Background:Type 1 Diabetes Mellitus (T1DM) is partly driven by autoimmune destruction of the pancreatic beta cell, facilitated by the release of inflammatory cytokines, including IFN-γ, TNF-α and IL-1β by cells of the innate immune system. Mesenchymal Stem Cells (MSCs) have been used to counteract autoimmunity in a range of therapeutic settings due to their secretion of trophic and immunomodulatory factors that ameliorate disease independently of the cells themselves.Objective:The aim of this study was to assess the effect of the secretome of human bone-marrow derived MSCs on cytokine-driven beta cell apoptosis.Methods:All experiments were conducted in two insulin-secreting islet cell lines (BRIN-BD11 and βTC1.6) with selected experiments confirmed in primary islets. MSC secretome was generated by conditioning serum-free media (MSC-CM) for 24 hours on sub-confluent MSC populations. The media was then removed and filtered in readiness for use.Results:Exposure to IFN-γ, TNF-α and IL-1β induced apoptosis in cell lines and primary islets. The addition of MSC-CM to cell lines and primary islets partially reversed cytokine-driven apoptosis. MSC-CM also restored glucose-stimulated insulin secretion in cytokine-treated cell lines, which was linked to improved cell viability following from cytokine challenge. Characterization of MSC-CM revealed significant concentrations of IL-4, IL-10, PIGF and VEGF. Of these, IL-10 alone prevented cytokine-driven apoptosis. Furthermore, the inhibition of IL-10 through the addition of a blocking antibody reversed the anti-apoptotic effects of MSC-CM.Conclusion:Overall, the protective effects of MSC-CM on islet beta cell survival appear to be largely IL-10-dependent.
Background: Normothermic Machine Perfusion (NMP) has been established in the field of solid organ transplantation for both liver and kidney allografts. The ability to perfuse organs at body temperature enables viability assessment as well as optimisation prior to implantation. Discussion: A recent in vitro report of the use of Mesenchymal Stem Cells (MSCs) in the use of a normothermic lung perfusion circuit has raised the possibility of their use in solid organ transplantation. The aim of this short review is to outline the potential uses of bone marrow derived MSCs for their use in renal allograft ex vivo NMP. An overview is provided of current literature of NMP as well as theorised uses for MSCs.
Background:Despite recent advances in culture techniques for undifferentiated human Embryonic Stem Cells (hESCs), further improvements are required to facilitate research and translation of these cells in clinical settings. We have previously derived hESC lines that can be cultured in their undifferentiated state on regular plastic culture dishes, without the need for feeder cells or other coating supports, denoted Matrix-Free Growth hESCs (MFG-hESCs).Objective:In this study, we further characterize and compare MFG-hESCs to hESCs in order to understand the molecular differences responsible for the unique ability of MFG-hESCs.Results:Microarray analysis demonstrated that MFG-hESCs highly resemble feeder-cultured hESCs in global gene expression profile. Two identified groups of genes with differential expression were those encoding for ribosomal proteins and attachment proteins, such as the RGD (Arg-Gly-Asp)-associated proteins. Real-time PCR and flow cytometry corroborated the microarray results. Culture of MFG-hESCs in the presence of RGD peptides resulted in decreased attachment ability compared to cells cultured in the presence of RGES (Arg-Gly-Asp-Ser) peptides.Conclusion:This study demonstrates that MFG-hESC lines overexpress cell attachment proteins but retain the typical characteristics of undifferentiated feeder-cultured hESCs. The ability to culture high-quality pluripotent stem cells in feeder- and matrix-free conditions creates a new opportunities for their large-scale manufacturing for experimental research and translational applications.
Background:According to WHO, 285 million people are visually impaired out of which, 39 million are classified as blind and the remaining 246 million people have low vision which comprises of moderate vision impairment and severe vision impairment. Therapies to treat major disorders leading to visual impairment like Age-related Macular Degeneration (AMD), Stargardt’s Disease (STGD), Retinitis Pigmentosa (RP) and corneal scarring are required.In the last decade, many advances have been made to treat these disorders using stem cell therapy. For corneal damage by accidental burns, scarring or limbal stem cell deficiencies which can lead to partial or total blindness, are treated with a risky intervention like keratoplasty. To overcome issues like graft rejection caused by keratoplasty as well as have a better outcome, limbal stem cell therapy has been introduced. Similarly, Retinal Pigment Epithelium (RPE) is a supporting tissue essential in nutrient transport, production of growth factors, phagocytosis of the photoreceptors and retinol cycling.Discussion and Conlusion:Degeneration of this monolayer causes many diseases that have no prevailing treatment; however, research is being carried out to replace this simple epithelial monolayer primarily with an autologous source of cells and currently using stem cells. This review discusses the advances made in the field of ocular stem cell therapy with regards to development, cultivation and novel methods used to deliver these cells to replace the corneal and retinal epithelium as a new standard for treatment.
