Follicular Mesenchymal Stem Cells Over-expressing Release-controlled Human Insulin Gene Efficiently Reversed Hyperglycemia In Type1Diabetes

Background:Type1diabetes is caused by immune-mediated destruction of insulin-secretingislet β-cells in the pancreas. The introduction into the body of exogenous genes thatexpress therapeutic proteins (gene therapy) provides an option to treat patients withtype1diabetes.Human hair follicles serve as a convenient source of mesenchymal stem cells(HF-MSCs), because they are readily accessible, and harvesting hair follicles causesonly minor adverse effects. Moreover, transplanted HF-MSCs survive in the humanbody for extended periods because of their low immunogenicity. HF-MSCs are a richsource of autologous stem cells that can be used for cell-based gene therapy.Effective temporal control of insulin gene expression is a crucial element of type1diabetes gene therapy. To mimic the physiological postprandial kinetics of insulinsecretion to transiently correct hyperglycemia, a system was developed thatpharmacologically controls the rapid secretion of insulin in pulses, thus avoiding theslow time course of expression systems that rely on gene transcription in response toglucose to mitigate impaired glucose tolerance or hypoglycemia.Aims:The aimis of this study were to examine the potential for HF-MSCs to act asgene target cells that over-express a release-controllable insulin gene and then to testthe ability of the transgenic HF-MSCs to reverse hyperglycemia in experimental type1diabetes. Methods:1. Isolation and cultivation of HF-MSCsHairs were physically removed from the occipital region of the scalp of thevolunteers. When the spindle shaped cells migrated from the hair follicles, they wereexpanded in vitro. Flow cytometry and immunostaining were used to detect thesurface markers of HF-MSCs. Multilineage differentiation potential was assessedusing Oil Red O and Alizarin Red S staining.2. Virus preparation and cell transductionLentiviral vector, which encodes an insulin gene that is induced usingrapamycin, was cotransfected with pMDL-g/p, pSRV-rev, and pMD2.G into humanembryonic kidney (HEK)293T cells. Viral supernatants were concentrated byultracentrifugation. After transduction, when expressing green-fluorescent protein(GFP) cells appeared, they were sorted using fluorescence-activated cell sorting anddesignated insulin-producing cells (In-HF-MSCs). The HF-MSCs transduced with alentiviral vector expressing GFP under the control of the CMV promoter weredesignated G-HF-MSCs and served as controls.3. Measurement of C-peptide and Insulin levelsIn-HF-MSCs were seeded. At the indicated times, culture supernatant wascollected and the amount of C-peptide in the supernatant was determined byenzyme-linked immunosorbent assay (ELISA) using a human C-peptide kit andimmunostaining.4. Implantation of Transgenic HF-MSCs and analysis of hyperglycemiaTransgenic HF-MSCs were injected subcutaneously into the back. Seven daysafter cell implantation, diabetic mice were fasted for3h and then injectedintraperitoneally with100μl rapamycin (30mg/kg body weight). Blood glucoselevels, body weight, and death rate were assessed at the indicated times.5. In vivo live image detection and histology Diabetic mice at days3,60,90,120postimplantation of In-HF-MSCs wereanaesthetized with isoflurane and subjected to in vivo live image analysis.Graft-bearing tissues were embedded in paraffin, and stained with hematoxylin/eosin(HE) or incubated with antibodies for immunofluorescence assays.Results:1. Isolation and characterization of HF-MSCsCells that migrated from the dermal sheath and papilla that exhibitedfibroblast-like morphology characteristic of MSCs were pooled and expanded. Thecells finally differentiated into adipocytes and osteoblasts. Immunofluorescence andflow cytometric analyses showed the spindle-shaped fibroblast-like cells were MSCs,because they were highly positive for CD44, CD73, CD90, and CD105. Thespindle-shaped fibroblast-like cells were therefore designated HF-MSCs.2. Generation of transgenic HF-MSCs over-expressing the human insulin geneWe genetically engineered HF-MSCs to overexpress the human insulin gene. Thecells were observed using a live-cell microscopy imaging systems to confirm that thecultures contained>90%GFP+/DAPI+cells. We next determined whether thetransduced HF-MSCs retained the MSC phenotype. The transgenic HF-MSCsdifferentiated into adipogenic and osteogenic lineages as efficiently as HF-MSCs.Immunofluorescence and flow cytometric analyses showed they expressed CD44,CD73, CD90, and CD105.3. In vitro release of insulin from In-HF-MSCs in response to RapamycinIn-HF-MSCs release human insulin in a time and dose-dependent manner inresponse to rapamycin.4. The effect of engrafted In-HF-MSCs on hyperglycemia in mice with type1diabetesWhen implanted into mice with STZ-induced type1diabetes and treated withrapamycin, the In-HF-MSCs released human insulin, dramatically reversedhyperglycemia, and significantly increased body weight to the same levels as normalmice. 5. In vivo imaging and histology of In-HF-MSCsWe next used in vivo imaging to determine the survival of In-HF-MSCs indiabetic mice. The fluorescence intensity of In-HF-MSCs in diabetic mice graduallyweakened with time and no signal was detected on day120. Moreover, histologicalanalysis failed to detect the presence of tumors.Conclusions:HF-MSCs can be used as gene target cells for safe and efficient over-expressionof a therapeutic gene, holding promise for cell-based gene therapy of human disease.