Source Of Pluripotent Stem Cells Research Articles

Prospects for the use of the achievements of regenerative medicine in otorhinolaryngology

Regenerative medicine is focusing on searching for stem cells, which can be efficiently and safely used for regeneration of damaged tissues and organs. Pluripotent stem cells would be ideal for this purpose. It is because they have the ability to differentiate into cells of all three germ layers (ecto-, meso- and endoderm). One of the sources of their isolation are embryos. For many years, they are made unsuccessful attempts to use of very controversial embryonic stem cells that are isolated from embryos. So strong ethical controversy forced scientists to look for other, undoubted ethically, sources of pluripotent stem cells. Induced pluripotent stem cells are proposed, as a more promising alternative to cells isolated from embryos. Unfortunately, both embryonic stem cells and induced pluripotent stem cells tend to genetic instability leading to the formation of teratomas. In parallel studies scientists try to use of stem cells isolated from adult tissues (e.g. bone marrow cells or adipose tissue) in the regeneration of parenchymal organs. Unfortunately, there is no convincing evidence for most of these cells that can regenerate damaged parenchymal organs. Regenerative medicine more frequently is employed in the otorhinolaryngological therapies. More and more researchers’ efforts are put into the development of an effective method of stimulation (in vitro) of pluripotent stem cells isolated from adult tissue for differentiation of the renewable progenitor stem cells which can keep their potential after transplantation into the recipient (e.g. in the treatment of imbalances or hearing loss). Moreover, there are promising methods for employing of the stem cells potential in tissue engineering as they are more effectively introduced as a clinical therapies.

The study of bone marrow cells in rats according to their age and caloric intake

The bone marrow is a central organ of haemopoiesis and a source of pluripotent hemopoietic stem cells. The age-related changes in bone marrow induce the disorder of coordination between parenchyma and stroma of the organ that negatively influences proliferative potential and differentiation of the cells. That is why it is urgent to study the age-related changes of bone marrow and influence of caloric intake limitation on it.Aim. The aim of presented work was the study of proliferative potential, index of viability and percentage ratio of the bone marrow cells in rats depending on their age and caloric intake.Methods. The following methods were used in the study: selection and cultivation of the bone marrow stem cells, assessment of proliferative potential and viability of cells, analysis of percentage ratio of cellular types.Result. It was established, that the number of cells in bone marrows increases with age. The limitation of caloric intake favors the decrease of cells number in bone marrow despite the rats’ age. The received data demonstrated that the limitation of caloric intake increases the proliferative potential of the bone marrow only in old animals. It was established, that the viability of bone marrow cells was high during all period of cultivation. The morphological population of bone marrow cells in rats of different age was heterogenic, not depending on the conditions of their feeding. It was demonstrated, that the limitation of feeding caused the increase of the number of non-differentiated blasts in bone marrow of old animals that can be connected with the activation of the processes of self-activation of stem cells pool and non-differentiated predecessors of immune system cells. The morphological analysis of the bone marrow cells culture of all animal groups despite the age and nutrition regime demonstrated that the cultivation favors the selective decrease of cells heterogeneity. At that the cells of erythroid and megakaryocytic sprouts were absent in cellular cultures.Conclusions. As a result, the limitation of caloric intake of old rats favors the restoration or activation of proliferative potential of immunocompetent cell of bone marrow in vitro system

Ex vivo gene therapy for the treatment of neurological disorders.

Ex vivo gene therapy involves the genetic modification of cells outside of the body to produce therapeutic factors and their subsequent transplantation back into patients. Various cell types can be genetically engineered. However, with the explosion in stem cell technologies, neural stem/progenitor cells and mesenchymal stem cells are most often used. The synergy between the effect of the new cell and the additional engineered properties can often provide significant benefits to neurodegenerative changes in the brain. In this review, we cover both preclinical animal studies and clinical human trials that have used ex vivo gene therapy to treat neurological disorders with a focus on Parkinson's disease, Huntington's disease, Alzheimer's disease, ALS, and stroke. We highlight some of the major advances in this field including new autologous sources of pluripotent stem cells, safer ways to introduce therapeutic transgenes, and various methods of gene regulation. We also address some of the remaining hurdles including tunable gene regulation, in vivo cell tracking, and rigorous experimental design. Overall, given the current outcomes from researchers and clinical trials, along with exciting new developments in ex vivo gene and cell therapy, we anticipate that successful treatments for neurological diseases will arise in the near future.

