Characterization of beta cell-like cells converted from human urine stem cells
Professor Xin-Fu Zhou
University of South Australia
Type 1 diabetes (T1D) is a life-long condition usually diagnosed in childhood. The desired therapy for T1D is to replenish the lost beta cells with functional healthy beta cells. Islet replacement therapy is a possible attractive solution and the success with clinical islet transplantation demonstrates the general approach has the potential to restore normal glucose control in T1D patients. In this project, we propose a novel approach to address the problem of low availability of islets for replacement therapy and the immune mediated rejection response by using patients own stem cells.
Professor Xin-Fu Zhou, Dr Larisa Bobrovskaya, Associate Professor Grigori Rychkov.
Type I diabetes (T1D) causes sever complication that partially treated with insulin replacement. However instabilities in blood glucose levels continue in the patients. Scientist hypothesised that pancreatic islets transplantation represents ideal clinical treatment for patients with severe complication and sustainable therapy for T1D patients. Pancreatic beta cells differentiated from stem cells are a good source of cells. We tested the patient-derived urine stem cells potency to derive induced pancreatic beta cells. The cells prove their safety and readiness to response to the induction protocols. The induced pancreatic cells were partially similar to the normal pancreatic cells and some of the produced cell line rescued the diabetic animal model. Our research is aiming to develop a protocol to generate identical pancreatic cells from isolated urine stem cells and to generate high numbers of the induced cells.
Type I diabetes (T1D) is an incurable disease. Current insulin replacement therapy only partially solves the problem as fluctuations in blood glucose levels is noticed in the insulin treated patients. Infusion of pancreatic islets represent potent clinical treatment for patients with severe complication. Pancreatic beta cells differentiated from pluripotent stem cells are a good source of cells for transplantation. We hypothesised that patient-derived urine stem cells are an excellent source for conversion into beta cells.
We isolated several urine stem cells with different method of isolation and established a technique that succeeds in deriving urine stem cells from 70% of the samples. The ability of the isolated cells to multiply efficiently were tested and optimised with several media formula and the generated cells were tested for safety by Karyotyping to eliminate the risk of tumour generation. The stem cell character of the urine cells were further verified depending on series of putative markers such as oct3/4. We developed protocol to drive the urine cells to induced pancreatic cells. These cells were provisionally tested and it was revealed that they produce low levels of insulin and c peptide by ELISA.
Induced pancreatic cells intravenous injection into diabetic mice model showed the ability of three lines out of the total six lines to reduce the blood glucose levels (BGL) over 19 days to levels similar to the and compared to USC transplanted cells. The transplants were able to reduce BGLs to less than 11 mmol/L. These levels concurred with our results that recorded when similar number of human islet cells were injected in the animal model. On the other hand injection the urine cells did not have the reduction effect on the BGL. Nevertheless, the injected cells were not detected in pancreatic sections and few of the cells were detected in the livers of the mice.
RNA-seq transcriptome libraries were prepared following TruSeqTM RNA sample preparation Kit from Illumina (San Diego, CA), using total RNA extracted from non-induced urine cells, induced Pancreatic-like cells and Human pancreas tissue. Then the clean reads were separately aligned to reference genome and to the read of human pancreas tissue with orientation mode using tophat software. Gene differential expression reveal the increment of expression of several key pancreatic cells marker in the induced Pancreatic-like cells (ex: Somatostatin, SLC2A2, MAFA MAFB and PAX6) also it reveals the inhibition of gene expression of several markers in urine stem cells to resemble those of the pancreatic tissue. However the insulin, ISL1 and PCSK2) genes did not elevated to the positive control levels.
Calcium imaging fail to reveal ions oscillation in the induced pancreatic cells. These results together with the results of RNA sequencing provoked the need to analyse more urine cells samples from different donors and that reveals the variation between the isolated cells not only in the number and proliferation potency but also in the morphology and respond to the small molecules inducers therefore we increase the stringency of our induction protocol to reduce the variation between different isolated line. We introduce new small molecules to the media and optimise the concentrations for these molecules. The cell lines variation was reduced to the minimum and we started characterisation by real time PCR using cell markers such as Pax1, Sox17, Ins, Isl1, FoxA2, NKX6.1.
The manuscript entitled “Generation of insulin-secreting cells from urine for the treatment of type 1 diabetes ” is under preparation.