Background:Osteosarcoma cancer stem cells (CSCs) are defined as a subpopulation of osteosarcoma cells, which have the ability of self-renewal, proliferation and differentiation. This study aimed to identify CSCs from human osteosarcomain vitro.Methods:Osteosarcoma CSCs were isolated and cultured with sphere-forming assay technique on an ultra-low well attachment surface plate. After sarcosphere colonies were formed, we conducted reverse transcriptase-polymerase chain reaction (RT-PCR) to detect the expression of genes of embryonic stem cells such asNANOG, Oct3/4, STAT3 and gene of MSC CD133. Immunofluorescence analysis (IFA) of alkaline phosphatase (ALP), osteocalcin, and CD 133 was also performed to see the expression of osteosarcoma CSC surface protein with immuno-enzymatic staining principle. We also performed alizarin red staining to evaluate calcification in osteosarcoma CSCs.Results:The culture sphere-of the osteosarcoma cells showed three dimension round shaped colonies (sarcospheres) in slightly hypoxicand serum free condition which was not attached to the substrate with tight density. RT-PCR demonstrated that sarcospheres expressed genes which encodeNANOG, Oct3/4 STAT 3, but not for CD 133. IFA showed positive protein expression of ALP, osteocalcin and CD 133 which was moderate, strong, and weak positive respectively. Sarcospheres also had a positive reaction toward alizarin red staining.Conclusion:Osteosarcoma CSCs could be isolated from human osteosarcoma by sphere-forming assay technique and characterized by the expression of genes of embryonic stem cells,such asNANOG, Oct3/4, STAT3 and IFA of ALP, osteocalcin, and CD 133.
Adult mesenchymal stem cells (MSCs) obtainable from autologous bone marrow aspirates have generated tremendous interest in the medical and scientific communities in the last two decades and are currently being investigated by a of interested physicians for use in point-of-care stem cell therapies due to their great potential to differentiate into multiple cell lineages such as bone, cartilage, muscle, tendon, and nerve.However, as these stem cells are found in very low numbers in adult tissue, centrifugal concentration or expansion through in vitro culturing has been pursued to obtain higher numbers of efficacious regenerative therapeutic applications.More recently, some physicians and scientists have chosen to explore use for direct injection of un-fractionated, native whole bone marrow aspirate as a strategy in regenerative treatment regimes.This review examines the potential merits and disadvantages of using either concentrated and culture expanded MSCs versus native whole bone marrow aspirate as key proliferant in direct regenerative injection therapy (RIT).Results from a number of published investigations have clearly shown high potential of various deleterious effects on manipulating MSCs obtained from native bone marrow aspirate either by centrifugal forces or expansion through in vitro culturing; moreover, currently used centrifugal concentration techniques do not significantly concentrate MSCs from bone marrow aspirate, thus, defeating the purpose of this manipulative step.On the other hand, preliminary results and observations of using un-fractionated whole bone marrow injection for treatment of various musculoskeletal joint diseases (for example, osteoarthritic joints) suggest that the procedure is safe and potentially efficacious, with no known deleterious effects as yet reported.
Dental pulp stem cells (DPSCs) from permanent teeth and stem cells from human exfoliated deciduous teeth (SHED) have attracted tremendous interest recently by playing a major role in tissue engineering and regenerative medicine. However, since stem cell technology is still in its infancy, interdisciplinary cooperation between medicine, ba- sic biological research, nanotechnology and materials science is needed to achieve successful clinical applications. Similar to mesenchymal stem cells, DPSCs and SHED can undergo self-renewal and have multipotent differentiation ability, but unlike other sources of stem cells such as embryonic stem cells, which involves the destruction of human embryo, DPSCs and SHED have limited ethical concerns as they are readily and easily accessible, non-invasive and disposed off naturally. Not only DPSCs and SHED can be used for cell based therapies and bio-artificial tissue constructs but also made to differentiate into other cell types. Here, we discuss on definitions, opportunities, advantages and limitations of DPSCs and SHED in tissue engineering and regenerative medicine.
