Comparison of in vitro Neuronal Differentiation Capacity Between Mouse Epiblast Stem Cells Derived From Nuclear Transfer and Naturally Fertilized Embryos

AbstractThe pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

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Hypoxic Culture Promotes Dopaminergic-Neuronal Differentiation of Nasal Olfactory Mucosa Mesenchymal Stem Cells via Upregulation of Hypoxia-Inducible Factor-1α

Cell Transplantation ◽

10.1177/0963689717720291 ◽

2017 ◽

Vol 26 (8) ◽

pp. 1452-1461 ◽

Cited By ~ 8

Author(s):

Yi Zhuo ◽

Lei Wang ◽

Lite Ge ◽

Xuan Li ◽

Da Duan ◽

...

Keyword(s):

Stem Cells ◽

Mesenchymal Stem Cells ◽

Neuronal Differentiation ◽

Hypoxia Inducible Factor ◽

Cell Voltage ◽

Ambient Air ◽

Olfactory Mucosa ◽

Hypoxia Inducible Factor 1Α ◽

Downstream Target

Olfactory mucosa mesenchymal stem cells (OM-MSCs) display significant clonogenic activity and may be easily propagated for Parkinson’s disease therapies. Methods of inducing OM-MSCs to differentiate into dopaminergic (DAergic) neurons using olfactory ensheathing cells (OECs) are thus an attractive topic of research. We designed a hypoxic induction protocol to generate DAergic neurons from OM-MSCs using a physiological oxygen (O2) level of 3% and OEC-conditioned medium (OCM; HI group). The normal induction (NI) group was cultured in O2 at ambient air level (21%). The role of hypoxia-inducible factor-1α (HIF-1α) in the differentiation of OM-MSCs under hypoxia was investigated by treating cells with an HIF-1α inhibitor before induction (HIR group). The proportions of β-tubulin- and tyrosine hydroxylase (TH)-positive cells were significantly increased in the HI group compared with the NI and HIR groups, as shown by immunocytochemistry and Western blotting. Furthermore, the level of dopamine was significantly increased in the HI group. A slow outward potassium current was recorded in differentiated cells after 21 d of induction using whole-cell voltage-clamp tests. A hypoxic environment thus promotes OM-MSCs to differentiate into DAergic neurons by increasing the expression of HIF-1α and by activating downstream target gene TH. This study indicated that OCM under hypoxic conditions could significantly upregulate key transcriptional factors involved in the development of DAergic neurons from OM-MSCs, mediated by HIF-1α. Hypoxia promotes DAergic neuronal differentiation of OM-MSCs, and HIF-1α may play an important role in hypoxia-inducible pathways during DAergic lineage specification and differentiation in vitro.

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Intramuscular Injection of Botox Induces Tendon Atrophy and Senescence of Tendon-Derived Stem Cells

10.21203/rs.3.rs-70180/v1 ◽

2020 ◽

Author(s):

Peilin Chen ◽

Ziming Chen ◽

Christopher Mitchell ◽

Junjie Gao ◽

Lianzhi Chen ◽

...

Keyword(s):

