scholarly journals miRNA-Based Rapid Differentiation of Purified Neurons from hPSCs Advancestowards Quick Screening for Neuronal Disease Phenotypes In Vitro

Cells ◽  
2020 ◽  
Vol 9 (3) ◽  
pp. 532 ◽  
Author(s):  
Mitsuru Ishikawa ◽  
Takeshi Aoyama ◽  
Shoichiro Shibata ◽  
Takefumi Sone ◽  
Hiroyuki Miyoshi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neuronal Differentiation ◽  
Pluripotent Stem Cells ◽  
Dermal Fibroblasts ◽  
Neuronal Maturation ◽  
Calcium Oscillation ◽  
Induction Method ◽  
Disease Phenotypes ◽  
Neuronal Disease

Obtaining differentiated cells with high physiological functions by an efficient, but simple and rapid differentiation method is crucial for modeling neuronal diseases in vitro using human pluripotent stem cells (hPSCs). Currently, methods involving the transient expression of one or a couple of transcription factors have been established as techniques for inducing neuronal differentiation in a rapid, single step. It has also been reported that microRNAs can function as reprogramming effectors for directly reprogramming human dermal fibroblasts to neurons. In this study, we tested the effect of adding neuronal microRNAs, miRNA-9/9*, and miR-124 (miR-9/9*-124), for the neuronal induction method of hPSCs using Tet-On-driven expression of the Neurogenin2 gene (Ngn2), a proneural factor. While it has been established that Ngn2 can facilitate differentiation from pluripotent stem cells into neurons with high purity due to its neurogenic effect, a long or indefinite time is required for neuronal maturation with Ngn2 misexpression alone. With the present method, the cells maintained a high neuronal differentiation rate while exhibiting increased gene expression of neuronal maturation markers, spontaneous calcium oscillation, and high electrical activity with network bursts as assessed by a multipoint electrode system. Moreover, when applying this method to iPSCs from Alzheimer’s disease (AD) patients with presenilin-1 (PS1) or presenilin-2 (PS2) mutations, cellular phenotypes such as increased amount of extracellular secretion of amyloid β42, abnormal oxygen consumption, and increased reactive oxygen species in the cells were observed in a shorter culture period than those previously reported. Therefore, it is strongly anticipated that the induction method combining Ngn2 and miR-9/9*-124 will enable more rapid and simple screening for various types of neuronal disease phenotypes and promote drug discovery.

scholarly journals Pluripotent Stem Cells as an In Vitro Model of Neuronal Differentiation

10.5772/24695 ◽  
2011 ◽  
Author(s):  
Irina Kerkis ◽  
Mirian A. F. Hayashi ◽  
Nelson F. ◽  
Antonio C. ◽  
Lygia V. ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neuronal Differentiation ◽  
Pluripotent Stem Cells ◽  
In Vitro Model ◽  
Vitro Model

scholarly journals A Monolayer System for the Efficient Generation of Motor Neuron Progenitors and Functional Motor Neurons from Human Pluripotent Stem Cells

Cells ◽  
2021 ◽  
Vol 10 (5) ◽  
pp. 1127
Author(s):  
Alessandro Cutarelli ◽  
Vladimir A. Martínez-Rojas ◽  
Alice Tata ◽  
Ingrid Battistella ◽  
Daniela Rossi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Motor Neuron ◽  
Motor Neurons ◽  
Pluripotent Stem Cells ◽  
Disease Modelling ◽  
Disease Phenotypes ◽  
In Vitro Systems ◽  
Induced Pluripotent

Methods for the conversion of human induced pluripotent stem cells (hiPSCs) into motor neurons (MNs) have opened to the generation of patient-derived in vitro systems that can be exploited for MN disease modelling. However, the lack of simplified and consistent protocols and the fact that hiPSC-derived MNs are often functionally immature yet limit the opportunity to fully take advantage of this technology, especially in research aimed at revealing the disease phenotypes that are manifested in functionally mature cells. In this study, we present a robust, optimized monolayer procedure to rapidly convert hiPSCs into enriched populations of motor neuron progenitor cells (MNPCs) that can be further amplified to produce a large number of cells to cover many experimental needs. These MNPCs can be efficiently differentiated towards mature MNs exhibiting functional electrical and pharmacological neuronal properties. Finally, we report that MN cultures can be long-term maintained, thus offering the opportunity to study degenerative phenomena associated with pathologies involving MNs and their functional, networked activity. These results indicate that our optimized procedure enables the efficient and robust generation of large quantities of MNPCs and functional MNs, providing a valid tool for MNs disease modelling and for drug discovery applications.

