Abstract 293: Embryological Origin of Human Smooth Muscle Cells Influences Their Ability to Support Vasculogenesis.

2013 ◽  
Vol 33 (suppl_1) ◽  
Author(s):  
Lucinda Low ◽  
Christine Cheung ◽  
Martin R Bennett ◽  
Sanjay Sinha
Keyword(s):  
Network Formation ◽  
Embryonic Stem ◽  
Fold Increase ◽  
Confocal Imaging ◽  
Paraxial Mesoderm ◽  
Hesc Line ◽  
Vitro Assay ◽  
Matrigel Assay ◽  
Network Area

Lineage-tracking studies in avian and mouse embryos have revealed that SMCs in different regions of the vasculature are derived from different embryological origins. We have developed an in vitro protocol to differentiate human embryonic stem cells (hESCs) into SMCs through different embryonic lineages, namely neuroectoderm, lateral plate mesoderm (LM) and paraxial mesoderm. As mural cells are thought to augment the formation of functional blood vessels during revascularisation, this study aims to determine whether embryologically-distinct SMCs differ in their ability to support vasculogenesis. We assessed the hypothesis that LM-SMCs are superior in supporting endothelial network formation in vitro. Using an mStrawberry-expressing hESC line, we derived the three origin-specific SMCs and co-cultured them with GFP-expressing HUVECs in a 3D in vitro matrigel assay. Endothelial network formation was assessed using real-time confocal imaging. Quantitative analysis revealed that LM-SMCs alone had a supportive effect on network formation and survival, with an increase in HUVEC network area after 4 days (3.23-fold increase ± 1.04-fold vs HUVECs alone; p<0.05; n=3) and 8 days (6.38-fold increase ± 0.39-fold vs HUVECs alone; p<0.001; n=3). In addition, LM-SMCs appear to facilitate more complex endothelial networks, with narrower cords and more branch points, compared with the other SMC types and HUVECs alone. To identify whether the LM-SMC-specific influence on network formation and survival was a paracrine effect, the three SMC types were tested in a paracrine 3D in vitro assay, where they shared media with HUVECs seeded in an adjacent well. Preliminary results reveal that the supportive effect of LM-SMCs is, at least partly, paracrine, with an increase in HUVEC network area after 4 days (7.08-fold increase vs HUVECs alone; n=1) and 8 days (21.39-fold increase vs HUVECs alone; n=1). Currently, we are exploring possible soluble factors that may be responsible and investigating this effect in an in vivo matrigel assay. In conclusion, the embryological origin of SMCs influences their functional ability to support vasculogenesis. This research will provide insight into which SMC type will be most effective in future revascularisation therapy.

scholarly journals Grafts Derived from an α-Synuclein Triplication Patient Mediate Functional Recovery but Develop Disease-Associated Pathology in the 6-OHDA Model of Parkinson’s Disease

2020 ◽  
pp. 1-14
Author(s):  
Shelby Shrigley ◽  
Fredrik Nilsson ◽  
Bengt Mattsson ◽  
Alessandro Fiorenzano ◽  
Janitha Mudannayake ◽  
...  
Keyword(s):  
Parkinson’S Disease ◽  
Parkinson's Disease ◽  
Cell Lines ◽  
Functional Recovery ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Alternative Source ◽  
And Function

Background: Human induced pluripotent stem cells (hiPSCs) have been proposed as an alternative source for cell replacement therapy for Parkinson’s disease (PD) and they provide the option of using the patient’s own cells. A few studies have investigated transplantation of patient-derived dopaminergic (DA) neurons in preclinical models; however, little is known about the long-term integrity and function of grafts derived from patients with PD. Objective: To assess the viability and function of DA neuron grafts derived from a patient hiPSC line with an α-synuclein gene triplication (AST18), using a clinical grade human embryonic stem cell (hESC) line (RC17) as a reference control. Methods: Cells were differentiated into ventral mesencephalic (VM)-patterned DA progenitors using an established GMP protocol. The progenitors were then either terminally differentiated to mature DA neurons in vitro or transplanted into 6-hydroxydopamine (6-OHDA) lesioned rats and their survival, maturation, function, and propensity to develop α-synuclein related pathology, were assessed in vivo. Results: Both cell lines generated functional neurons with DA properties in vitro. AST18-derived VM progenitor cells survived transplantation and matured into neuron-rich grafts similar to the RC17 cells. After 24 weeks, both cell lines produced DA-rich grafts that mediated full functional recovery; however, pathological changes were only observed in grafts derived from the α-synuclein triplication patient line. Conclusion: This data shows proof-of-principle for survival and functional recovery with familial PD patient-derived cells in the 6-OHDA model of PD. However, signs of slowly developing pathology warrants further investigation before use of autologous grafts in patients.

