Chuan Tong

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.

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.

Controlled release of thymosin beta 4 using a collagen-chitosan sponge scaffold augments cutaneous wound healing and increases angiogenesis in diabetic rats with hindlimb ischemia.

It is important to establish an efficient vascularization for the long-term acceptance of bioengineered skin equivalents treating the cutaneous wounds of diabetic rats with hindlimb ischemia. This study investigates the possible use of a collagen-chitosan sponge scaffold encapsulated with thymosin beta 4 (CCSS-eTβ4), an angiogenic factor, to accelerate cutaneous wound healing in streptozotocin (STZ)-induced diabetic rats with hindlimb ischemia. CCSSs-eTβ4 was fabricated using a freeze-drying method. The scaffolds were analyzed by scanning electron microscopy, swelling and degradation assays, mechanical properties, and scaffolds of 50:50 collagen-chitosan were selected and applied. The controlled release of Tβ4 from the scaffolds elicited localized and prolonged effects over 12 days, as shown by an enzyme-linked immunosorbent assay (ELISA). In vivo, CCSSs-eTβ4 improved diabetic cutaneous wound healing, with faster wound reepithelialization, better dermal reorganization, and higher wound vascularization. Furthermore, CCSSs-eTβ4 downregulated inflammatory genes and upregulated angiogenic genes in the wound tissue. Significant increases in CD31-positive endothelial cells and new vessel density were also observed. In vitro, Tβ4 increased the migratory and proliferative activity of high glucose (HG)-treated human umbilical vein endothelial cells (HUVECs). Meanwhile, we found that Tβ4 could promote HG-treated HUVECs migration and improve angiogenesis by activation of the VEGF/AKT pathway. Overall, these findings demonstrated the promising potential of CCSSs-eTβ4 to promote more effective wound healing and suggest its possible application for diabetic cutaneous wound treatment.

Transdifferentiation of Umbilical Cord- Derived Mesenchymal Stem Cells Into Epidermal-Like Cells by the Mimicking Skin Microenvironment

Human umbilical cord–derived mesenchymal stem cells (UC-MSCs) are multipotent, primitive, and have been widely used for skin tissue engineering. Their transdifferentiation is determined by the local microenvironment. In this study, we investigated the potential epidermal differentiation of UC-MSCs and the formation of epidermis substitutes in a 3-dimensional (3D) microenvironment, which was fabricated by UC-MSCs embedded into collagen–chitosan scaffolds (CCSs) combined with an air–liquid interface (ALI) culture system. Using fluorescence microscope, we observed that UC-MSCs were spindle-shaped and evenly distributed in the scaffold. Methyl thiazolyl blue tetrazolium bromide assay and Live/Dead assay indicated that the CCSs have good biocompatibility with UC-MSCs. Immunohistochemistry and western blotting assay showed that UC-MSCs on the surface of the CCSs were positive for the epidermal markers cytokeratin 19 and involucrin at 14 days. In addition, hematoxylin–eosin staining indicated that multilayered epidermis substitutes were established. The constructed epidermis substitutes were applied to treat full-thickness wounds in rats and proved to promote wound healing. In conclusion, manipulating the 3D microenvironment is a novel method for inducing the epidermal differentiation of MSCs to engineer epidermal substitutes, which provides an alternative strategy for skin tissue engineering.

CD133-directed CAR T cells for advanced metastasis malignancies: A phase I trial

ABSTRACT Expressed by cancer stem cells of various epithelial cell origins, CD133 is an attractive therapeutic target for cancers. Autologous chimeric antigen receptor-modified T-cell directed CD133 (CART-133) was first tested in this trial. The anti-tumor specificity and the postulated toxicities of CART-133 were first assessed. Then, we conducted a phase I clinical study in which patients with advanced and CD133-positive tumors received CART-133 cell-infusion. We enrolled 23 patients (14 with hepatocellular carcinoma [HCC], 7 with pancreatic carcinomas, and 2 with colorectal carcinomas). The 8 initially enrolled patients with HCC were treated by a CART-133 cell dose escalation scheme (0.05–2 × 106/kg). The higher CAR-copy numbers and its reverse relationship with the count of CD133+ cells in peripheral blood led to the determination of an acceptable cell dose is 0.5–2 × 106/kg and reinfusion cycle in 23 patients. The primary toxicity is a decrease in hemoglobin/platelet (≤ grade 3) that is self-recovered within 1 week. Of 23 patients, three achieved partial remission, and 14 achieved stable disease. The 3-month disease control rate was 65.2%, and the median progression-free survival was 5 months. Repeated cell infusions seemed to provide a longer period of disease stability, especially in patients who achieved tumor reduction after the first cell-infusion. 21 out of 23 patients had not developed detectable de novo lesions during this term. Analysis of biopsied tissues by immunohistochemistry showed CD133+ cells were eliminated after CART-133 infusions. This trial showed the feasibility, controllable toxicities, and effective activity of CART-133 transfer for treating patients with CD133-postive and late-stage metastasis malignancies.