Andrographolide Promotes Neural Differentiation of Rat Adipose Tissue-Derived Stromal Cells through Wnt/β-Catenin Signaling Pathway

Adipose tissue-derived stromal cells (ADSCs) are a high-yield source of pluripotent stem cells for use in cell-based therapies. We explored the effect of andrographolide (ANDRO, one of the ingredients of the medicinal herb extract) on the neural differentiation of rat ADSCs and associated molecular mechanisms. We observed that rat ADSCs were small and spindle-shaped and expressed multiple stem cell markers including nestin. They were multipotent as evidenced by adipogenic, osteogenic, chondrogenic, and neural differentiation under appropriate conditions. The proportion of cells exhibiting neural-like morphology was higher, and neurites developed faster in the ANDRO group than in the control group in the same neural differentiation medium. Expression levels of the neural lineage markers MAP2, tau, GFAP, and β-tubulin III were higher in the ANDRO group. ANDRO induced a concentration-dependent increase in Wnt/β-catenin signaling as evidenced by the enhanced expression of nuclear β-catenin and the inhibited form of GSK-3β (pSer9). Thus, this study shows for the first time how by enhancing the neural differentiation of ADSCs we expect that ANDRO pretreatment may increase the efficacy of adult stem cell transplantation in nervous system diseases, but more exploration is needed.

Pluripotent stem cells and their utility in treating photoreceptor degenerations.

Age-related macular degeneration and inherited retinal degenerations represent the leading causes of blindness in industrialized countries. Despite different initiating causes, they share a common final pathophysiology, the loss of the light sensitive photoreceptors. Replacement by transplantation may offer a potential treatment strategy for both patient populations. The last decade has seen remarkable progress in our ability to generate retinal cell types, including photoreceptors, from a variety of murine and human pluripotent stem cell sources. Driven in large part by the requirement for renewable cell sources, stem cells have emerged not only as a promising source of replacement photoreceptors but also to provide in vitro systems with which to study retinal development and disease processes and to test therapeutic agents.

Progress towards generation of human haematopoietic stem cells.

De novo generation of haematopoietic stem cells from different human pluripotent stem cell sources remains a high priority for haematology and regenerative medicine. At present, efficient derivation of functional haematopoietic stem cells with the capability for definitive in vivo engraftment and multi-lineage potential remains challenging. Here, we discuss recent progress and strategies to overcome obstacles that have thwarted past efforts. In addition, we review promising advances in the generation of mature blood lineages and the potential of induced pluripotent stem cells.

Hedgehog Promotes Production of Inhibitory Interneurons in Vivo and in Vitro from Pluripotent Stem Cells.

Loss or damage of cortical inhibitory interneurons characterizes a number of neurological disorders. There is therefore a great deal of interest in learning how to generate these neurons from a pluripotent stem cell source so they can be used for cell replacement therapies or for in vitro drug testing. To design a directed differentiation protocol, a number of groups have used the information gained in the last 15 years detailing the conditions that promote interneuron progenitor differentiation in the ventral telencephalon during embryogenesis. The use of Hedgehog peptides and agonists is featured prominently in these approaches. We review here the data documenting a role for Hedgehog in specifying interneurons in both the embryonic brain during development and in vitro during the directed differentiation of pluripotent stem cells.

Amniotic membrane mesenchymal stem cells can differentiate into germ cells in vitro.