Generation of T Cells from Human Embryonic Stem Cells.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1527-1527
Author(s):  
Frank Timmermans ◽  
Imke Velghe ◽  
Lieve Van Walleghem ◽  
Magda De Smedt ◽  
Stefanie Van Coppernolle ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Embryonic Stem Cells ◽  
Human Embryonic Stem Cells ◽  
Early Stage ◽  
Embryonic Stem ◽  
Hesc Line ◽  
Double Positive

Abstract Background: Human embryonic stem cells (hESC) are derived from early stage blastocysts and are characterized by the ability to both self-renew and to generate differentiated functional cell types. One of the major challenges in the field of hESC research, is to set up a culture system that drives hESC down a particular lineage fate. To date, studies reporting hematopoietic development have not provided evidence on the differentiation capacity of hESC into T lineage cells in vitro. Material and Methods: hESC line H1 (National Institutes of Health [NIH] code: WA01), Wisconson, Madison, USA) was used (Passage 30–60) in all experiments. The hESC line was kept in an undifferentiated state on MEFs as previously described. OP9 cells and OP9 cells that express high levels of the Notch ligand Delta-like 1 (OP9-DLL1, a gift from J. C. Zuniga-Pflücker, University of Toronto, Canada) were cultured as previously described in MEM-α with 20 % FCS. Results: Our data show that T cells can be generated in vitro from hESC in a robust and highly reproducible manner using the sequential exposure of hESC to the murine OP9 cell line and OP9-DLL1. On OP9 stromal layers, a CD34highCD43dim hematopoietic precursor population is generated that is confined to vascular-like structures, reminiscent of blood islands that emerge during in vivo embryonic development. This precursor population becomes T lineage committed when exposed to OP9-DLL1 monolayers, passing sequentially through a CD34+CD7+ phenotype, a CD4+CD8+ double positive intermediate stage and eventually differentiates into a mature T cells. Polyclonal T cells are generated, cell receptor (TCR) alpha-beta and TCRgamma-delta which are functional based on proliferative capacity and production of cytokines after TCR crosslinking. Conclusion: We show that mature and functional T cells can be generated from hESC using well defined in vitro conditions. This protocol in combination with the recently described induced pluripotent cells may find clinical applicability in tumor immunology.

scholarly journals Species-specific pace of development is associated with differences in protein stability

Science ◽  
2020 ◽  
Vol 369 (6510) ◽  
pp. eaba7667 ◽  
Author(s):  
Teresa Rayon ◽  
Despina Stamataki ◽  
Ruben Perez-Carrasco ◽  
Lorena Garcia-Perez ◽  
Christopher Barrington ◽  
...  
Keyword(s):  
Protein Stability ◽  
Motor Neurons ◽  
Molecular Mechanisms ◽  
Genomic Sequence ◽  
Embryonic Stem ◽  
Fold Increase ◽  
Regulatory Elements ◽  
Cycle Duration ◽  
Species Specific

Although many molecular mechanisms controlling developmental processes are evolutionarily conserved, the speed at which the embryo develops can vary substantially between species. For example, the same genetic program, comprising sequential changes in transcriptional states, governs the differentiation of motor neurons in mouse and human, but the tempo at which it operates differs between species. Using in vitro directed differentiation of embryonic stem cells to motor neurons, we show that the program runs more than twice as fast in mouse as in human. This is not due to differences in signaling, nor the genomic sequence of genes or their regulatory elements. Instead, there is an approximately two-fold increase in protein stability and cell cycle duration in human cells compared with mouse cells. This can account for the slower pace of human development and suggests that differences in protein turnover play a role in interspecies differences in developmental tempo.

