Change in Viability and Function of Pancreatic Islets after Coculture with Mesenchymal Stromal Cells: A Systemic Review and Meta-Analysis.

BACKGROUND: There is no clear consensus on the effect of coculture of islets with mesenchymal stem cells (MSCs) on islet function and viability. METHODS: We conducted a meta-analysis of relevant studies to evaluate the effect of coculture of islets with MSCs on the function and viability of islets, both in vitro and in vivo. We searched PubMed, Embase, and Web of Science databases for all relevant studies that compared the effect of coculture of islets with MSCs on the function and viability of islets (language of publication: English; reference period: January 2000-May 2019). Data pertaining to islet function and viability, concentrations of some cytokines, and in vivo experimental outcomes were extracted and compared. RESULTS: Twenty-four articles were included in the meta-analysis. In comparison to islets cultured alone, coculture of islets with MSCs was associated with a significantly higher islet viability [weighted mean difference (WMD), -15.59; -22.34 to -8.83; P < 0.00001], insulin level (WMD, -5.74; -9.29 to -2.19; P = 0.002), insulin secretion index (WMD, -2.45; -3.70 to -1.21; P = 0.0001), and higher concentrations of interleukin-6 (WMD, -1225.66; -2044.47 to -406.86; P = 0.003) and vascular endothelial growth factor (WMD, -1.19; -2.25 to -0.14; P = 0.03). Direct coculture of islets and MSCs significantly increased islet viability (WMD, -19.82; -26.56 to -13.07; P < 0.00001). In the in vivo experiments, coculture of islets with MSCs induced lower fasting blood glucose level (on postoperative days 21 and 28, WMD, 102.60; 27.14 to 178.05; P = 0.008 and WMD, 121.19; 49.56 to 192.82; P = 0.0009) and better glucose tolerance (blood glucose at 30 minutes after intraperitoneal injection of glucose, WMD, 85.92; 5.33 to 166.51; P = 0.04). CONCLUSION: Coculture of islets with MSCs improves insulin secretory function of islets and enhances islet viability. Direct coculture of two cells significantly increased islet viability. MSC-based strategy may be beneficial for clinical islet transplantation for type 1 diabetes in the future.

Novel Mouse miRNA Chr13_novelMiR7354-5p Improves Bone-Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells.

MicroRNAs (miRNAs) that play key roles in the generation of insulin-producing cells from stem cells provide a cell-based approach for insulin replacement therapy. In this study, we used next-generation sequencing to detect the miRNA expression profile of normal mouse pancreatic ß cells, non-ß cells, bone marrow mesenchymal stem cells (BM-MSCs), and adipose-derived stem cells (ADSCs) and determined relative miRNA expression levels in mouse pancreatic ß cells. After the novel mouse miRNA candidates were identified using miRDeep 2.0, we found that Chr13_novelMiR7354-5p, a novel miRNA candidate, significantly promoted the differentiation of BM-MSCs into insulin-producing cells in vitro. Furthermore, Chr13_novelMiR7354-5p-transfected BM-MSCs reversed hyperglycemia in streptozotocin (STZ)-treated diabetic mice. In addition, bioinformatics analyses, a luciferase reporter assay, and western blotting demonstrated that Chr13_novelMiR7354-5p targeted Notch1 and Rbpj. Our results provide compelling evidence of the existence of 65 novel mouse miRNA candidates and present a new treatment strategy to generate insulin-producing cells from stem cells.

Conditioned medium derived from human amniotic stem cells delays H2O2­induced premature senescence in human dermal fibroblasts.

Stem cells derived from human amniotic membrane (hAM) are promising targets in regenerative medicine. A previous study focused on human amniotic stem cells in skin wound and scar­free healing. The present study aimed to investigate whether hydrogen peroxide (H2O2)­induced senescence of human dermal fibroblasts (hDFs) was influenced by the anti­aging effect of conditioned medium (CdM) derived from human amniotic stem cells. First, the biological function of two types of amniotic stem cells, namely human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs), on hDFs was compared. The results of cell proliferation and wound healing assays showed that CdM promoted cell proliferation and migration. In addition, CdM from hAECs and hAMSCs significantly promoted proliferation of senescent hDFs induced by H2O2. These results indicated that CdM protects cells from damage caused by H2O2. Treatment with CdM decreased senescence­associated ß­galactosidase activity and improved the entry of proliferating cells into the S phase. Simultaneously, it was found that CdM increased the activity of superoxide dismutase and catalase and decreased malondialdehyde by reducing H2O2­induced intracellular reactive oxygen species production. It was found that CdM downregulated H2O2­stimulated 8­hydroxydeoxyguanosine and γ­H2AX levels and decreased the expression of the senescence­associated proteins p21 and p16. In conclusion, the findings indicated that the paracrine effects derived from human amniotic stem cells aided delaying oxidative stress­induced premature senescence.