This is the first report on differentiation of mouse amniotic membrane mesenchymal stem cells (AM-MSCs) into male germ cells (GCs). AM-MSCs have the multipotent differentiation capacity and can be differentiated into various cell types. In the present study, AM-MSCs were induced for differentiation into GCs. AM-MSCs were isolated from mouse embryonic membrane by enzymatic digestion. AM-MSCs were characterized with osteogenic and adipogenic differentiation test and flow cytometric analysis of some CD-markers. AM-MSCs were induced to differentiate into GCs using a creative two-step method. Passage-3 AM-MSCs were firstly treated with 25ng/ml bone morphogenetic protein 4 (BMP4) for 5d and in continuing with 1μM retinoic acid (RA) for 12d (total treatment time was 17d). At the end of the treatment period, real-time reverse transcription (RT)-PCR was performed to evaluate the expression of GC-specific markers-Itgb1, Dazl, Stra8, Piwil2, Mvh, Oct4, and c-Kit- in the cells. Moreover, flow cytometry and immunofluorescence staining were performed to evaluate the expression of Mvh and Dazl at protein level. Real-time RT-PCR showed that most of the tested markers were upregulated in the treated AM-MSCs. Furthermore, flow cytometric and immunofluorescence analyses both revealed that a considerable part of the treated cells expressed GC-specific markers. The percentage of positive cells for Mvh and Dazl was about 23 and 46%, respectively. Our results indicated that a number of AM-MSCs successfully differentiated into the GCs. Finally, it seems that AM-MSCs would be a potential source of adult pluripotent stem cells for in vitro generation of GCs and cell-based therapies for treatment of infertility.

Adult Human Peripheral Blood Mononuclear Cells Are Capable of Producing Neurocyte or Photoreceptor-Like Cells That Survive in Mouse Eyes After Preinduction With Neonatal Retina.

This study provides support for the use of peripheral blood mononuclear cells (PBMCs) as a potential source of pluripotent stem cells for treating retinal degeneration. First, this study demonstrated that PBMCs can differentiate into retinal neuron-like cells in vitro and in vivo. Second, some transplanted cells expressed markers for neural progenitors, mature neurons, or photoreceptors at 1, 3, and 6 months after subretinal injection. Finally, this study showed that PBMC transplantation can improve the function of a degenerated retina.

Comparative study of human embryonic stem cells (hESC) and human induced pluripotent stem cells (hiPSC) as a treatment for retinal dystrophies

Retinal dystrophies (RD) are major causes of familial blindness and are characterized by progressive dysfunction of photoreceptor and/or retinal pigment epithelium (RPE) cells. In this study, we aimed to evaluate and compare the therapeutic effects of two pluripotent stem cell (PSC)-based therapies. We differentiated RPE from human embryonic stem cells (hESCs) or human-induced pluripotent stem cells (hiPSCs) and transplanted them into the subretinal space of the Royal College of Surgeons (RCS) rat. Once differentiated, cells from either source of PSC resembled mature RPE in their morphology and gene expression profile. Following transplantation, both hESC- and hiPSC-derived cells maintained the expression of specific RPE markers, lost their proliferative capacity, established tight junctions, and were able to perform phagocytosis of photoreceptor outer segments. Remarkably, grafted areas showed increased numbers of photoreceptor nuclei and outer segment disk membranes. Regardless of the cell source, human transplants protected retina from cell apoptosis, glial stress and accumulation of autofluorescence, and responded better to light stimuli. Altogether, our results show that hESC- and hiPSC-derived cells survived, migrated, integrated, and functioned as RPE in the RCS rat retina, providing preclinical evidence that either PSC source could be of potential benefit for treating RD.

Reprogramming of Human Fibroblasts to Induced Pluripotent Stem Cells with Sleeping Beauty Transposon-Based Stable Gene Delivery.

Human induced pluripotent stem (iPS) cells are a source of patient-specific pluripotent stem cells and resemble human embryonic stem (ES) cells in gene expression profiles, morphology, pluripotency, and in vitro differentiation potential. iPS cells are applied in disease modeling, drug screenings, toxicology screenings, and autologous cell therapy. In this protocol, we describe how to derive human iPS cells from fibroblasts by Sleeping Beauty (SB) transposon-mediated gene transfer of reprogramming factors. First, the components of the non-viral Sleeping Beauty transposon system, namely a transposon vector encoding reprogramming transcription factors and a helper plasmid expressing the SB transposase, are electroporated into human fibroblasts. The reprogramming cassette undergoes transposition from the transfected plasmids into the fibroblast genome, thereby resulting in stable delivery of the reprogramming factors. Reprogramming by using this protocol takes ~4 weeks, after which the iPS cells are isolated and clonally propagated.