Novel in vitro assay to investigate radiation induced changes in the functionality of human embryonic stem cell-derived neurospheres

2020 ◽  
Vol 79 ◽  
pp. 40-47
Author(s):  
Margot Mayer ◽  
Onetsine Arrizabalaga ◽  
Manuel Ciba ◽  
Insa S. Schroeder ◽  
Sylvia Ritter ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
In Vitro Assay ◽  
Vitro Assay ◽  
Radiation Induced ◽  
Induced Changes

scholarly journals Recapitulating early development of mouse musculoskeletal precursors of the paraxial mesoderm in vitro

2017 ◽  
Author(s):  
Jérome Chal ◽  
Ziad Al Tanoury ◽  
Masayuki Oginuma ◽  
Philippe Moncuquet ◽  
Bénédicte Gobert ◽  
...  
Keyword(s):  
Neural Tube ◽  
Es Cells ◽  
Horse Serum ◽  
Muscle Fibers ◽  
Embryonic Stem ◽  
Paraxial Mesoderm ◽  
Mouse Muscle ◽  
Efficient System

AbstractIn vertebrates, body skeletal muscles and axial skeleton derive from the paraxial mesoderm which flanks the neural tube and notochord. The paraxial mesoderm forms in the posterior region of the embryo as presomitic mesoderm (PSM), which generates the embryonic segments called somites. Here, we characterized gene signatures identified using microarray series from the mouse PSM and compared the PSM transcriptome dynamics to that of the developing neural tube. In contrast to the PSM where an abrupt transcriptome reorganisation occurs at the level of the determination front, we show that transcriptome changes are progressive during parallel stages of neural tube differentiation. We show that these early differentiation stages of the paraxial mesoderm can be efficiently recapitulated in monolayer culture in vitro using murine Embryonic Stem (ES) cells. We describe a serum-containing protocol which parallels in vivo tissue maturation allowing differentiation of ES cells towards a paraxial mesoderm fate. We show that R-spondin treatment or Wnt activation alone can induce posterior PSM markers in both mouse and human ES/iPS cells but acquisition of a committed posterior PSM fate requires BMP inhibition to prevent induced cells to drift to a lateral plate mesoderm identity. We show that posterior PSM-like cells induced from mouse ES cells can be further differentiated in vitro to acquire an anterior PSM Pax3-positive identity. When grafted into injured adult muscle, these induced PSM-like precursors generated large numbers of immature muscle fibers. We further show that exposing ES-derived PSM-like cells to a brief FGF inhibition step followed by culture in horse serum-containing medium allows efficient recapitulation of the myogenic program. Differentiating ES cells first produce mononucleated embryonic myocytes and subsequently multinucleated myotubes, as well as Pax7-positive cells. The protocol described here results in improved differentiation and maturation of mouse muscle fibers differentiated in vitro over serum-free protocols. It provides an efficient system for the study of myogenic processes otherwise difficult to study in vivo such as fusion or satellite cell differentiation.

scholarly journals Emergence of a node-like population within an in vitro derived Neural Mesodermal Progenitors (NMPs) population

2018 ◽  
Author(s):  
Shlomit Edri ◽  
Penelope Hayward ◽  
Wajid Jawaid ◽  
Alfonso Martinez Arias
Keyword(s):  
Stem Cells ◽  
Single Cell Analysis ◽  
Embryonic Stem ◽  
Paraxial Mesoderm ◽  
Classification Model ◽  
Support Vector ◽  
Epiblast Stem Cells ◽  
Sequence Of Events

