured with addition of the conditioned medium generated by either diabetic or non-diabetic MSCs and inoculated on LB-agar plates overnight. Bacterial colonies were counted. 2. Human macrophages were isolated from umbilical cord blood and co-cultured with either diabetic or non-diabetic MSCs, in a trans-well system, for 24 hours. The macrophages were then cultured with heat-inactivated E. coli for one hour. After extensive washing, macrophage and bacteria were stained with Pappenheim method. Bacterial phagocytosis of macrophages, after co- cultured with diabetic or non-diabetic MSCs, was assessed under a microscope. Results: There was no statistical difference in the number of E. coli colonies when regular medium produced by diabetic and non- diabetic MSCs was added into the bacterial culture. When the diabetic and non-diabetic MSCs were treated with LPS and the conditioned medium was collected and added into bacterial cultures, E. coli colonies increased in the diabetic group, about 3 fold, as compared with the non-diabetic group (p &lt; 0.05). Macrophages were counted in defined areas of the Petri dishes and designated as infected or uninfected, according to the presentation of bacterial bodies or not. While the infection rate of macrophages co-cultured with non-diabetic MSCs was 85% (±5.5%), it was 70% (±6.6%) when macrophages were co-cultured with diabetic MSCs (p = 0.006). Conclusion: MSCs-produced paracrine factors suppressed the growth of E. coli but diabetic and non-diabetic MSCs had no difference in such a function. Activation with LPS did not augment the non-diabetic MSCs but weakened diabetic MSCs in suppression of bacterial growth. MSCs regulate macrophages in bacterial phagocytosis. Diabetic MSCs, however, had a limited role in regulation of macrophages. This study demonstrated that MSCs in diabetic patients are compromised in anti-bacterial infection. The results not only deepen the understanding of bacterial infection in diabetes but also open up new strategy to control bacterial infection in diabetic patients.

Abstract 120: Reversal of Nox 4-Induced Oxidative Stress in Type 2 Diabetic Mesenchymal Stem Cells Restores Their Capacity to Augment Postischemic Neovascularization in db/db Mice

Rationale: We have recently shown that experimental type 2 diabetes (db/db mouse) restricts mesenchymal stem cell’ (MSCs) differentiation capacity through a hyperinsulinemia-dependent induction of Nox 4 expression that promotes adipocytic and inhibits endothelial cell differentiation both in vitro and in vivo. We also showed that transplantation of type 2 diabetic MSCs into wild type (WT) recipients reduced post-ischemic neovascularization significantly below that of WT recipients receiving WT MSCs. Objective: This study tested the hypothesis that pretreatment of db/db MSCs with siRNA Nox4 prior to their transplantation and infusion 24 hrs after the induction of hindlimb ischemia restores their differentiation capacity in vivo and rescues their impaired capacity to promote neovascularization in wild type mice. Methods and Results: To reverse oxidative stress in db/db MSCs, we knockdown NOX 4 expression by a siRNA under in vitro conditions. In vitro inhibition of Nox 4 expression of db/db MSCs significantly increased their differentiation capacity towards endothelial cells and reduced their propensity to differentiate into adipocytes. Db/db MSCs also showed significantly increased tubular formation ability after Nox 4 knockdown. We transplanted NOX 4 siRNA transduced db/db MSCs into WT recipients 24 hours after induction of hindlimb ischemia. Blood flow recovery was measured at different time points by laser Doppler scanning. Reversal of oxidative stress in db/db MSCs by pretreatment with Nox4 siRNA prior to transplantation reversed their impaired capacity to augment post-ischemic neovascularization as evidenced by blood flow recovery that increased to that of WT mice. In addition, knockdown of NOX 4 expression in db/db MSCs also increased collateral artery diameter and capillary density by 50%. Conclusions: Pretreatment of db/db MSCs with siRNA against Nox 4 significantly increased their differentiation capacity towards endothelial cells rather than adipocytes and also their capacity to augment neovascularization after induction of hindlimb ischemia. Reversal of MSC oxidant stress induced by type 2 diabetes might permit greater leverage of the therapeutic potential of MSC transplantation to treat cardiovascular diseases.

Diabetic mesenchymal stem cells lack expression of the pluripotency factor KLF4

Diabetic mesenchymal stem cells lack expression of the pluripotency factor KLF4