Markers of Pluripotency in Human Amniotic Epithelial Cells and Their Differentiation to Progenitor of Cortical Neurons.

Human pluripotent stem cells (hPSC) have promise for regenerative medicine due to their auto-renovation and differentiation capacities. Nevertheless, there are several ethical and methodological issues about these cells that have not been resolved. Human amniotic epithelial cells (hAEC) have been proposed as source of pluripotent stem cells. Several groups have studied hAEC but have reported inconsistencies about their pluripotency properties. The aim of the present study was the in vitro characterization of hAEC collected from a Mexican population in order to identify transcription factors involved in the pluripotency circuitry and to determine their epigenetic state. Finally, we evaluated if these cells differentiate to cortical progenitors. We analyzed qualitatively and quantitatively the expression of the transcription factors of pluripotency (OCT4, SOX2, NANOG, KLF4 and REX1) by RT-PCR and RT-qPCR in hAEC. Also, we determined the presence of OCT4, SOX2, NANOG, SSEA3, SSEA4, TRA-1-60, E-cadherin, KLF4, TFE3 as well as the proliferation and epigenetic state by immunocytochemistry of the cells. Finally, hAEC were differentiated towards cortical progenitors using a protocol of two stages. Here we show that hAEC, obtained from a Mexican population and cultured in vitro (P0-P3), maintained the expression of several markers strongly involved in pluripotency maintenance (OCT4, SOX2, NANOG, TFE3, KLF4, SSEA3, SSEA4, TRA-1-60 and E-cadherin). Finally, when hAEC were treated with growth factors and small molecules, they expressed markers characteristic of cortical progenitors (TBR2, OTX2, NeuN and β-III-tubulin). Our results demonstrated that hAEC express naïve pluripotent markers (KLF4, REX1 and TFE3) as well as the cortical neuron phenotype after differentiation. This highlights the need for further investigation of hAEC as a possible source of hPSC.

Challenges in Translating Germinal Stem Cell Research and Therapy.

The field of stem cells research is one of the most exciting and fast developing fields with a great potential to heal different degenerative diseases in regenerative medicine, to better understand cancer and to improve quality of human life. In spite of great enthusiasm and effort, there are several general obstacles and questions about stem cells, which still need to be resolved to enable safe and efficient treatment of degenerative diseases in the future. There are more interesting types of stem cells in humans, as also evidenced by thirteen manuscripts published in this special issue. Very small embryonic-like stem cells (VSELs) represent one of the most interesting types of stem cells in humans. These small stem cells are suggested to originate in epiblast of an embryo and to persist in a quiescent state in adult human tissues and organs (e.g., bone marrow) from embryonic period of life. They are mobilized into peripheral blood to regenerate the damaged area. It is not excluded that they are involved in tumor formation at inappropriate condition. They have been proven to be multipotent with a degree of pluripotency and able to differentiate into different cell types of all three germ layers. In the manuscript by H. Sovalat et al. it is shown that VSELs have been present in the peripheral blood of healthy volunteers in the same numbers regardless of their age, ranging from 20 to 70 years. Interestingly, endurance exercise such as long-distance running mobilizes VSELs into peripheral blood and increases their number in bone marrow in favor of regeneration, as found by K. Marycz et al. The VSELs have also been identified in bone marrow of other mammalian species such as rat (A. Labedz-Maslowska et al.). Moreover, the VSELs have been proposed by D. Bhartiya et al. to exist in adult mammalian ovaries and to participate in primordial follicle assembly and postnatal oogenesis under the regulation of follicle-stimulating hormone (FSH). This idea has been experimentally supported by M. Zbucka-Kretowska et al. who evidenced the effective release of VSELs into peripheral blood of women treated by FSH hormonal therapy to retrieve the oocytes in the in vitro fertilization program. The VSELs are related to germinal lineage, especially primordial germ cells (PGCs), and it is not excluded that they are related to germinal stem cells (GSCs) and may be even their progenitors. The PGCs, precursors of gametes, can be generated from stem cells, thereafter, result in birth of mouse pups, and represent an exciting new tool to treat infertility in the future, as reviewed by A. Nikolic et al. The female GSCs, namely, oogonial stem cells, can be efficiently isolated from adult mouse ovaries by a new method provided by Z. Lu et al. and might be also applied to human ovaries. B. Fereydouni et al. have demonstrated the development of oocyte-like cells in long-term cell cultures of neonatal marmoset monkey ovary expressing several genes related to the germinal lineage. The ovarian GSCs are very interesting to regenerate the nonfunctional ovaries although also some other types of cells such as human amniotic epithelial cells can express a beneficial paracrine effect on function of chemotherapy-treated ovaries in a mouse model (X. Yao et al.). The GSCs can also be retrieved from adult human testicles but are heterogeneous and, along with genes related to the germinal lineage, express more or less pluripotency, as reported by S. Conrad et al. In a mouse model, derivation of embryonic-like stem cells from spermatogonial stem cells (SSCs) depends on animal age (up to 7 weeks of age) and special time window in long-term SSC cultures (H. Azizi et al.). Not only reproductive tissues but also parthenogenetic embryos may represent an interesting source of pluripotent stem cells after activation of nonfertilized oocytes from the in vitro fertilization program, as reviewed by A. Bos-Mikich et al. Last but not least, the microRNAs, noncoding RNA molecules mediating the translational suppression and posttranscriptional control of gene expression, have been exposed by I. Virant-Klun et al. as extremely important regulators of developmental processes, pluripotency, differentiation, and also shift to cancer. The manuscripts published in this special issue bring an important new knowledge to the field and arise some new interests and challenges for the future. Irma Virant-Klun Hong-Thuy Bui Mariusz Z. Ratajczak