Stem Cells ◽

Mechanical Loading ◽

Patellar Tendon ◽

Intramuscular Injection ◽

Vastus Lateralis ◽

Collagen Fibre ◽

Differentiation Capacity ◽

Botox Injection

Abstract Background: Botulinum toxin (Botox) injection is in widespread clinical use for the treatment of muscle spasms and tendinopathy but the mechanism of action is poorly understood. Hypothesis: We hypothesised that the reduction of patellar-tendon mechanical-loading following intra-muscular injection of Botox results in tendon atrophy that is at least in part mediated by the induction of senescence of tendon-derived stem cells (TDSCs). Study Design: Controlled laboratory study Methods: A total of 36 mice were randomly divided in 2 groups (18 Botox-injected and 18 vehicle-only control). Mice were injected into to right vastus lateralis of quadriceps muscles either with Botox to induce mechanical stress deprivation of the patellar tendon or with normal saline as control. At 2 weeks post-injection, animals were euthanized prior to tissues harvest for either evaluation of tendon morphology or in vitro studies. TDSCs were isolated by cell-sorting prior to determination of viability, differentiation capacity and senescence markers, as well as assessing their response to mechanical loading in a bioreactor. Finally, to examine the mechanism of tendon atrophy in vitro, key proteins in the PTEN/AKT pathway were evaluated in TDSCs in both groups. Results: Two weeks after Botox injection, patellar tendons displayed atrophic features including tissue volume reduction and collagen fibre misalignment and increased degradation. The colony formation assay revealed the significantly reduced colony units of TDSCs in Botox injected group compared to controls. Multipotent differentiation capacity of TDSCs has also diminished after Botox injection. To examine if mechanical deprived TDSC is capable of forming tendon tissue, we used an isolated bioreactor system to culture 3D TDSCs constructs. The result showed that TDSCs from the Botox-treated group failed to restore tenogenic differentiation after appropriate mechanical loading. Examination of PTEN/AKT signalling pathway revealed that injection of Botox into quadriceps muscle causes PTEN/AKT mediated cell senescence of TDSCs. Conclusion: Intramuscular injection of Botox interferes with tendon homeostasis by inducing tendon atrophy and senescence of TDSCs. Botox injection may have long-term adverse consequences for the treatment of tendinopathy. Clinical relevance: Intramuscular Botox injection for tendinopathy and tendon injury could cause adverse effects in human tendons and re-evaluation of its long-term efficacy is warranted.

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Premature neuronal differentiation in familial Alzheimer’s disease human stem cells in vitro and in postmortem brain tissue

Alzheimer s & Dementia ◽

10.1002/alz.039793 ◽

2020 ◽

Vol 16 (S3) ◽

Author(s):

Charles Arber ◽

Christopher E.J. Lovejoy ◽

Nanet Willumsen ◽

Argyro Alatza ◽

Jackie M. Casey ◽

...

Keyword(s):

Alzheimer’S Disease ◽

Stem Cells ◽

Neuronal Differentiation ◽

Brain Tissue ◽

Postmortem Brain ◽

Human Stem Cells ◽

Familial Alzheimer’S Disease ◽

Postmortem Brain Tissue ◽

Familial Alzheimer's Disease

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Nuclear Transfer-Derived Epiblast Stem Cells Are Transcriptionally and Epigenetically Distinguishable from Their Fertilized-Derived Counterparts

Stem Cells ◽

10.1002/stem.400 ◽

2010 ◽

Vol 28 (4) ◽

pp. 743-752 ◽

Cited By ~ 22

Author(s):

Julien Maruotti ◽

Xiang Peng Dai ◽

Vincent Brochard ◽

Luc Jouneau ◽

Jun Liu ◽

...

Keyword(s):

Stem Cells ◽

Nuclear Transfer ◽

Epiblast Stem Cells

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47 DIFFERENTIAL DEVELOPMENTAL ABILITY OF EMBRYOS CLONED FROM TISSUE-SPECIFIC STEM CELLS

Reproduction Fertility and Development ◽

10.1071/rdv19n1ab47 ◽

2007 ◽

Vol 19 (1) ◽

pp. 142

Author(s):

K. Inoue ◽

N. Ogonuki ◽

H. Miki ◽

S. Noda ◽

S. Inoue ◽

...

Keyword(s):

Stem Cells ◽

Nuclear Transfer ◽

Embryonic Stem ◽

Donor Cell ◽

Fibroblast Cell ◽

High Rate ◽

Full Potential ◽

Cell Nuclei ◽

Cell Stage

Although cloning animals by somatic cell nuclear transfer is generally an inefficient process, use of appropriate donor cell types may improve the cloning outcome significantly. Among the donor cells tested so far, mouse embryonic stem cells have given the best efficiency in terms of the development of reconstructed embryos into offspring. In this study, we examined whether 2 in vitro-produced pluripotent stem cells—neural stem cells (NSCs) and mesenchymal stem cells (MSCs)—could be better nuclear donors than other differentiated cells. Embryos were reconstructed by transfer of nuclei from NSCs or MSCs with full potential for differentiation in vitro. Most (76%) of the 2-cell NCS embryos developed to the 4-cell stage; 43% implanted and 1.6% developed to term after transfer to pseudopregnant recipients. These rates were very similar to those of embryos cloned from fibroblast cell nuclei. Interestingly, in the patterns of zygotic gene expression, NSC embryos were more similar to in vitro-fertilized embryos than fibroblast cloned embryos. By contrast, embryos reconstructed using MSC nuclei showed lower developmental ability and no implantation was obtained after embryo transfer. Chromosomal analysis of the donor MSCs revealed very high frequencies of monosomy and trisomy, which might have caused the very poor post-implantation development of embryos following nuclear transfer. Thus, in vitro-produced pluripotent cells can serve as donors of nuclei for cloning mice, but may be prone to chromosomal aberrations leading to a high rate of cloned embryo death.