Cancellation of c-MYC Silencing in Human Induced Pluripotent Stem Cells Contributes to the Efficient in Vitro Production of Platelets with the Ability of Hemostasis In Vivo.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 1488-1488 ◽  
Author(s):  
Naoya Takayama ◽  
Sou Nakamura ◽  
Satoshi Nishimura ◽  
Ryoko Ohnishi ◽  
Kazutoshi Takahashi ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Blood Platelets ◽  
Dermal Fibroblasts ◽  
Efficient Production ◽  
Nog Mice ◽  
Induced Pluripotent

Abstract Abstract 1488 Poster Board I-511 Human induced pluripotent stem cells (hiPSCs) generated from somatic cells by introduction of OCT3/4, SOX2, KLF4 and c-MYC represent a potential source of hematopoietic cells for transfusion without the risk of immune rejection. We recently established an in vitro culture system with which hiPSCs could be differentiated into the unique structure of an “in vitro hematopoietic niche” containing hematopoietic progenitors. Upon further cultivation under appropriate conditions, these hematopoietic progenitor cells differentiated into megakaryocytes, which could then generate platelets with morphologies indistinguishable from peripheral blood platelets regardless of either 4-factor iPSCs (n=8, 4-factor hiPSC clones generated from adult dermal fibroblasts through induction with c-MYC) or 3-factor iPSC clones (without c-MYC, n=3). It is well known that iPSC differentiation yields a heterogeneous population of clones. To select the best hiPSC clone for platelet production, we quantified thrombopoiesis with 11 independent hiPSC clones by comparison with human embryonic stem cells (hESCs) evaluated previously (Takayama et al., Blood, 2008) as a reference. Particularly noteworthy is our finding that 4-factor iPSCs have an advantage over 3-factor iPSCs or hESCs (P<0.01) that is mediated through cancellation of c-MYC silencing (re-activation) over the course of differentiation evidenced by RT-PCR studies. Indeed, ectopic expression of c-MYC, but not OCT3/4, SOX2 or KLF4, using a retroviral vector in hESC-derived progenitors accelerated both megakaryopoiesis and thrombopoiesis. By contrast, the platelet activation statuses (i.e., PAC-1 ligation with activated integrin αIIbβ3 following agonist stimulation) were comparable for platelets obtained from 4-factor hiPSCs and hESCs, though levels of c-MYC clearly differed, indicating that at least integrin activation is independent of c-MYC. To further estimate the in vivo functionality of iPSC-derived platelets, we developed a mouse model for transfusion. Irradiation (2.0 Gy, 9 days beforehand) induced thrombocytopenia in NOG (nod-scid/IL-2 γc-null) mice. Subsequent flow cytometry showed that 2 hrs after transfusion (1.0∼1.2×107 platelets per a mouse) of NOG mice via the tail vein, the circulating levels of selected 4-factor iPSC-derived platelets were similar to those of human adult platelets (platelet chimerism of human CD41/mouse CD41; 4∼10%). Moreover, by using our recently established in vivo imaging system, which enables observation of single platelet behavior, we observed that 4-factor iPSC-derived platelets circulate in NOG mice and contribute to the development thrombi within their vessels, suggesting the in vivo functionality of iPSC-derived platelets is intact. A number of studies have suggested that c-MYC can have deleterious effects leading to in vivo oncogeneity after transplantation in vivo. By contrast, our data strongly indicate the importance of c-MYC for platelet generation from hiPSCs and hESCs. Given that anucleate platelets are routinely irradiated before transfusion in clinical settings, use of c-MYC for hiPSCs generation may contribute to the efficient production of HLA-matched platelet concentrates for those requiring repeated transfusion. Disclosures: No relevant conflicts of interest to declare.