The preservation of the genetic and epigenetic integrity of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) during in vitro propagation is critical for their use in both research and future therapeutic applications.It has been reported that hESCs and iPSCs have the ability to adapt to various culture conditions.However, human pluripotent stem cells cultured in serum free media can frequently accumulate point mutations, aneuploidy, and progressive epigenetic changes over prolonged culture in vitro for reasons that are poorly understood.The phenotypic and epigenetic changes brought about by the culture conditions can have significant impacts on their use in research and in clinical applications.An increased understanding of the potential effects of in vitro culture environments on pluripotent stem cell growth can enhance the development of improved culture systems for hESCs or iPSCs, and facilitate any future therapeutic applications using these cells.In this review, we first focus on the occurrence, potential causes and consequences of genetic and epigenetic unstable human pluripotent stem cells in vitro.We further discuss the current methods for detection and characterization of abnormal pluripotent stem cells that involve simply traditional karyotype analysis.All these observations highlight the need for novel screening strategies to determine the safety of hESCs or iPSCs and optimization and standardization of procedures for the generation and culture of pluripotent stem cells that minimize culture-induced epigenetic and genetic instability.
Previous studies have demonstrated that glucagon-like peptide-1 (GLP-1) stimulates β-cell formation and insulin secretion.Currently, there has been no report in understanding the effect of GLP-1 / its agonist exendin-4 in differentiation of human embryonic stem cells (hESCs) to definitive endodermal (DE).We hypothesized that exendin-4 signaling in hESCs via GLP-1 receptor (GLP-1R) may have potential role in DE differentiation.The effect of Ex-4 on pluripotent hESCs and the combined effect of Ex-4 and activin A-treated hESC-derived DE were examined.Analysis by quantitative real-time PCR (qPCR) demonstrates that Ex-4 alone was not sufficient to enhance DE formation in hESCs.On the other hand, a combinatorial treatment with activin A and Ex-4 resulted in significant decrease in expression levels of DE markers.The miRNA expression profiles between activin A-treated hESCs and activin A/Ex-4-treated hESCs after 5 days of treatment demonstrated similar expression levels of endoderm and pancreas-associated miRNAs.However, it was shown that the levels of pluripotency-associated miRNAs, miR-302a* and miR302c*, were upregulated in the presence of Ex-4.Furthermore, it was observed that exposure to bFGF and Ex-4 in apoptosis-inducing medium resulted in downregulation of CASP3 and p53.Taken together, these data revealed the possibility of Ex-4 in maintaining pluripotency and inhibiting apoptosis.The knowledge of GLP-1 signaling pathways could be useful for understanding the mechanism of GLP-1R-ligand interactions and their relevance to hESC development.
Generation of mature differentiated cells is essential for the treatment of several diseases that depend on cell replacement therapies.With the growing knowledge of transcriptome diversity in cells, increased understanding now exists on the potential to inter-convert a specialized cell type into a differentiated cell of another lineage (transdifferentiation).Transcription in terminally committed cells is controlled by many extracellular and intracellular components and the intrinsic structure and confirmation of the DNA itself (the epigenome).The patterns of these modifications in differentiated cells are generally stable and heritable with characteristic modification patterns reflecting the phenotype they acquire during differentiation.Adult tissue-derived stem or progenitor cells possess inherent traits that result in "commitment" to a particular phenotype, demonstrated by their relatively restricted differentiation capacity.Adult tissue-derived stem cell populations represent a source of cells that would predictably require fewer manipulations to achieve an alternative, differentiated phenotype.By characterising cells with respect to epigenetic patterns, it may be possible to identify stem / progenitor cells that are poised to differentiate towards a particular lineage.Assessing the chromatin compactness at gene promoter regions may assist in identifying mechanisms for inducing cells to adopt a specific phenotype with increased efficiency of differentiation.