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64 VALPROIC ACID ENHANCES IN VITRO DEVELOPMENT OF SOMATIC CELL NUCLEAR TRANSFER EMBRYOS IN PIGS

Reproduction Fertility and Development ◽

10.1071/rdv22n1ab64 ◽

2010 ◽

Vol 22 (1) ◽

pp. 190

Author(s):

Y. J. Kim ◽

K. S. Ahn ◽

M. J. Kim ◽

H. Shim

Keyword(s):

Stem Cells ◽

Valproic Acid ◽

Somatic Cell Nuclear Transfer ◽

Nuclear Transfer ◽

Developmental Competence ◽

Control Group ◽

In Vitro Development ◽

Vitro Development ◽

And Control

Epigenetic modification influences reprogramming and subsequent development of somatic cell nuclear transfer embryos. Such modification includes an increase of histone acetylation and a decrease of DNA methylation in transferred donor nuclei. Histone deacetylase inhibitors (HDACi) such as trichostatin A (TSA) and valproic acid (VPA) have been known to maintain high cellular levels of histone acetylation. Hence, the treatment of HDACi to NT embryos may increase efficiency of cloning. Indeed, TSA treatment has significantly enhanced the developmental competence of nuclear transfer embryos in several species including pigs (Zhang et al. 2007 Cloning Stem Cells 9, 357-363; Li et al. 2008 Theriogenology 70, 800-808). Valproic acid, another type of HDACi, has often been used to assist reprogramming of somatic cells into induced pluripotent stem cells in mice. In the present study, we tested the potency of VPA compared with TSA on the enhancement of in vitro development in porcine nuclear transfer embryos. Reconstructed embryos were produced by transferring nuclei of adult ear skin fibroblasts into enucleated oocytes. After electrical activation, these embryos were cultured in PZM-3 containing no HDACi (control), 5 mM VPA, or 50 nM TSA for 24 h, and another 5 days thereafter without HDACi. At least 3 replicates were conducted for the following experiments. The rates of cleavage were not different among the VPA, TSA, and control groups. However, the rate of blastocyst development was significantly higher (P < 0.05) in embryos treated with VPA than in those treated with TSA and without HDACi (125/306, 40.8% v. 94/313, 30.0% v. 80/329, 24.3%). Differential staining of inner cell mass (ICM) and trophectoderm (TE) also supported the beneficial effect of VPA treatment in NT embryos. Compared with the control group, the number of TE cells was significantly increased (P < 0.05) in the VPA and TSA treatment groups (79.3 ± 7.4 v. 74.6 ± 9.2 v. 40.0 ± 6.7). Moreover, VPA treatment significantly increased (P < 0.05) the number of ICM cells compared with the control (15.6 ± 1.7 v. 10.8 ± 2.6), whereas no differences were observed between the TSA treatment and control group (12.9 ± 3.0 v. 10.8 ± 2.6). The present study demonstrates that VPA enhances in vitro development of nuclear transfer embryos, in particular by an increase of blastocyst formation and the number of ICM cells, suggesting that VPA may be more potent than TSA in supporting developmental competence of cloned embryos. However, long-term effects of different HDACi in the development of nuclear transfer embryos, including any adverse outcome from destabilizing epigenetic condition, remain to be determined by further in vivo embryo transfer studies.

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An optimized method for neuronal differentiation of embryonic stem cells in vitro

Journal of Neuroscience Methods ◽

10.1016/j.jneumeth.2019.108486 ◽

2020 ◽

Vol 330 ◽

pp. 108486 ◽

Cited By ~ 2

Author(s):

Yuan Li ◽

Xiang Mao ◽

Xianyi Zhou ◽

Yuting Su ◽

Xiangyu Zhou ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Neuronal Differentiation ◽

Embryonic Stem

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

Cells ◽

10.3390/cells9030532 ◽

2020 ◽

Vol 9 (3) ◽

pp. 532 ◽

Cited By ~ 5

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.

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