Lost in translation: pluripotent stem cell-derived hematopoiesis.

Pluripotent stem cells (PSCs) such as embryonic stem cells or induced pluripotent stem cells represent a promising cell type to gain novel insights into human biology. Understanding the differentiation process of PSCs in vitro may allow for the identification of cell extrinsic/intrinsic factors, driving the specification process toward all cell types of the three germ layers, which may be similar to the human in vivo scenario. This would not only lay the ground for an improved understanding of human embryonic development but would also contribute toward the generation of novel cell types used in cell replacement therapies. In this line, especially the developmental process of mesodermal cells toward the hematopoietic lineage is of great interest. Therefore, this review highlights recent progress in the field of hematopoietic specification of pluripotent stem cell sources. In addition, we would like to shed light on emerging factors controlling primitive and definitive hematopoietic development and to highlight recent approaches to improve the differentiation potential of PSC sources toward hematopoietic stem/progenitor cells. While the generation of fully defined hematopoietic stem cells from PSCs remains challenging in vitro, we here underline the instructive role of cell extrinsic factors such as cytokines for the generation of PSC-derived mature hematopoietic cells. Thus, we have comprehensively examined the role of cytokines for the derivation of mature hematopoietic cell types such as macrophages, granulocytes, megakaryocytes, erythrocytes, dendritic cells, and cells of the B- and T-cell lineage.

185. Targeted Correction and Restored Function of CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

Recently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources – potentially useful for the development of new therapeutic approaches. The primary defect in Cystic Fibrosis (CF), an autosomal recessive disorder, is the regulation of epithelial chloride transport by a chloride channel protein encoded by the cystic fibrosis transmembrane conductance regulator (CFTR) gene. Recurrent pulmonary infections are responsible for 80 to 90% of the deaths in CF patients. Starting with skin fibroblasts from patients diagnosed with CF, we have derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele vs. the other. Differentiation for a total of 19 days in a protocol designed to derive anterior foregut endoderm, subsequently up-regulated expression of NKX2-1, SOX9, TP63, FOXP2, FOXA2, and CFTR, suggesting commitment of at least a sub-population of cells within the endodermal culture to a lung epithelial cell fate.Once differentiated, mutant CF iPSCs yielded neither mature CFTR protein nor CFTR-specific chloride channel activity (as assayed in Ussing chamber experiments) – whereas corrected CF iPSCs and the normal control WA09 hESCs yielded mature CFTR protein and CFTR-specific chloride channel activity.In vitro differentiation of the mutant CF iPSCs into lung epithelial cells and tissue, controlled for by the parallel differentiation of the otherwise isogenic corrected CF iPSCs, may provide a valuable tool for drug screening and examining the functional consequence of mutant CFTR expression. Furthermore, corrected CF iPSCs present a potential source of patient-specific cells capable, in vitro, of differentiation into various lung stem/progenitor cells – either for transplantation of autologous lung cells or for seeding de-vitalized lung scaffolds ex vivo to generate autologous lungs.

