In situ generation of human brain organoids on a micropillar array

Lab on a Chip ◽  
2017 ◽  
Vol 17 (17) ◽  
pp. 2941-2950 ◽  
Author(s):  
Yujuan Zhu ◽  
Li Wang ◽  
Hao Yu ◽  
Fangchao Yin ◽  
Yaqing Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Human Brain ◽  
Pluripotent Stem Cells ◽  
3D Culture ◽  
In Situ Generation ◽  
Induced Pluripotent ◽  
High Throughput Manner

We present a simple and high throughput manner to generate brain organoids in situ from human induced pluripotent stem cells on micropillar arrays and to investigate long-term brain organogenesis in 3D culture in vitro.

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.

scholarly journals Forced HoxB4 Sustains CD45-c-kit+ Pre-Hematopoieitc Stem Cells (HSCs) Derived from Murine Induced-Pluripotent Stem Cells, Which Develop Long-Term and Short-Term Repopulating HSCs According to GATA2 Expression Level

Blood ◽  
2014 ◽  
Vol 124 (21) ◽  
pp. 1567-1567
Author(s):  
Kiyoko Izawa ◽  
Tomoya Kakegawa ◽  
Masayuki Yamamoto ◽  
Arinobu Tojo
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Research Funding ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Donor Cells ◽  
Induced Pluripotent ◽  
Cell Fractions

Abstract Background: Hematopoietic stem cells (HSCs) constitute a rare population of bone marrow cells (BMCs) and their ex vivo expansion with stemness for a prolonged period is quite unlikely. It is also difficult to efficiently produce HSCs from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). HoxB4 is a positive regulator of murine HSC self-renewal when ectopically expressed. Moreover, it was reported that HoxB4 overexpression could promote differentiation of ESCs to definitive HSCs. We established iPSCs (GGH-iPSCs) from BMCs of a C57BL/6J-Ly5.2 GATA2-GFP knock-in mouse (PNAS 103:2202, 2006) and transduced them with a 4-hydroxytamoxyfen (4-HT)-inducible HoxB4 construct. Using GGH-iPSCs, we showed that enforced HoxB4 activity throughout their culture period resulted in the long-term maintenance of iPSC-derived HSCs, which could repopulate in recipient mice over 30 weeks after transplantation, in vitro (Blood 122:2418, 2013). In the present study, we tried to elucidate the precise stage of hematopoietic differentiation governed by enforced HoxB4 in this iPSC model, especially in association with GATA2 expression. Experimental procedures and Results: GGH-iPSCs were induced toward hematopoietic differentiatin under enforced HoxB4, and after 2 months of culture, almost all the resulting cells expressed c-kit but not CD45, and approximately half of them were highly positive for GFP (GATA2). We also found that these CD45- cell populations expressed HSC marker genes such as SCL and LMO2, and that they produced CD45+ blood cells after HoxB4 was switched off. These results suggest that CD45- pre-HSCs, which can develop LT-HSCs, may be included in HoxB4-sustained iPSC-derived cell populations. Next, according to the GFP (GATA2) expression level, we divided CD45-c-kit+ cells on HoxB4 into two fractions; CD45-c-kit+GATA2high and CD45-c-kit+GATA2low/- cells, and cultured FACS-sorted cells over an OP9 monolayer with cytokines and without 4-HT to switch off HoxB4. Then, CD45+ blood cells emerged from both cell fractions over time, and CD45-c-kit+GATA2low/- cells produced much more CD45+ cells than CD45-c-kit+GATA2high cells one month later (Figure Left). Furthermore, to test a long-term repopulating activity in mice, each of cell fractions was cultured without 4-HT for 4 days after sorting, and then subjected to transplanttion into sublethally-irradiated C57BL/6-Ly5.1 recipient mice. Two weeks after transplantation, peripheral blood Ly5.2+ donor cells were much more frequent in mice transplanted with CD45-c-kit+GATA2low/- cells, compared to those with CD45-c-kit+GATA2high cells, whereas the CD45-c-kit+GATA2low/- fraction-derived donor cells rapidly decreased and disappeared around 2 months later. In contrast, CD45-c-kit+GATA2high donor cells could repopulate in recipient mice over 20 weeks after transplantation (Figure Right). These results suggest that CD45-c-kit+GATA2low/- and CD45-c-kit+GATA2high cell fractions include short-term (ST) and long-term (LT) -repopulating HSCs, respectively. Conclusion: Taken together, in a hematopoietic differentiation model of murine GGH-iPSCs, long-term repopulating hematopoietic stem cells (LT-HSCs) emerge from CD45-c-kit+GATA2high pre-HSC fraction which can be sustained in vitro by enforced HoxB4. In this experimental setting, enforced HoxB4 is likely to hinder differentiation from CD45- pre-HSCs to CD45+ HSCs. Figure 1 Figure 1. Figure 2 Figure 2. Disclosures Tojo: Novartis: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Research Funding; Chugai: Research Funding, Speakers Bureau; Dainippon Sumitomo: Research Funding; Pfizer: Research Funding; Celgene: Consultancy.

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.

scholarly journals Ammonia-based enrichment and long-term propagation of zone I hepatocyte-like cells

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Ruri Tsuneishi ◽  
Noriaki Saku ◽  
Shoko Miyata ◽  
Saeko Akiyama ◽  
Palaksha Kanive Javaregowda ◽  
...  
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Selection Strategy ◽  
Ammonia Metabolism ◽  
Cytochrome P450 Genes ◽  
Induced Pluripotent ◽  
Selection Agent

AbstractAmmonia has a cytotoxic effect and can therefore be used as a selection agent for enrichment of zone I hepatocytes. However, it has not yet been determined whether ammonia-treated hepatocyte-like cells are able to proliferate in vitro. In this study, we employed an ammonia selection strategy to purify hepatocyte-like cells that were differentiated from human embryonic stem cells (ESCs) and from induced pluripotent stem cells (iPSCs). The resistance to cytotoxicity or cell death by ammonia is likely attributable to the metabolism of ammonia in the cells. In addition to ammonia metabolism-related genes, ammonia-selected hepatocytes showed increased expression of the cytochrome P450 genes. Additionally, the ammonia-selected cells achieved immortality or at least an equivalent life span to human pluripotent stem cells without affecting expression of the liver-associated genes. Ammonia treatment in combination with in vitro propagation is useful for obtaining large quantities of hepatocytes.

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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