Histological, immunohistochemical, and genomic evaluation of excisional and diabetic wounds treated with human Wharton's jelly stem cells with and without a nanocarrier.

We showed in previous studies that human umbilical cord Wharton's jelly stem cells (hWJSCs) improved the healing rates of excisional and diabetic wounds in the mouse model. As an extension of those studies, we report here the more detailed quantitative histological, immunohistochemical, and genomic evaluation of biopsies from those excisional and diabetic wounds in an attempt to understand the mechanisms of the enhanced wound healing aided by hWJSCs. Bright-field microscopic observations and ImageJ software analysis on histological sections of the excisional and diabetic wound biopsies collected at different time points showed that the thickness of the epidermis and dermis, and positive picrosirius-red stained areas for collagen, were significantly greater in the presence of hWJSCs compared with controls (P < 0.05). Immunohistochemistry of the diabetic wound biopsies showed increased positive staining for the vascular endothelial marker CD31 and cell proliferation marker Ki67 in the presence of hWJSCs and its conditioned medium (hWJSC-CM). Quantitative real-time polymerase chain reaction showed upregulation of groups of genes involved in extracellular matrix regulation, collagen biosynthesis, angiogenesis, antifibrosis, granulation, and immunomodulation in the presence of hWJSCs. Taken together, the results demonstrated that hWJSCs and hWJSC-CM that contains the paracrine secretions of hWJSCs, enhance the healing of excisional and diabetic wounds via re-epithelialization, collagen deposition, angiogenesis, and immunomodulation. The inclusion of an Aloe vera-polycaprolactone (AV/PCL) nanocarrier did not significantly change the effect of the hWJSCs. However, the topical application of an AV/PCL nanocarrier impregnated with hWJSCs is convenient and less invasive than the administration of hWJSC injections into wounds.

Improving hematopoietic recovery through modeling and modulation of the mesenchymal stromal cell secretome.

BACKGROUND: Efficient and sustained hematopoietic recovery after hematopoietic stem cell or bone marrow transplantation is supported by paracrine signaling from specific subpopulations of mesenchymal stromal cells (MSCs). Here, we considered whether in vitro mechanopriming of human MSCs could be administered to predictively and significantly improve in vivo hematopoietic recovery after irradiation injury. METHODS: First, we implemented regression modeling to identify eight MSC-secreted proteins that correlated strongly with improved rescue from radiation damage, including hematopoietic recovery, in a murine model of hematopoietic failure. Using these partial least squares regression (PLSR) model parameters, we then predicted recovery potential of MSC populations that were culture expanded on substrata of varying mechanical stiffness. Lastly, we experimentally validated these predictions using an in vitro co-culture model of hematopoiesis and using new in vivo experiments for the same irradiation injury model used to generate survival predictions. RESULTS: MSCs grown on the least stiff (elastic moduli ~ 1 kPa) of these polydimethylsiloxane (PDMS) substrata secreted high concentrations of key proteins identified in regression modeling, at concentrations comparable to those secreted by minor subpopulations of MSCs shown previously to be effective in supporting such radiation rescue. We confirmed that these MSCs expanded on PDMS could promote hematopoiesis in an in vitro co-culture model with hematopoietic stem and progenitor cells (HSPCs). Further, MSCs cultured on PDMS of highest stiffness (elastic moduli ~ 100 kPa) promoted expression of CD123+ HSPCs, indicative of myeloid differentiation. Systemic administration of mechanoprimed MSCs resulted in improved mouse survival and weight recovery after bone marrow ablation, as compared with both standard MSC expansion on stiffer materials and with biophysically sorted MSC subpopulations. Additionally, we observed recovery of white blood cells, platelets, and red blood cells, indicative of complete recovery of all hematopoietic lineages. CONCLUSIONS: These results demonstrate that computational techniques to identify MSC biomarkers can be leveraged to predict and engineer therapeutically effective MSC phenotypes defined by mechanoprimed secreted factors, for translational applications including hematopoietic recovery.

Oxygen-regulated gene expression in murine cumulus cells.

