Insulin producing cells established using non-integrated lentiviral vector harboring PDX1 gene

Abstract

AIM:To investigate reprogramming of human adipose tissue derived stem cells into insulin producing cells using non-integrated lentivirus harboring PDX1 gene. METHODS:In this study, human adipose tissue derived stem cells (hADSCs) were obtained from abdominal adipose tissues by liposuction, selected by plastic adhesion, and characterized by flow cytometric analysis. Human ADSCs were differentiated into adipocytes and osteocytes using differentiating medium to confirm their multipotency. Non-integrated lentiviruses harboring PDX1 (Non-integrated LV-PDX1) were constructed using specific plasmids (pLV-HELP, pMD2G, LV-105-PDX1-1). Then, hADSCs were transduced with non-integrated LV-PDX1. After transduction, ADSCs PDX1+ were cultured in high glucose DMEM medium supplement by B27, nico- were implanted into hyperglycemic rats. RESULTS:Human ADSCs exhibited their fibroblast-like morphology and made colonies after 7-10 d of culture. Determination of hADSCs identified by FACS analysis showed that hADSCs were positive for mesenchymal cell markers and negative for hematopoietic cell markers that guaranteed the lack of hematopoietic contamination. In vitro differentiation of hADSCs into osteocytes and adipocytes were detected by Alizarin red and Oil red O staining and confirmed their multilineage differentiation ability. Transduced hADSCs +PDX1 became round and clusters in the differentiation medium. The appropriate expression of PDX1 and insulin proteins was confirmed using immunocytochemistry analysis.Significant expressions of PDX1 , Ngn3, glucagon, Glut2 and somatostatin were detected by quantitative RT-PCR. hADSCs PDX1+ revealed the glucose sensing ability by expressing Glut2 when they were cultured in the medium containing high glucose concentration. The insulin secretion of hADSCs PDX1+ in the high glucose medium was 2.32 μU/mL. hADSCs PDX1+ implantation into hyperglycemic rats cured it two days after injection by reducing blood glucose levels from 485 mg/dL to the normal level. CONCLUSION: Human ADSCs can differentiate intoIPCs by non-integrated LV-PDX1 transduction and have the potential to be used as a resource in type 1 diabetes cell therapy.

Directed differentiation of human iPSC into insulin producing cells is improved by induced expression of PDX1 and NKX6.1 factors in IPC progenitors

“…The effect of Pdx1 exogenous expression on differentiation or transdifferentiation processes of various cells into IPCs was previously reported, either as a sole introduced factor [47–49] or in combination with NeuroD1 and MafA [50], but not with Nkx6.1. Our results demonstrate for the first time that differences in insulin secretion depend on the differentiation stage at which transcription factors expression was induced.…”

Tailor-made generation of insulin-producing cells from canine mesenchymal stem cells derived from bone marrow and adipose tissue

“…Alternatively, cAD-MSCs have been proposed as the potential MSC candidate for regenerative diabetes therapy as mentioned in previous reports 18,51,76,[78][79][80] . We showed in this study that cAD-MSC-derived IPC colonies could efficiently be generated from low attachment culture with the expression of crucial pancreatic mRNA markers.…”

“…PDX1-positive cells were considered as the pancreatic progenitors for three pancreatic lineages, comprising endocrine, exocrine, and ductal cells 10 . It has been shown that the overexpression of PDX1 by lentiviral vector into mouse MSCs could enhance IPC generation by triggering the morphological change from adherent spindle fibroblast-like cells toward the ball-like cell colonies 33,76 . For cBM-MSCs, we found that 3D culture condition was required to form the IPC colony which was considered as the native pancreatic islet morphology 35,63,65,77 .…”

Regenerative Therapy of Type 1 Diabetes Mellitus: From Pancreatic Islet Transplantation to Mesenchymal Stem Cells

“…Higher expression of Pdx1, Ngn3, Glut2, and somatostatin was detected. After transplantation of these cells in diabetic mice, hyperglycemia was reverted [ 64 ].…”

“…Additionally, whether immunosuppressive properties of MSCs cause adverse effects is discussed, such as infections or graft versus host disease. A series of clinical trials using MSCs for treatment purpose is already going on [ 64 , 166 , 177 – 180 ]. It seems that if MSCs pose a risk of tumor formation, it can be hypothesized to be a lot weaker and perhaps better controllable as in the case of ESCs and iPS cells.…”