Generation Of Human Stem Cells Research Articles

Human Induced Pluripotent Stem Cells in the Curative Treatment of Diabetes and Potential Impediments Ahead.

The successful landmark discovery of mouse and human inducible pluripotential stem cells (iPSC's) by Takahashi and Yamanaka in 2006 and 2007 has triggered a revolution in the potential generation of self-compatible cells for regenerative medicine, and further opened up a new avenue for "disease in dish" drug screening of self-target cells (Neofytou et al. 2015). The introduction of four 'Yamanaka' transcription factors through viral or other transfection of mature cells can induce pluripotency and acquired plasticity. These factors include transduction with octamer-binding transcription factor-4 (Oct-4), nanog homeobox (Nanog), sex-determining region Y-box-2 (Sox-2) and MYC protooncogene (cMyc). Such cells become iPSC's (Takahashi and Yamanaka 2006). These reprogrammed cells exhibit increased telomerase activity and have a hypomethylated gene promotor region similar to embryonic stem cells (ESC's). These milestone discoveries have generated immense hope that diseases such as diabetes could be treated and effectively cured by transplantation of self-compatible, personalized autologous stem cell transplantation of β-cells that release physiological insulin under glycemic control (Maehr et al. 2009; Park et al. 2008) (Fig. 1). Diabetes is a profligate disease of disordered glucose metabolism resulting from an absolute or relative deficiency of insulin, the consequences of which lead to immense socio-economic societal burden. While there are many different types of diabetes, the two major types (type 1 diabetes (T1DM) and type 2 diabetes (T2DM) are caused respectively by immune-mediated destruction (T1DM) or malfunctioning (T2DM) insulin-producing β-cells within the endocrine pancreas, the islets of Langerhans (Atkinson et al. 2011; Holman et al. 2015; You and Henneberg 2016). Almost 425million people are affected by the global burden of diabetes, and this is predicted to increase by 48% (629million) by 2045 (International Diabetes Federation Atlas 8th Ed 2018). Whole pancreas or islet cell transplantation offer an effective alternative to injected insulin, but both require lifelong potent immunosuppression to control both allo-and autoimmunity. Whole pancreas transplantation involves invasive complex surgery and is associated with greater morbidity and occasional mortality, while islet transplantation involves a minimally invasive intraportal hepatic infusion. Generally, whole pancreas transplantation provides greater metabolic reserve, but this may be matched by cumulative multiple islet infusions to achieve insulin independence. An additional challenge of islet transplantation is progressive loss of complete insulin independence over time, which may be multifactorial, the dominant factor however being ineffective control of autoimmunity. Both whole pancreas and islet transplantation are restricted to patients at risk of severe hypoglycemia that cannot be stabilized by alternate means, or in recipients that are already immunosuppressed in order to sustain a kidney or other solid organ transplant. The risks of chronic immunosuppression and the scarcity of human organ donors mean that both of these transplantation therapies cannot presently be extended to the broader diabetic population (Shapiro 2011; Shapiro et al. 2006). Recent progress in xenotransplantation of multiple knock-out 'humanized' pig islets could offer one potential solution, perhaps aided by clustered regularly interspaced short palindromic repeats/CRISPR associated-9 (CRISPR/Cas-9) gene editing approaches, but this remains to be proven in practice. Human stem cell derived new β-cell products could effectively address the global supply challenge for broad application across all forms of diabetes, but recurrent autoimmunity may still remain an insurmountable challenge. Considerable progress in the generation of human stem cell derived SC-β cells from ESC, iPS and other adult cell sources such as mesenchymal stem cells (MSCs) offer huge hope that a personalized, 'syngeneic' cell could be transplanted without risk of alloimmunity, thereby securing sufficient supply to meet future global demand (Cito et al. 2018).

Establishment of a xeno-free culture system that preserves the characteristics of placenta mesenchymal stem cells.

Although stem cells are promising candidates for cell replacement therapies, the vast majority are derived using animal sera, which has risk of being contaminated by animal viruses or toxins. To overcome these potential problems, we initially established multiple lines of stem cells from first-trimester human placenta (fPMSC), which were cultivated using human follicular fluid (hFF) instead of fetal bovine serum (FBS). FF provides a very important microenvironment for the development of oocytes. No differences were found in the general morphology, growth rate, karyotype, gene and surface expressions between placental MSCs cultured in 5% hFF-supplemented medium (fPMSC-X) or 10% FBS-supplemented medium (fPMSC). Differentiation experiments confirmed similar levels of potency in cells grown in either condition. Since hFF preserved the unique features of the stem cells and is free from potential pathogens, it should be considered as the main culture medium supplement for the propagation of human stem cells for clinical applications.

Connecting the Cell Cycle with Cellular Identity

The remarkable ability of oocytes to reinstate the totipotent state from a unipotent somatic cell, allowing the cloning of animals and the generation of human stem cells, has fascinated scientists for decades. Due to the complexity of oocytes, it has remained challenging to understand the rapid reprogramming following nuclear transfer at a molecular level. Conversely, the detailed characterization of molecular mechanisms is also often insufficient to comprehend the functional relevance of a complex molecular process, such as the dissociation of transcription factors from chromatin during cell division, the role of chromatin modifications in cellular memory, or of cell type-specific DNA replication. This review attempts to bridge the gap between nuclear transfer and molecular biology by focusing on the role of the cell cycle in reprogramming.

Isolation of three stem cell lines from human sacrococcygeal teratomas

Sacrococcygeal teratomas (SCTs) are benign tumours of the newborn with absolute indication for surgery directly after birth. We recently described the presence of stem cells positive for the stem cell markers nanog and Oct4 in SCTs. Here we report the isolation of three stem cell lines from three different SCTs. Cells were propagated in mesenchymal or in embryonic stem cell medium. Non-clonal homogeneous stem cell lines were obtained after two to three passages and characterized in vitro by immunocytochemistry, RT-PCR, western blot, FACS analysis, and metaphase spreads. The differentiation potential was tested in vitro and in vivo. The isolated cell lines, which we refer to as human sacrococcygeal teratoma stem cells (hSctSCs), express nanog, Oct4 and stella, and are negative for malignancy markers alpha-fetoprotein and carcinoembryonic antigen. They can be induced in vitro to express neuronal, osteogenic, and chondrogenic traits. After grafting in vivo, spontaneous integration into the neural crest of the chick embryo and teratoma formation in the nude mouse were obtained. Our results indicate that SCTs are derived from remnants of the epiblast-derived primitive streak, which in the human embryo normally regresses but forms teratomas in children affected with SCT. The hSctSCs therefore may be comparable to mouse epiblast-derived stem cells (EpiSCs) and share characteristic features with human embryonic stem (hES) cells. Thus, SCT tissue obtained after surgery appears to be a novel source for the generation of human stem cells without the ethical implications associated with hES cells.

From embryos to embryonic stem cells: biopolitics and therapeutic potential.

The inner cell mass of the preimplantation blastocyst, from which all the cells of the body develop, is a source of embryonic stem cells. These cells can be maintained in their undifferentiated state over long periods in culture and yet retain their pluripotency. The generation of human stem cells capable of differentiating into all the cell types of the human body opens the way for the use of these cells in therapeutic transplantation for a myriad of diseases. However, as discussed here, there are a number of logistical, biological, and clinical hurdles that must be overcome prior to the use of these cells in routine clinical practice.