Human Novel MicroRNA Seq-915_x4024 in Keratinocytes Contributes to Skin Regeneration by Suppressing Scar Formation.

Early in gestation, wounds in fetal skin heal by regeneration, in which microRNAs play key roles. Seq-915_x4024 is a novel microRNA candidate confirmed by deep sequencing and mirTools 2.0. It is highly expressed in fetal keratinocytes during early gestation. Using an in vitro wound-healing assay, Transwell cell migration assay, and MTS proliferation assay, we demonstrated that keratinocytes overexpressing seq-915_x4024 exhibited higher proliferative activity and the ability to promote fibroblast migration and fibroblast proliferation. These characteristics of keratinocytes are the same biological behaviors as those of fetal keratinocytes, which contribute to skin regeneration. In addition, seq-915_x4024 suppressed the expression of the pro-inflammatory markers TNF-α, IL-6, and IL-8 and the pro-inflammatory chemokines CXCL1 and CXCL5. We also demonstrated that seq-915_x4024 regulates TGF-ß isoforms and the extracellular matrix. Moreover, using an in vivo wound-healing model, we demonstrated that overexpression of seq-915_x4024 in keratinocytes suppresses inflammatory cell infiltration and scar formation. Using bioinformatics analyses, luciferase reporter assays, and western blotting, we further demonstrated that Sar1A, Smad2, TNF-α, and IL-8 are direct targets of seq-915_x4024. Furthermore, the expression of phosphorylated Smad2 and Smad3 was reduced by seq-915_x4024. Seq-915_x4024 could be used as an anti-fibrotic factor for the treatment of wound healing.

The differentiation of human MSCs derived from adipose and amniotic tissues into insulin-producing cells, induced by PEI@Fe3O4 nanoparticles-mediated NRSF and SHH silencing.

Type 1 diabetes involves the immunologically mediated destruction of insulin­producing cells (IPCs) in the pancreatic islet. Mesenchymal stem cells (MSCs) have the ability to differentiate into IPCs and have become the most promising means for diabetes therapy. The present study demonstrated that human adipose­derived stem cells (hADSCs) and human amniotic MSCs (hAMSCs) are able to differentiate into functional IPCs by knocking down neuronal restrictive silencing factor (NRSF) and Sonic hedgehog (SHH). In the current study, PEI@Fe3O4 nanoparticles (NPs) were used to deliver NRSF small interfering (si)RNA and SHH siRNA to hADSCs and hAMSCs. Following infection with PEI@Fe3O4 NPs containing NRSF siRNA and SHH siRNA, the MSCs were induced to differentiate into IPCs. Four specific genes for islet cells were expressed in the differentiated cells. These cells also produced and released insulin in a glucose­responsive manner. These findings indicated that hADSCs and hAMSCs may be induced to differentiate into IPCs via PEI@Fe3O4 NP­mediated NRSF and SHH silencing.

Repression of COUP-TFI Improves Bone Marrow-Derived Mesenchymal Stem Cell Differentiation into Insulin-Producing Cells.

Identifying molecular mechanisms that regulate insulin expression in bone marrow-derived mesenchymal stem cells (bmMSCs) can provide clues on how to stimulate the differentiation of bmMSCs into insulin-producing cells (IPCs), which can be used as a therapeutic approach against type 1 diabetes (T1D). As repression factors may inhibit differentiation, the efficiency of this process is insufficient for cell transplantation. In this study, we used the mouse insulin 2 (Ins2) promoter sequence and performed a DNA affinity precipitation assay combined with liquid chromatography-mass spectrometry to identify the transcription factor, chicken ovalbumin upstream promoter transcriptional factor I (COUP-TFI). Functionally, bmMSCs were reprogrammed into IPCs via COUP-TFI suppression and MafA overexpression. The differentiated cells expressed higher levels of genes specific for islet endocrine cells, and they released C-peptide and insulin in response to glucose stimulation. Transplantation of IPCs into streptozotocin-induced diabetic mice caused a reduction in hyperglycemia. Mechanistically, COUP-TFI bound to the DR1 (direct repeats with 1 spacer) element in the Ins2 promoter, thereby negatively regulating promoter activity. Taken together, the data provide a novel mechanism by which COUP-TFI acts as a negative regulator in the Ins2 promoter. The differentiation of bmMSCs into IPCs could be improved by knockdown of COUP-TFI, which may provide a novel stem cell-based therapy for T1D.

MicroRNA-149 contributes to scarless wound healing by attenuating inflammatory response.