AbstractThe mammalian embryos Caudal Lateral Epiblast (CLE) harbours bipotent progenitors, called Neural Mesodermal Progenitors (NMPs), that contribute to the spinal cord and the paraxial mesoderm throughout axial elongation. Here we performed a single cell analysis of different in vitro NMPs populations produced either from embryonic stem cells (ESCs) or epiblast stem cells (EpiSCs) and compared them to E8.25 CLE mouse embryos. In our analysis of this region our findings challenge the notion that NMPs should coexpress Sox2 and T. We built a Support Vector Machine (SVM) based on the embryo CLE and use it as a classification model to analyse the in vitro NMP-like populations. We showed that ESCs derived NMPs are heterogeneous and contain few NMP-like cells, whereas EpiSCs derived NMPs, produce a high proportion of cells with the embryo NMP signature. Importantly, we found that the population from which the Epi-NMPs are derived in culture, contains a nodelike population, which is responsible for maintaining the expression of T in vitro. These results mimic the events in vivo and suggest a sequence of events for the NMPs emergence.

Abstract 137: Pharmacologically Controlled IGF-1 Release For Paracrine Support Of Engineered Heart Muscle

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
POH LOONG SOONG ◽  
Malte Tiburcy ◽  
Jan Christoph ◽  
Stefan Luther ◽  
Fawad Jebran ◽  
...  
Keyword(s):  
Heart Muscle ◽  
Single Cell Analysis ◽  
Single Cells ◽  
Embryonic Stem ◽  
Fold Increase ◽  
Cardiomyocyte Hypertrophy ◽  
Akt Phosphorylation ◽  
Heart Repair

Tissue engineered heart repair is developing rapidly, but needs refinement before clinical translation. We tested the hypothesis that force generating human engineered heart muscle (EHM) can be enhanced by integration of insulin-like growth factor-1 (IGF-1) secreting fibroblasts. Methods: TetOn lentiviral particles encoding for IGF-1 and the Tet-transactivator (tTA) were cloned and used to stably transduce human foreskin fibroblasts (HFF). Baseline and doxycycline induced IGF-1 release from HFFTetOn+IGF1 was quantified by ELISA. HFFwt and HFFTetOn+IGF1 conditioned medium was layered over human embryonic stem cell (HES2) derived cardiomyocytes followed by an analysis of AKT-phosphorylation. EHMs were assembled from HES2-derived cardiomyocytes and HFF (HFFwt or HFFTetOn+IGF1) at 70:30 ratio. Transgene activation was induced by addition of doxycycline (10 ng/ml) for 7 days. Twitch forces and response to pharmacological stimuli were measured to assess the functional consequences of IGF-1 release. EHMs were subsequently subjected to morphological analysis or dissociated into single cells to assess cellular composition of EHMs. Results: HFFTetOn+IGF1 released IGF-1 upon doxycycline stimulation (3.3x10-6 vs 8.3x10-8 [HFFwt] ng/ml/cell/day. Secreted IGF-1 from HFFTetOn+IGF1 induced Akt phosphorylation in HES2-derived cardiomyocytes (2.4±0.6 fold increase of [HFFwt]; n=3). EHMs with HFFTetOn+IGF1 developed significantly higher twitch forces than EHMs with HFFwt (0.24±0.03 vs 0.15±0.02 mN; n=10) under baseline conditions (IGF-1 leak). Doxycycline induced IGF-1 release further enhanced (P<0.05) EHM twitch force (0.26±0.03 mN; n=10). Single cell analysis from EHMs demonstrated cardiomyocyte hypertrophy in response to paracrine IGF-1 release (154±11% of [HFFwt] control; n=4). Histological analyses demonstrated that HFFTetOn+IGF1 supplemented EHMs contained thicker muscle bundles and enlarged cardiomyocytes. Conclusion: EHM can be functionally enhanced by integration of drug-controllable IGF-1 release. Drug controllable, cell based paracrine release of protective factors may not only be exploited to enhance tissue engineered myocardium in vitro but also to achieve better survival and integration of EHM grafts in vivo.