Electroporation is a common method of gene transfer that has recently been used to efficiently transfect mesenchymal stem (stromal) cells (MSCs); however, the electrical stimulus has the potential to alter cell state.This study examines possible negative effects of electroporation of human embryonic stem cell (hESC)-derived MSCs including, loss of potency or induction of differentiation.Immunofluorescence and PCR were used to quantify protein and RNA expression of CD73 (an MSC marker) and markers of mature mesenchymal cell types following electroporation.The relative fraction of cells expressing CD73 protein was not altered in cells exposed to a 20 ms pulse at 1000 V or 1500 V compared to controls even after three passages, suggesting MSCs retain multipotency following electroporation.In addition, RNA expression of markers indicative of mature cells of bone, fat and cardiac muscle did not differ from unmanipulated controls soon after electroporation.Taken together, these results indicate electroporation under conditions favorable for MSC transfection does not significantly alter stem cell state.
The generation of insulin producing cells from human embryonic stem cells (hESCs) has shown great promise as a cellular replacement therapy for the treatment of Type 1 Diabetes. Mature functional β-cell surrogates however, have yet to be successfully generated in vivo. One approach to potentially improve current differentiation protocols is the use of 3 dimensional (3D) scaffolds, which has been shown to enhance cellular function and differentiation potential. The present study aimed to explore the feasibility of using single cell preparations of pluripotent hESCs seeded onto laminin or Matrigel coated 3D poly(lactic-co-glycolic) acid (PLGA) scaffolds to derive definitive endoderm, the first vital stage of endoderm tissue differentiation. Our results demonstrated that hESCs which were induced to differentiate on laminin or Matrigel coated 3D scaffolds can be successfully coaxed to differentiate into definitive endoderm. The cells that were cultured on laminin or Matrigel coated 3D scaffolds expressed significantly higher levels of the key endoderm transcription factors SOX17 and FOXA2 in comparison to those differentiated on 2D monolayers. On Matrigel coated 3D scaffolds, the differentiated cells expressed lower levels of the endoderm surface marker CXCR4 and anterior endoderm marker CER in comparison to its monolayer counterpart. Together, the results of this study demonstrated the positive effect of 3D cultures on endoderm commitment from hESCs over traditional monolayer cultures. Furthermore, the definitive endoderm produced on Matrigel coated scaffolds may have a more posterior phenotype in comparison to those derived from monolayers. This may have an effect on later stages of pancreatic differentiation and warrants further detailed investigations.
The recent breakthrough in reprogramming somatic cells has invigorated the prospect that disease mechanisms that underpin various human diseases particularly the neurodegenerative disorders could be unravelled by using the disease-specific pluripotent stem cells. A number of studies have demonstrated that such disease-specific induced pluripotent stem cell (iPSC) could be generated relatively easy. Some recent studies have substantiated the utility of this technology in describing the initial characterization of neurodegenerative patient-derived iPSC as a proof of concept. However, as it is becoming evident now that the cell type of origin influences the molecular and functional properties of derived iPSC. The indications that reprogramming may erase the cell memory also raises the question if the disease phenotype may not be correctly represented or also erased in iPSC unless coaxed by further perturbation in vitro culture conditions. Other associated difficulties in iPSC research such as culture variability, selective adaptation of such cultures and the lack of robust protocols to generate homogeneous population of desired cell type may have compounding affects in the use of these cells in disease modelling. Unless these issues are addressed properly the prospects of iPSC in disease modelling may remain a slippery slop.
We are in the beginning of the era of regenerative medicine and many researchers are testing adult stem cells to be used for tissue repair and regeneration in the human body.Many adult stem cells have been discovered since the late 1990's with more recently a novel adult stem cell described in menstrual blood.The menstrual blood is derived from shedding of the endometrial lining, specifically the functionalis layer, which contains highly proliferative cells used to prepare the female body for implementation of a fertilized egg.Cell characterization experiments of stromal stem cells discovered in menstrual blood have demonstrated cells to be multipotent which can successfully differentiate in vitro into cell lineages derived from the mesoderm and the ectoderm.When menstrual blood cells were seeded in culture the average number of adherent cells was 8.50 % with a range of 0.48% to 47.76%.Demonstrating longevity one cell line allowed to grow was subcultured 47 times before complete senescence and death.The menstrual blood stromal stem cell phenotypic analysis incorporates mesenchymal cell markers such as CD13, CD29, CD44, CD49f, CD73, CD90, CD105, CD166, MHC Class I and pluripotent embryonic stem cell markers SSEA-4, Nanog and Oct-4.Karyotypic analysis demonstrated the maintenance of diploid cells without chromosomal abnormalities.In conclusion preliminary studies have demonstrated menstrual stem cells are easily expandable to clinical relevance.Pivotal pre-clinical studies are now underway to test the safety and efficacy of menstrual stem cells in several different animal models including one for neuroprotection following transplantation into an experimental stroke model.The study demonstrates menstrual stem cells are a novel cell population that may be routinely and safely isolated to provide a renewable source of stem cells from child-bearing women.