A fibrotic or pathological scar is an undesired consequence of skin wound healing and may trigger a series of problems. An attenuated inflammatory response is a significant characteristic of fetal skin wound healing, which can contribute to the scarless healing of fetal skin. According to deep sequencing data, microRNA­149 (miR­149) expression was increased in mid-gestational compared with that in late­gestational fetal skin keratinocytes. It was demonstrated that overexpression of miR­149 in HaCaT cells can downregulate the expression of pro­inflammatory cytokines interleukin (IL)­1α, IL­1ß, and IL­6 at basal levels and in inflammatory conditions. Furthermore, miR­149 was revealed to indirectly accelerate transforming growth factor­ß3 and collagen type III expression in fibroblasts, which are essential cells in extracellular matrix remodeling. In a rat skin wound model, miR­149 improved the quality of the arrangement of collagen bundles and reduced inflammatory cell infiltration during skin wound healing. These results indicate that miR­149 may be a potential regulator in improving the quality of skin wound healing.

Comparison of the proliferation, migration and angiogenic properties of human amniotic epithelial and mesenchymal stem cells and their effects on endothelial cells.

In vivo studies have shown that amnion-produced growth factors participate in many diseases that involve angiogenesis, re-epithelialization and immunomodulation. Although human amniotic epithelial cells (hAECs) and human amniotic mesenchymal stem cells (hAMSCs) can be obtained from amniotic membranes, there is little information regarding their biological differences. The aim of the present study was to isolate and characterize cells from human amnions, to investigate the biological potential and behavior of these cells on the function of endothelial cells in vivo and in vitro and to examine variations in the expression profile of growth factors in different human amnion-derived cell types. Amnion fragments were enzymatically digested into two cell fractions, which were analyzed by mesenchymal and epithelial cell markers. Human aortic endothelial cells (hAoECs) were cultured with conditioned medium (CdM) collected from hAECs or hAMSCs. We used scratch and Transwell assays to evaluate migration ability; Cell Counting Kit-8 (CCK-8) and cell cycle analysis to evaluate proliferation ability; and a Matrigel tube formation assay to evaluate angiogenesis ability. To detect expression of angiogenesis-related genes, qPCR and enzyme-linked immunosorbent assay (ELISA) analyses were conducted. As stem cells, hAECs and hAMSCs all expressed the stem cell markers SSEA-4, OCT-4 and SOX-2. CdM obtained from hAECs promoted cell migration; CdM obtained from hAMSCs promoted cell proliferation; CdM obtained from hAECs and hAMSCs both promoted angiogenesis in hAoECs. Amnion-derived cells secreted significant amounts of angiogenic factors including HGF, IGF-1, VEGF, EGF, HB-EGF and bFGF, although differences in the cellular expression profile of these soluble factors were observed. Our results highlight that human amniotic epithelial and mesenchymal stem cells, which showed differences in their soluble factor secretion and angiogenic functions, could be ideal cell sources for regenerative medicine.

Dynamic Expression of Novel MiRNA Candidates and MiRNA-34 Family Members in Early- to Mid-Gestational Fetal Keratinocytes Contributes to Scarless Wound Healing by Targeting the TGF-ß Pathway.

BACKGROUND: Early- to mid-gestational fetal mammalian skin wounds heal rapidly and without scarring. Keratinocytes (KCs) have been found to exert important effects on the regulation of fibroblasts. There may be significant differences of gestational fetal KCs at different ages. The advantages in early- to mid-gestational fetal KCs could lead to fetal scarless wound healing. METHODS: KCs from six human fetal skin samples were divided into two groups: a mid-gestation group (less than 28 weeks of gestational age) and a late-gestation group (more than 28 weeks of gestational age). RNA extracted from KCs was used to prepare a library of small RNAs for next-generation sequencing (NGS). To uncover potential novel microRNA (miRNAs), the mirTools 2.0 web server was used to identify candidate novel human miRNAs from the NGS data. Other bioinformatical analyses were used to further validate the novel miRNAs. The expression levels of the miRNAs were further confirmed by real-time quantitative RT-PCR. RESULTS: A total of 61.59 million reads were mapped to 1,170 known human miRNAs in miRBase. Among a total of 202 potential novel miRNAs uncovered, 106 candidates have a higher probability of being novel human miRNAs. A total of 110 miRNAs, including 22 novel miRNA candidates, were significantly differently expressed between mid- and late-gestational fetal KCs. Thirty-three differentially expressed miRNAs and miR-34 family members are correlated with the transforming growth factor-ß (TGF-ß) pathway. CONCLUSIONS: Taken together, our results provide compelling evidence supporting the existence of 106 novel miRNAs and the dynamic expression of miRNAs that extensively targets the TGF-ß pathway at different gestational ages in fetal KCs. MiRNAs showing altered expression at different gestational ages in fetal KCs may contribute to scarless wound healing in early- to mid-gestational fetal KCs, and thus may be new targets for potential scar prevention and reduction therapies.