Derivation of Hematopoietic Progenitor Cells From Vector-Free Human Induced Pluripotent Stem Cells

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 4746-4746
Author(s):  
Friedrich Schuening ◽  
Michail Zaboikin ◽  
Tatiana Zaboikina ◽  
Narasimhachar Srinivasakumar
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Dermal Fibroblasts ◽  
Embryoid Bodies ◽  
Chromosomal Anomalies ◽  
Hematopoietic Stem ◽  
Induced Pluripotent

Abstract Abstract 4746 Induced pluripotent stem cells (iPSCs), due to their self-renewal and differentiation capability, have tremendous potential in regenerative medicine. Differentiation of IPSCs in vitro to obtain sufficient number of hematopoietic stem cells (HSCs) and their progenitors (HPCs) from iPSCs for therapeutic purposes is a holy grail of cellular therapy. To this end, we are comparing different in vitro differentiation approaches for generation of HSCs/HPCs from IPSCs. We have generated iPSCs from human adult dermal fibroblasts using two different reprogramming methods: 1) Transduction with retroviral vectors encoding human Klf4, Oct3/4, Sox2 and cMyc or 2) Electroporation with Epstein–Barr virus (EBV) based episomal plasmid vectors encoding Klf4, Oct3/4, Sox2, L-Myc and p53 targeting shRNA. The transduced/electroporated cells were reprogrammed on SNL5 mouse feeder cells. Putative iPSC-like colonies were cloned and adapted to grow under feeder-free conditions on Matrigel (BD) in mTeSR1 (Stem Cell Technologies) medium. From over 30 individual clones isolated, six were further characterized for: 1) expression of pluripotency markers (Tra-1–60, SSEA-3, SSEA-4, Nanog and Oct3/4) by immunofluorescence; 2) endogenous and total mRNA expression by quantitative real-time reverse-transcriptase PCR (RT-qPCR) for Klf4, Oct3/4, Sox2 and cMyc to distinguish between cellular and vector derived expression of reprogramming factors; 3) RT-qPCR to determine expression of other markers of pluripotency such as Nanog and DNA methyl transferease; 4) karyotype analysis to determine chromosomal anomalies. The vector-free IPSC clones were also tested for residual integrated EBV plasmid DNA by qPCR. Trilineage differentiation ability of the clones was determined through embryoid body formation in suspension cultures, and subsequent staining of resulting embryoid bodies after adherence to gelatin coated dishes for makers of ectoderm, mesoderm and endoderm. HSCs/HPCs were obtained from IPSCs by 1) coculture with OP9 stromal cells, or 2) step-wise differentiation in feeder-free conditions on Matrigel under defined conditions in the presence of appropriate growth factors [Niwa A et al. PLoS One. (2011); 6(7):e22261.]. The resultant HSCs/HPCs were subjected to colony forming assays in semi-solid medium containing hematopoietic cytokines. Both erythroid and myelomonocytic colonies could be readily identified. The influence of ambient oxygen concentration on the HSC/HPC derivation procedure is being investigated. The results of these studies will be presented. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Maintenance and Neuronal Differentiation of Chicken Induced Pluripotent Stem-Like Cells

2014 ◽  
Vol 2014 ◽  
pp. 1-14 ◽  
Author(s):  
Rui Dai ◽  
Ricardo Rossello ◽  
Chun-chun Chen ◽  
Joeran Kessler ◽  
Ian Davison ◽  
...  
Keyword(s):  
Stem Cells ◽  
Neuronal Differentiation ◽  
Pluripotent Stem Cells ◽  
Vocal Learning ◽  
Culture Conditions ◽  
Differentiated Cells ◽  
Induced Pluripotent ◽  
Stem Cell State

Pluripotent stem cells have the potential to become any cell in the adult body, including neurons and glia. Avian stem cells could be used to study questions, like vocal learning, that would be difficult to examine with traditional mouse models. Induced pluripotent stem cells (iPSCs) are differentiated cells that have been reprogrammed to a pluripotent stem cell state, usually using inducing genes or other molecules. We recently succeeded in generating avian iPSC-like cells using mammalian genes, overcoming a limitation in the generation and use of iPSCs in nonmammalian species (Rosselló et al., 2013). However, there were no established optimal cell culture conditions for avian iPSCs to establish long-term cell lines and thus to study neuronal differentiationin vitro. Here we present an efficient method of maintaining chicken iPSC-like cells and for differentiating them into action potential generating neurons.

