Jiejie Liu

Infusion of Mesenchymal Stem Cells Ameliorates Hyperglycemia in Type 2 Diabetic Rats Identification of a Novel Role in Improving Insulin Sensitivity

Infusion of mesenchymal stem cells (MSCs) has been shown to effectively lower blood glucose in diabetic individuals, but the mechanism involved could not be adequately explained by their potential role in promoting islet regeneration. We therefore hypothesized that infused MSCs might also contribute to amelioration of the insulin resistance of peripheral insulin target tissues. To test the hypothesis, we induced a diabetic rat model by high-fat diet/streptozotocin (STZ) administration, performed MSC infusion during the early phase (7 days) or late phase (21 days) after STZ injection, and then evaluated the therapeutic effects of MSC infusion and explored the possible mechanisms involved. MSC infusion ameliorated hyperglycemia in rats with type 2 diabetes (T2D). Infusion of MSCs during the early phase not only promoted β-cell function but also ameliorated insulin resistance, whereas infusion in the late phase merely ameliorated insulin resistance. Infusion of MSCs resulted in an increase of GLUT4 expression and an elevation of phosphorylated insulin receptor substrate 1 (IRS-1) and Akt (protein kinase B) in insulin target tissues. This is the first report of MSC treatment improving insulin sensitivity in T2D. These data indicate that multiple roles and mechanisms are involved in the efficacy of MSCs in ameliorating hyperglycemia in T2D.

Multiple intravenous infusions of bone marrow mesenchymal stem cells reverse hyperglycemia in experimental type 2 diabetes rats

The worldwide rapid increase in diabetes poses a significant challenge to current therapeutic approaches. Single-dose mesenchymal stem cell (MSC) infusion ameliorates hyperglycemia but fails to restore normoglycemia in diabetic animals. We therefore hypothesized that multiple intravenous MSC infusions may reverse hyperglycemia in type 2 diabetes (T2D) rats. We administered serial allogenous bone-marrow derived MSC infusions (1 × 10(6)cells/infusion) via the tail vein once every 2 weeks to T2D rats, induced by high-fat diet and streptozocin (STZ) administration. Hyperglycemia decreased only transiently after a single infusion in early-phase (1 week) T2D rats, but approximated normal levels after at least three-time infusions. This normal blood level was maintained for at least 9 weeks. Serum concentrations of both insulin and C-peptide were dramatically increased after serial MSC infusions. Oral glucose tolerance tests revealed that glucose metabolism was significantly ameliorated. Immunofluorescence analysis of insulin/glucagon staining revealed the restoration of islet structure and number after multiple MSC treatments. When multiple-MSC treatment was initiated in late-phase (5 week) T2D rats, the results were slightly different. The results of this study suggested that a multiple-MSC infusion strategy offers a viable clinical option for T2D patients.

Hypoxia Pretreatment of Bone Marrow Mesenchymal Stem Cells Facilitates Angiogenesis by Improving the Function of Endothelial Cells in Diabetic Rats with Lower Ischemia

Endothelial dysfunction induced by unordered metabolism results in vascular reconstruction challenges in diabetic lower limb ischemia (DLLI). Mesenchymal stem cells (MSCs) are multipotent secretory cells that are suitable for clinical DLLI treatment, but their use has been hampered by poor survival after injection. Hypoxia can significantly enhance the capacity of MSCs to secrete angiogenic factors. We investigated transient hypoxia pretreatment of MSCs to facilitate revascularization in DLLI. Rat bone marrow MSCs (BM-MSCs) were cultured at different oxygen concentrations for varying time periods. The results indicated that transient pretreatment (5% O2, 48 h) not only increased the expression of VEGF-1α, ANG, HIF-1α and MMP-9 in BM-MSCs as assessed by real-time RT-PCR, but also increased the expression of Bcl-2 as determined by western blotting. The transplantation of pretreated BM-MSCs into rats with DLLI demonstrated accelerated vascular reconstruction when assayed by angiography and immunohistochemistry. CM-Dil-labeled tracer experiments indicated that the survival of BM-MSCs was significantly improved, with approximately 5% of the injected cells remaining alive at 14 days. The expression levels of VEGF-1α, MMP-9 and VEGF-R were significantly increased, and the expression of pAKT was up-regulated in ischemic muscle. Double immunofluorescence studies confirmed that the pretreated BM-MSCs promoted the proliferation and inhibited the apoptosis of endothelial cells. In vitro, pretreated BM-MSCs increased the migratory and tube forming capacity of endothelial cells (ECs). Hypoxia pretreatment of BM-MSCs significantly improved angiogenesis in response to tissue ischemia by ameliorating endothelial cell dysfunction and is a promising therapeutic treatment for DLLI.