Oxygen is an important component of the environment of the cumulus-oocyte complex (COC), both in vivo within the ovarian follicle and during in vitro oocyte maturation (IVM). Cumulus cells have a key role in supporting oocyte development, and cumulus cell function and gene expression are known to be altered when the environment of the COC is perturbed. Oxygen-regulated gene expression is mediated through the actions of the transcription factors, the hypoxia-inducible factors (HIFs). In the present study, the effect of oxygen on cumulus cell gene expression was examined following in vitro maturation of the murine COC at 2%, 5% or 20% oxygen. Increased expression of HIF-responsive genes, including glucose transporter-1, lactate dehydrogenase A and BCL2/adenovirus E1B interacting protein 3, was observed in cumulus cells matured at 2% or 5%, compared with 20% oxygen. Stabilisation of HIF1α protein in cumulus cells exposed to low oxygen was confirmed by western blot and HIF-mediated transcriptional activity was demonstrated using a transgenic mouse expressing green fluorescent protein under the control of a promoter containing hypoxia response elements. These results indicate that oxygen concentration influences cumulus cell gene expression and support a role for HIF1α in mediating the cumulus cell response to varying oxygen.

A nanoscaffold impregnated with human wharton's jelly stem cells or its secretions improves healing of wounds.

Wound healing is a major problem in diabetic patients and current methods of treatment have met with limited success. Since skin cell renewal is under the control of mesenchymal stem cells (MSCs) treatment of wounds has been attempted with the application of exogenous bone marrow MSCs (hBMMSCs). However, hBMMSCs have the limitations of painful harvest, low cell numbers and short-lived stemness properties unlike MSCs from the Wharton's jelly of human umbilical cords (hWJSCs). Since nanoscaffolds provide three dimensional architectural patterns that mimic in vivo stem cell niches and aloe vera has antibacterial properties we evaluated the use of an aloe vera-polycaprolactone (AV/PCL) nanoscaffold impregnated with green fluorescent protein (GFP)-labeled hWJSCs (GFP-hWJSCs + AV/PCL) or its conditioned medium (hWJSC-CM + AV/PCL) for healing of excisional and diabetic wounds. In skin fibroblast scratch-wound assays exposed to GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL, fibroblasts migrated significantly faster from edges of scratches into vacant areas together with increased secretion of collagen I and III, elastin, fibronectin, superoxide dismutase, and metalloproteinase-1 (MMP-1) compared to controls. After one application of GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL excisional and diabetic wounds in mice showed rapid wound closure, reepithelialization, and increased numbers of sebaceous glands and hair follicles compared to controls. The same wounds exposed to GFP-hWJSCs + AV/PCL or hWJSC-CM + AV/PCL also showed positive keratinocyte markers (cytokeratin, involucrin, filaggrin) and increased expression of ICAM-1, TIMP-1, and VEGF-A compared to controls. AV/PCL nanoscaffolds in combination with hWJSCs appear to have synergistic benefits for wound healing.

Human Wharton's jelly stem cells and its conditioned medium enhance healing of excisional and diabetic wounds.

Wound healing is a major problem in diabetic patients and current treatments have met with limited success. We evaluated the treatment of excisional and diabetic wounds using a stem cell isolated from the human umbilical cord Wharton's jelly (hWJSC) that shares unique properties with embryonic and adult mesenchymal stem cells. hWJSCs are non-controversial, available in abundance, hypo-immunogenic, non-tumorigenic, differentiate into keratinocytes, and secrete important molecules for tissue repair. When human skin fibroblasts (CCD) in conventional scratch-wound assays were exposed to hWJSC-conditioned medium (hWJSC-CM) the fibroblasts at the wound edges migrated and completely covered the spaces by day 2 compared to controls. The number of invaded cells, cell viability, total collagen, elastin, and fibronectin levels were significantly greater in the hWJSC-CM treatment arm compared to controls (P < 0.05). When a single application of green fluorescent protein (GFP)-labeled hWJSCs (GFP-hWJSCs) or hWJSC-CM was administered to full-thickness murine excisional and diabetic wounds, healing rates were significantly greater compared to controls (P < 0.05). Wound biopsies collected at various time points showed the presence of green GFP-labeled hWJSCs, positive human keratinocyte markers (cytokeratin, involucrin, filaggrin) and expression of ICAM-1, TIMP-1, and VEGF-A. On histology, the GFP-hWJSCs and hWJSC-CM treated wounds showed reepithelialization, increased vascularity and cellular density and increased sebaceous gland and hair follicle numbers compared to controls. hWJSCs showed increased expression of several miRNAs associated with wound healing compared to CCDs. Our studies demonstrated that hWJSCs enhance healing of excisional and diabetic wounds via differentiation into keratinocytes and release of important molecules.

Pharmacologic synergy between dual phosphoinositide-3-kinase and mammalian target of rapamycin inhibition and 5-fluorouracil in PIK3CA mutant gastric cancer cells.