Mini Review; Differentiation of Human Pluripotent Stem Cells into Oocytes

2020 ◽  
Vol 15 (4) ◽  
pp. 301-307 ◽  
Author(s):  
Gaifang Wang ◽  
Maryam Farzaneh
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Human Pluripotent Stem Cells ◽  
Human Oocyte ◽  
Differentiation Potential ◽  
Cell Lineages ◽  
Cell Based Therapy ◽  
Induced Pluripotent

Primary Ovarian Insufficiency (POI) is one of the main diseases causing female infertility that occurs in about 1% of women between 30-40 years of age. There are few effective methods for the treatment of women with POI. In the past few years, stem cell-based therapy as one of the most highly investigated new therapies has emerged as a promising strategy for the treatment of POI. Human pluripotent stem cells (hPSCs) can self-renew indefinitely and differentiate into any type of cell. Human Embryonic Stem Cells (hESCs) as a type of pluripotent stem cells are the most powerful candidate for the treatment of POI. Human-induced Pluripotent Stem Cells (hiPSCs) are derived from adult somatic cells by the treatment with exogenous defined factors to create an embryonic-like pluripotent state. Both hiPSCs and hESCs can proliferate and give rise to ectodermal, mesodermal, endodermal, and germ cell lineages. After ovarian stimulation, the number of available oocytes is limited and the yield of total oocytes with high quality is low. Therefore, a robust and reproducible in-vitro culture system that supports the differentiation of human oocytes from PSCs is necessary. Very few studies have focused on the derivation of oocyte-like cells from hiPSCs and the details of hPSCs differentiation into oocytes have not been fully investigated. Therefore, in this review, we focus on the differentiation potential of hPSCs into human oocyte-like cells.

scholarly journals Stability of Imprinting and Differentiation Capacity in Naïve Human Cells Induced by Chemical Inhibition of CDK8 and CDK19

Cells ◽  
2021 ◽  
Vol 10 (4) ◽  
pp. 876
Author(s):  
Raquel Bernad ◽  
Cian J. Lynch ◽  
Rocio G. Urdinguio ◽  
Camille Stephan-Otto Attolini ◽  
Mario F. Fraga ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Primordial Germ Cell ◽  
Normal Karyotype ◽  
Cell Types ◽  
Dna Demethylation ◽  
Starting State ◽  
Teratoma Formation

Pluripotent stem cells can be stabilized in vitro at different developmental states by the use of specific chemicals and soluble factors. The naïve and primed states are the best characterized pluripotency states. Naïve pluripotent stem cells (PSCs) correspond to the early pre-implantation blastocyst and, in mice, constitute the optimal starting state for subsequent developmental applications. However, the stabilization of human naïve PSCs remains challenging because, after short-term culture, most current methods result in karyotypic abnormalities, aberrant DNA methylation patterns, loss of imprinting and severely compromised developmental potency. We have recently developed a novel method to induce and stabilize naïve human PSCs that consists in the simple addition of a chemical inhibitor for the closely related CDK8 and CDK19 kinases (CDK8/19i). Long-term cultured CDK8/19i-naïve human PSCs preserve their normal karyotype and do not show widespread DNA demethylation. Here, we investigate the long-term stability of allele-specific methylation at imprinted loci and the differentiation potency of CDK8/19i-naïve human PSCs. We report that long-term cultured CDK8/19i-naïve human PSCs retain the imprinting profile of their parental primed cells, and imprints are further retained upon differentiation in the context of teratoma formation. We have also tested the capacity of long-term cultured CDK8/19i-naïve human PSCs to differentiate into primordial germ cell (PGC)-like cells (PGCLCs) and trophoblast stem cells (TSCs), two cell types that are accessible from the naïve state. Interestingly, long-term cultured CDK8/19i-naïve human PSCs differentiated into PGCLCs with a similar efficiency to their primed counterparts. Also, long-term cultured CDK8/19i-naïve human PSCs were able to differentiate into TSCs, a transition that was not possible for primed PSCs. We conclude that inhibition of CDK8/19 stabilizes human PSCs in a functional naïve state that preserves imprinting and potency over long-term culture.

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