Culturing on Wharton's Jelly Extract Delays Mesenchymal Stem Cell Senescence through p53 and p16INK4a/pRb Pathways

Mesenchymal stem cells (MSCs) hold great therapeutic potential. However, MSCs undergo replication senescence during the in vitro expansion process. Whartons jelly from the human umbilical cord harbors a large number of MSCs. In this study, we hypothesized that Whartons jelly would be beneficial for in vitro expansion of MSCs. Whartons jelly extract (WJEs), which is mainly composed of extracellular matrix and cytokines, was prepared as coating substrate. Human MSCs were isolated and cultured on WJE-coated plates. Although the proliferation capacity of cells was not augmented by WJE in early phase culture, adynamic growth in late-phase culture was clearly reduced, suggesting that the replicative senescence of MSCs was efficiently slowed by WJE. This was confirmed by β-galactosidase staining and telomere length measurements of MSCs in late-phase culture. In addition, the decreased differentiation ability of MSCs after long-term culture was largely ameliorated by WJE. Reactive oxygen species (ROS), p53, and p16INK4a/pRb expression increased with passaging. Analysis at the molecular level revealed that WJE-based culture efficiently suppressed the enhancement of intracellular ROS, p53, and p16INK4a/pRb in MSCs. These data demonstrated that WJE provided an ideal microenvironment for MSCs culture expansion in vitro preserved MSC properties by delaying MSCs senescence, and allowed large numbers of MSCs to be obtained for basic research and clinical therapies.

Human umbilical cord-derived mesenchymal stem cells elicit macrophages into an anti-inflammatory phenotype to alleviate insulin resistance in type 2 diabetic rats.

Insulin resistance, a major characteristic of type 2 diabetes (T2D), is closely associated with adipose tissue macrophages (ATMs) that induce chronic low‐grade inflammation. Recently, mesenchymal stem cells (MSCs) have been identified in alleviation of insulin resistance. However, the underlying mechanism still remains elusive. Thus, we aimed to investigate whether the effect of MSCs on insulin resistance was related to macrophages phenotypes in adipose tissues of T2D rats. In this study, human umbilical cord‐derived MSCs (UC‐MSCs) infusion produced significantly anti‐diabetic effects and promoted insulin sensitivity in T2D rats that were induced by a high‐fat diet combined with streptozotocin and directed ATMs into an alternatively activated phenotype (M2, anti‐inflammatory). In vitro, MSC‐induced M2 macrophages alleviated insulin resistance caused by classically activated macrophages (M1, pro‐inflammatory). Further analysis showed that M1 stimulated UC‐MSCs to increase expression of interleukin (IL)‐6, a molecule which upregulated IL4R expression, promoted phosphorylation of STAT6 in macrophages, and eventually polarized macrophages into M2 phenotype. Moreover, the UC‐MSCs effect on macrophages was largely abrogated by small interfering RNA (siRNA) knockdown of IL‐6. Together, our results indicate that UC‐MSCs can alleviate insulin resistance in part via production of IL‐6 that elicits M2 polarization. Additionally, human obesity and insulin resistance were associated with increased pro‐inflammatory ATMs infiltration. Thus, MSCs may be a new treatment for obesity‐related insulin resistance and T2D concerning macrophage polarized effects. Stem Cells 2016;34:627–639

Hypoxia pretreatment of bone marrow-derived mesenchymal stem cells seeded in a collagen-chitosan sponge scaffold promotes skin wound healing in diabetic rats with hindlimb ischemia.

Bone marrow—derived mesenchymal stem cells (BM‐MSCs) have properties that make them promising for the treatment of chronic nonhealing wounds. The major challenge is ensuring an efficient, safe, and painless delivery of BM‐MSCs. Tissue‐engineered skin substitutes have considerable benefits in skin damage resulting from chronic nonhealing wounds. Here, we have constructed a three‐dimensional biomimetic scaffold known as collagen‐chitosan sponge scaffolds (CCSS) using the cross‐linking and freeze‐drying method. Scanning electron microscopy images showed that CCSS had an interconnected network pore configuration about 100 μm and exhibited a suitable swelling ratio for maintaining morphological stability and appropriate biodegradability to improve biostability using swelling and degradation assays. Furthermore, BM‐MSCs were seeded in CCSS using the two‐step seeding method to construct tissue‐engineered skin substitutes. In addition, in this three‐dimensional biomimetic CCSS, BM‐MSCs secreted their own collagen and maintain favorable survival ability and viability. Importantly, BM‐MSCs exhibited a significant upregulated expression of proangiogenesis factors, including HIF‐1α, VEGF, and PDGF following hypoxia pretreatment. In vivo, hypoxia pretreatment of the skin substitute observably accelerated wound closure via the reduction of inflammation and enhanced angiogenesis in diabetic rats with hindlimb ischemia. Thus, hypoxia pretreatment of the skin substitutes can serve as ideal bioengineering skin substitutes to promote optimal diabetic skin wound healing.

Treatment of MSCs with Wnt1a-conditioned medium activates DP cells and promotes hair follicle regrowth

Hair loss (alopecia) is a common problem for people. The dermal papilla is the key signaling center that regulates hair growth and it engage in crosstalk with the microenvironment, including Wnt signaling and stem cells. In this study, we explored the effects of bone marrow mesenchymal stem cell overexpression of Wnt1a on mouse hair follicle regeneration. Wnt-CM accelerated hair follicle progression from telogen to anagen and enhanced the ALP expression in the DP area. Moreover, the hair induction-related genes were upregulated, as demonstrated by qRT-PCR. Wnt-CM treatment restored and increased DP cell expression of genes downregulated by dihydrotestosterone treatment, as demonstrated by qRT-PCR assays. Our study reveals that BM-MSC-generated Wnt1a promotes the DPs ability to induce hair cycling and regeneration.