Reports have shown that murine embryonic stem cells (mESC) can differentiate into primordial germ cells (PGC) and then to early gametes (oocytes and sperm) that after fertilization form blastocysts.Preliminary studies indicated that hESC also have the potential to differentiate into germ cells.Currently, there are no reports on directed differentiation of hESCs into oocytes.Here, we investigated the effects of human fetal testicular extracts (HFTE) and hormones i.e. follicle-stimulating hormone (FSH), human chorionic gonadotropin (hCG) to coax hESC to differentiate into oocytes.The embryoid bodies (EBs) derived from hESC formed ovarian-like structures (OLS) after treatment with HFTE for 7 days and with hormones (FSH + hCG) for 20-30 days.OLS exhibited typical oocyte-like spherical shape of variable sizes and some with zona pellucida-like covering RT-PCR, immunological fluorescence staining and flow cytometry analyses showed that these structures are positive for specific germ cell and oocyte markers such as OCT4, SCP3, C-KIT and DDX4, STELLA, FIGLA, GDF9, NANOG，FSHR, ZP1, ZP2 and ZP3.Quantitative RT-PCR analysis showed that FSHR, GDF9 and FIGLA were up-regulated during OLS differentiation from EB in a time-dependent manner.These results demonstrated that hESC have the ability to differentiate into ovarian structures.This study thus provides an in vitro model to study germ-cell formation from hESC and these germ cells may generate a potential source of oocytes for therapeutic cloning.
The seminal report from Takahashi and Yamanka in 2006 describing the reprogramming of somatic cells to induced pluripotent stem (iPS) cells [1] marked the beginning of a new field of research, resulting in hundreds of publications in a short 3 years.Among other things, the promise of iPS cells in cell therapy circumvents many of the ethical concerns associated with embryonic stem (ES) cell research, and autologous patient-specific cells can be generated.Nonetheless, the jury is still out on the extent to which iPS cells and ES cells are functionally equivalent.This review focuses on the genetic and functional comparisons between these two cells types.
The ethical debate surrounding human pluripotent stem (PS) cell research is mainly due to use of human embryonic stem (ES) cells. It has been suggested by many that human induced pluripotent stem (iPS) cells would end the debate due to their non-embryonic origin. This review examines the ethical issues surrounding the use of iPS cells and their ES cell counterparts, and argues that while iPS cells are in many ways ethically less contentious, they will certainly not end the debate.
In 2006, Yamanaka and Takahashi electrified the scientific community by discovering that mouse somatic cells can be converted into embryonic stem cell-like cells by retroviral transduction of four transcription factors: Oct4, Sox2, Klf4, and c-Myc (OSKM).The first generation of mouse induced pluripotent stem (iPS) cells was incompletely reprogrammed, and failed to contribute to germline transmission.Nearly one year later, three groups, including Yamanaka's, improved the reprogramming methodology and generated iPS cells that were in many respects, indistinguishable from ES cells, and also contributed to chimera formation and germline transmission.Shortly thereafter, the successful reprogramming of human somatic cells opened the gate for the development of patient-specific iPS cells for biomedical research and clinical application.Though human iPS cells resemble human ES cells in many aspects, the current iPS cell technologies showed several limitations for clinical usage.First, the efficiency of iPS cell generation is still low and the reprogramming process takes at least two weeks.Second, the virus-delivery of reprogramming factors introduces inconceivable risks of insertional mutagenesis in the genome.Third, given the various strategies for direct reprogramming, it remains difficult to assess the quality of iPS cells generated in each lab and for each patient.These issues should be addressed properly before any iPS cells could be translated into clinic.Here, we review recent progress in human iPS cell technologies, with a focus on the virus-free and integration-free iPS cell generation, which may lead towards the eventual goal of clinical applications.