Cardiotrophin-1 displays early expression in the murine heart tube and promotes cardiac myocyte survival

Development ◽  
1996 ◽  
Vol 122 (2) ◽  
pp. 419-428 ◽  
Author(s):  
Z. Sheng ◽  
D. Pennica ◽  
W.I. Wood ◽  
K.R. Chien
Keyword(s):  
Cardiac Myocyte ◽  
Embryonic Stem ◽  
Neuronal Cell ◽  
Fold Increase ◽  
Cell System ◽  
Neonatal Rat ◽  
Heart Tube ◽  
Related Family ◽  
Early Expression

We have recently isolated a novel cytokine, cardiotrophin-1 (CT-1), from an in vitro embryonic stem cell system of cardiogenesis that can activate embryonic markers in neonatal rat cardiac myocytes. CT-1 is a new member of the interleukin 6 (IL-6)/leukemia inhibitory factor (LIF) cytokines, which activate downstream signals via gp130-dependent pathways. To define the developmental pattern of expression of CT-1 during murine embryogenesis, we have developed antibodies directed against a CT-1 fusion protein. As assessed by immunolocalization, CT-1 is predominantly expressed in the early mouse embryonic heart tube (E8.5-10.5). In the heart, CT-1 is primarily expressed in myocardial cells, and not in endocardial cushion or outflow tract tissues. After E12.5, CT-1 expression is found in other tissues, including skeletal, liver and dorsal root ganglia. Given the effects of a related family member (ciliary neurotrophic factor, CNTF) on neuronal cell survival, we studied the ability of CT-1 to promote cardiac myocyte survival and proliferation in vitro. Both CT-1 and LIF, which share the same receptors, dramatically promote neonatal cardiac myocyte survival, while IL-6 and CNTF are without effect. A cell proliferation assay documents that CT-1 provokes an approximate 2-fold increase in embryonic cardiac myocyte proliferation. Thus, CT-1 may play an autocrine role during cardiac chamber growth and morphogenesis by promoting the survival and proliferation of immature myocytes, most likely via gp130-dependent signaling pathways.

scholarly journals Chronic Electrical Stimulation Promotes the Excitability and Plasticity of ESC-derived Neurons following Glutamate-induced Inhibition In vitro

2018 ◽  
Author(s):  
Charles-Francois V. Latchoumane ◽  
LaDonya Jackson ◽  
Mohammad .S Eslampanah Sendi ◽  
Kayvan F. Tehrani ◽  
Luke J. Mortensen ◽  
...  
Keyword(s):  
Electrical Stimulation ◽  
Network Formation ◽  
Embryonic Stem ◽  
Low Frequency ◽  
Pcr Analysis ◽  
Acute Administration ◽  
Cns Injury ◽  
Frequency Stimulation ◽  
Underlying Mechanisms

ABSTRACTFunctional electrical stimulation (FES) is rapidly gaining traction as a therapeutic tool for mediating the repair and recovery of the injured central nervous system (CNS). However, the underlying mechanisms and impact of these stimulation paradigms at a molecular, cellular and network level remain largely unknown. In this study, we used embryonic stem cell (ESC)-derived neuron and glial cocultures to investigate network maturation following acute administration of L-glutamate, which is a known mediator of excitotoxicity following CNS injury. We then modulated network maturation using chronic low frequency stimulation (LFS) and direct current stimulation (DCS) protocols. We demonstrated that L-glutamate impaired the rate of maturation of ESC-derived neurons and glia immediately and over a week following acute treatment. The administration of chronic LFS and DCS protocols individually following L-glutamate infusion significantly promoted the excitability of neurons as well as network synchrony, while the combination of LFS/DCS did not. qRT-PCR analysis revealed that LFS and DCS alone significantly up-regulated the expression of excitability and plasticity-related transcripts encoding N-methyl-D-aspartate (NMDA) receptor subunit (NR2A), brain-derived neurotrophic factor (BDNF) and Ras-related protein (RAB3A). In contrast, the simultaneous administration of LFS/DCS down-regulated BDNF and RAB3A expression. Our results demonstrate that LFS and DCS stimulation can modulate network maturation excitability and synchrony following the acute administration of an inhibitory dose of L-glutamate, as well as an upregulation of NR2A, BDNF and RAB3A gene expression. Our study also provides a novel framework for investigating the effects of electrical stimulation on neuronal responses and network formation/repair after traumatic brain injury.

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