Hypoxia Regulates the Therapeutic Potential of Mesenchymal Stem Cells Through Enhanced Autophagy

Bone marrow–derived mesenchymal stem cells (BM-MSCs)have great therapeutic potential for the repair of diabetic lower-limb ischemia because of their proangiogenic properties. However, cells transplanted into an ischemic environment have reduced cell survival rates and impaired angiogenic capacity in vivo. We explored hypoxia pretreatment as a method to promote BM-MSC survival by inducing autophagy. Our results showed that hypoxic pretreatment has no effect on the phenotype or differentiation capacity of BM-MSCs; however, hypoxia increased viability and reduced apoptosis in cells treated with lipopolysaccharide. Immunofluorescence and western blot results showed that hypoxia pretreatment enhances cell autophagy mediated by elevated expression of hypoxia inducible factor-1α (HIF-1α). The AMPK/mTOR (adenosine monophosphate–activated protein kinase/mammalian target of rapamycin) signaling pathway was also activated in BM-MSCs during hypoxia-enhanced autophagy. It is important to note that hypoxia pretreatment in BM-MSCs significantly enhanced cell survival and promoted angiogenesis in the lower limb of ischemic diabetic rats. In conclusion, hypoxia pretreatment enhances survival in BM-MSCs, promoting angiogenesis by increasing autophagy and significantly decreasing apoptosis. Therefore, modulation of autophagy with hypoxic pretreatment may provide a novel strategy to improve MSC-based therapies.

Mesenchymal Stem Cell–Conditioned Medium Improves the Proliferation and Migration of Keratinocytes in a Diabetes-Like Microenvironment

The impairment of wound healing in diabetic patients is an important clinical problem. Proper keratinocyte migration and proliferation are the crucial steps during reepithelialization, and these steps may be impaired in diabetes mellitus (DM) due to hyperglycemia and chronic inflammation in wound site. In this study, we explored the effects of diabetes-like microenvironment with high glucose (HG) and intense inflammation on the migration and proliferation of keratinocytes in vitro. We found that the migration and proliferation of rat keratinocytes were reduced with HG and lipopolysaccharide (LPS) stimulation via Erk signaling pathway in a reactive oxygen species (ROS)-dependent manner. Nevertheless, mesenchymal stem cell–conditioned medium (MSC-CM) counteracts the effects of HG and LPS. Treatment of rat keratinocyte with MSC-CM decreased HG- and/or LPS-induced ROS overproduction. Furthermore, MSC-CM reversed the downregulation of phosphorylation of MEK1/2 and Erk 1/2, which was induced by HG and/or LPS without affecting total levels. Our results may provide a possible mechanism for delayed wound healing in DM and provide a foundation to develop MSC-CM as an alternative therapeutic strategy to ameliorate the poor wound-healing conditions.

Bone marrow-derived mesenchymal stem cells ameliorate chronic high glucose-induced β-cell injury through modulation of autophagy

Chronic hyperglycemia causes a progressive decrease of β-cell function and mass in type 2 diabetic patients. Growing evidence suggests that augment of autophagy may be an effective approach to protect β cells against various extra-/intracellular stimuli. In this study, we thus investigated whether bone marrow-derived mesenchymal stem cells (BM-MSCs) could ameliorate chronic high glucose (HG)-induced β-cell injury through modulation of autophagy. Prolonged exposure to HG decreased cell viability, increased cell apoptosis and impaired basal insulin secretion and glucose-stimulated insulin secretion of INS-1 cells, but BM-MSC treatment significantly alleviated these glucotoxic alternations. In addition, western blotting displayed upregulated expression of Beclin1 and LC3-II in INS-1 cells co-cultured with BM-MSCs. Results from immunofluorescence staining and transmission electronic microscope analysis also revealed that BM-MSCs promoted autophagosomes and autolysosomes formation in HG-treated INS-1 cells. However, it should be noted that inhibition of autophagy significantly diminished the protective effects of BM-MSCs on HG-treated INS-1 cells, suggesting that the improvement of β-cell function and survival induced by BM-MSCs was mediated through autophagy. Furthermore, our results showed that BM-MSCs improved mitochondrial function and reduced reactive oxygen species production in HG-treated INS-1 cells, largely owing to autophagic clearance of impaired mitochondria. In vivo study was performed in rats with type 2 diabetes (T2D). BM-MSC infusion not only ameliorated hyperglycemia, but also promoted restoration of pancreatic β cells in T2D rats. Meanwhile, BM-MSC infusion upregulated LAMP2 expression and enhanced formation of autophagosomes and autolysosomes, combined with reduced β-cell apoptosis and increased number of insulin granules. These findings together indicated that BM-MSCs could protect β cells against chronic HG-induced injury through modulation of autophagy in vitro and in vivo. This study unveiled novel evidence of BM-MSCs as an ideal strategy to enhance autophagy for treatment of T2D mellitus.