Targeted Correction and Restored Function of the CFTR Gene in Cystic Fibrosis Induced Pluripotent Stem Cells

SummaryRecently developed reprogramming and genome editing technologies make possible the derivation of corrected patient-specific pluripotent stem cell sources—potentially useful for the development of new therapeutic approaches. Starting with skin fibroblasts from patients diagnosed with cystic fibrosis, we derived and characterized induced pluripotent stem cell (iPSC) lines. We then utilized zinc-finger nucleases (ZFNs), designed to target the endogenous CFTR gene, to mediate correction of the inherited genetic mutation in these patient-derived lines via homology-directed repair (HDR). We observed an exquisitely sensitive, homology-dependent preference for targeting one CFTR allele versus the other. The corrected cystic fibrosis iPSCs, when induced to differentiate in vitro, expressed the corrected CFTR gene; importantly, CFTR correction resulted in restored expression of the mature CFTR glycoprotein and restoration of CFTR chloride channel function in iPSC-derived epithelial cells.

Periostin conditions the matrix to generate a niche for islet regeneration.

Islet replacement via the Edmonton Protocol offers proof of concept that transient insulin independence can be achieved in patients with diabetes (1–4). However, the need for life-long immunosuppression, an extreme shortage of human islets, along with graft failure due to rejection and continued autoimmunity, limits the widespread application of this approach. Although recent progress has been made in the derivation of “ -like” cells from human pluripotent stem cell sources (embryonic stem cells or induced pluripotent stem cells) (5–10), inducing the replication of surviving -cells in situ (11, 12) and/or stimulating new islet formation from progenitor cells within the pancreas (islet neogenesis) (13–16) represents the most direct way to increase -cell mass. Studies in both rodents and humans suggest endogenous islet repair mechanisms may remain intact during diabetes and can be activated by cellor pharmacologically-mediated stimuli. Evidence of ongoing -cell regeneration has been observed in type 1 diabetes (T1D) patients at early onset (17) or many years after diagnosis (18). Indeed, recent analysis of T1D patients with disease duration of more than 50 years (medalists) has uncovered sustained c-peptide and proliferating -cells within the pancreas in the face of ongoing autoimmunity (19). Moreover, the increase in -cell mass in response to pregnancy suggests that the human endocrine pancreas has the capacity to regenerate if we can understand how to “tip the balance” in favor of islet regeneration vs destruction during diabetes. Interestingly, increased -cell replication was first suggested as the mechanism for increased -cell mass during pregnancy, but a recent autopsy study of human pancreases during pregnancy showed an increased proportion of small islets and increased number of insulin cells in the ducts but no change in -cell replication or apoptosis frequency (20). Nonetheless, novel strategies to optimize islet regeneration, either by stimulating -cell replication or by harnessing endogenous islet neogenic processes within the diabetic pancreas, remain under intense investigation for the treatment of diabetes. In initial publications documenting endogenous islet regeneration by stem cells, transplantation of bone marrow-derived c-kit cells reduced hyperglycemia in mice with streptozotocin (STZ)-induced -cell deletion (21). Importantly, transplanted stem cells did not acquire insulin expression as previously thought. In contrast, donor cells surrounded regenerating islets or engrafted in ductal regions and stimulated proliferation within recipient islets via undetermined paracrine activities (21). Subsequently, other preclinical and early clinical reports have shown that both human hematopoietic (22–26) or multipotent stromal cell (25–28), also known as mesenchymal stem cell (MSC) lineages, can enhance islet regeneration. Furthermore, islet regeneration and immune protection may be achieved by coinfusion of murine hematopoietic cells with MSC in autoimmune non-obese diabetic mice (29). Although this concept termed “stem cell-stimulated islet regeneration” has emerged as a central process for pancreas repair, either by stimulating -cell proliferation (11, 24– 26) or by initiating new islet formation from ductal or islet-derived precursors (14–16, 25, 26), the molecular signaling mechanisms by which distinct stem and progenitor cell types mediate islet recovery are not well understood. More importantly, recent controversy of the role of betatrophin in islet regeneration (30, 31) has underscored

Enabling consistency in pluripotent stem cell-derived products for research and development and clinical applications through material standards.