Phosphoinositide-3-kinase (PI3K) and mammalian target of rapamycin (mTOR) inhibitors are an emerging class of anti-cancer agents. Here, we tested the hypothesis that the dual PI3K/mTOR inhibitor, PI103, could synergize with the chemotherapeutic agent, 5-fluorouracil (5-FU) by inhibiting E2F1, thymidylate synthase (TS) and enhancing DNA damage. Drug combination effects were assessed in gastric cancer cells using the median-effect equation. The specific effects of inhibition of E2F1 and PIK3CA were examined by siRNA, and mTOR by rapamycin exposure. Protein expression and apoptosis pre- and post-treatment was measured using standard methods. PI103 and 5-FU was synergistic in 3/5 gastric cancer cell lines tested. Synergy was associated with PI3KCA mutation, reduced TS and E2F1 protein levels, increased H2AX phosphorylation and apoptosis. E2F1 siRNA enhanced sensitivity to 5-FU only in cells displaying synergy. Excess thymidine exposure converted synergism to antagonism in all cells. Inhibition of PI3K and mTOR alone enhanced 5-FU cytotoxicity in only 2/3 cell lines that displayed synergy each. In AGS cells, PI3K inhibition alone enhanced 5-FU sensitivity as much as dual PI3K/mTOR inhibition. In HGC27 cells, dual inhibition increased 5-FU sensitivity more than single PI3K or mTOR inhibition. Combined PI103 and 5-FU treatment reduced in vivo tumor growth more than treatment with single agents. PI3K/mTOR inhibitors can enhance 5-FU cytotoxicity in vitro and in vivo, especially in PIK3CA mutant tumor cells. Dual, rather than single, PI3K/mTOR inhibitors may combine better with 5-FU due to cellular heterogeneity in sensitivity to PI3K and mTOR inhibition.

Hormonally regulated follicle differentiation and luteinization in the mouse is associated with hypoxia inducible factor activity.

Hypoxia inducible factors (HIFs) are transcription factors that mediate physiological responses to hypoxia. Hypoxia is established as the major inducer of HIFs, but stimuli such as transition metals and hormones also induce HIF target genes. Whilst the ovarian granulosa cell layer is known to be avascular and the follicle is vascularised via the thecal cell layer, little is known about the role of hypoxia or HIFs in regulating ovarian function. In this study, we hypothesized that hypoxia as well as non-hypoxic stimuli cooperate in promoting follicle differentiation and luteinization via HIF activity and resultant gene regulation. We quantitatively measured the HIF1alpha protein response to hCG in ovarian granulosa cell cultures and in vivo and developed a transgenic (HRE(4)-SV40-EGFP) HIF reporter mouse line. We observed a time-dependent increase of HIF1alpha protein levels in granulosa cells post-hCG in vivo, maximal around time of ovulation. hCG alone was unable to promote HIF1alpha protein accumulation in cultured granulosa cells, but increased protein abundance was observed when combined with a hypoxic stimulus. HRE-EGFP ovaries showed no follicular EGFP in stages prior to antrum formation. However, HIF regulated EGFP was maximally induced in granulosa cells around the time of ovulation and readily observed in corpora lutea. There was also an increase in HIF regulated EGFP activity in the corpora lutea from functional to regressing stages. Taken together, these observations establish the notion that HIFs play a role during follicular differentiation and luteinization.

The effect of acute intermittent hypercapnic hypoxia treatment on IL-6, TNF-alpha, and CRP levels in piglets.

STUDY OBJECTIVES: Obstructive sleep apnea (OSA) is characterized by repeated episodes of upper-airway obstruction during sleep leading to significant hypercapnic hypoxic conditions. These conditions are associated with increased levels of proinflammatory cytokines (including interleukin [IL]-6, tumor necrosis factor [TNF]-alpha, and C-reactive protein [CRP]) and subsequent increased cardiovascular risk. It is unclear whether hypercapnic hypoxia itself causes inflammatory perturbations. DESIGN: We evaluated circulating IL-6, TNF- a and CRP in a piglet model of infant OSA, following exposure to acute intermittent hypercapnic hypoxia (IHH). Study groups comprised of treatment (n = 8) and control (n = 8) groups. Treatment was two 90-minute sessions of IHH with arterial blood sampled before and after each IHH session. MEASUREMENTS AND RESULTS: IL-6, TNF-alpha and CRP levels were measured before and after IHH treatment sessions. Results showed an increase in IL-6 following the first session of IHH that was neither sustained, nor repeated, during a subsequent exposure. Using mixed-modelling, TNF-alpha changed between time points and groups. There were no changes in CRP over the duration of the study. CONCLUSION: These results suggest that acute hypoxia causes a transient increase in IL-6 levels and has implications for the pathogenesis of increased cardiovascular disease in OSA, especially in childhood.