There is a need for physical standards (reference materials) to ensure both reproducibility and consistency in the production of somatic cell types from human pluripotent stem cell (hPSC) sources. We have outlined the need for reference materials (RMs) in relation to the unique properties and concerns surrounding hPSC-derived products and suggest in-house approaches to RM generation relevant to basic research, drug screening, and therapeutic applications. hPSCs have an unparalleled potential as a source of somatic cells for drug screening, disease modeling, and therapeutic application. Undefined variation and product variability after differentiation to the lineage or cell type of interest impede efficient translation and can obscure the evaluation of clinical safety and efficacy. Moreover, in the absence of a consistent population, data generated from in vitro studies could be unreliable and irreproducible. Efforts to devise approaches and tools that facilitate improved consistency of hPSC-derived products, both as development tools and therapeutic products, will aid translation. Standards exist in both written and physical form; however, because many unknown factors persist in the field, premature written standards could inhibit rather than promote innovation and translation. We focused on the derivation of physical standard RMs. We outline the need for RMs and assess the approaches to in-house RM generation for hPSC-derived products, a critical tool for the analysis and control of product variation that can be applied by researchers and developers. We then explore potential routes for the generation of RMs, including both cellular and noncellular materials and novel methods that might provide valuable tools to measure and account for variation. Multiparametric techniques to identify "signatures" for therapeutically relevant cell types, such as neurons and cardiomyocytes that can be derived from hPSCs, would be of significant utility, although physical RMs will be required for clinical purposes.

Pluripotent stem cells for Schwann cell engineering.

Tissue engineering of Schwann cells (SCs) can serve a number of purposes, such as in vitro SC-related disease modeling, treatment of peripheral nerve diseases or peripheral nerve injury, and, potentially, treatment of CNS diseases. SCs can be generated from autologous stem cells in vitro by recapitulating the various stages of in vivo neural crest formation and SC differentiation. In this review, we survey the cellular and molecular mechanisms underlying these in vivo processes. We then focus on the current in vitro strategies for generating SCs from two sources of pluripotent stem cells, namely embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different methods for SC engineering from ESCs and iPSCs are reviewed and suggestions are proposed for optimizing the existing protocols. Potential safety issues regarding the clinical application of iPSC-derived SCs are discussed as well. Lastly, we will address future aspects of SC engineering.

Induction of dental pulp-derived induced pluripotent stem cells in the absence of c-Myc for differentiation into neuron-like cells

BackgroundA recent research breakthrough has demonstrated that the ectopic expression of four genes is sufficient to reprogram human fibroblasts into inducible pluripotent stem cells (iPSCs). However, whether human dental pulp cells (DPCs) could be reprogrammed into iPSCs remains an open question. In this study, we demonstrated that DPCs from deciduous and permanent teeth can be reprogrammed into iPSCs without c-Myc and had the capacity to differentiate into neuron-like cells. MethodsDPCs were obtained from donors and reprogrammed into iPSCs using retroviral transduction with SOX2, OCT4, and KLF4. Then, these iPSCs were differentiated into neuron-like cells. Microarray and bioinformatics were used to compare the gene expression profile among these iPSCs and iPSC-derived neuron-like cells. ResultsThe DPCs displayed a high vitality and capability to quickly restart proliferation and expressed elevated pluripotency similar to mesenchymal stem cells. According to our results, DPC-derived iPSC colonies that could be subcultured and propagated were established as early as 10 days after transduction, in comparison with the skin fibroblast (DPC-derived iPSCs) without c-Myc presented embryonic stem cell-like properties and the pluripotent potential to differentiate into neuron-like cells, which resemble neurons both morphologically and functionally. ConclusionThe human DPCs from deciduous and permanent teeth can undergo reprogramming to establish pluripotent stem cell lines without c-Myc. These surgical residues, usually regarded as medical waste, can be used as an alternative source of pluripotent stem cells for personalized medicine.