scholarly journals Quantitative Analysis of Dopamine Neuron Subtypes Generated from Mouse Embryonic Stem Cells

2016 ◽  
Author(s):  
Yu-Ting L. Dingle ◽  
Katherine B. Xiong ◽  
Jason T. Machan ◽  
Kimberly A. Seymour ◽  
Debra Ellisor ◽  
...  
Keyword(s):  
Stem Cell ◽  
Sonic Hedgehog ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Systematic Random Sampling ◽  
Brain Science ◽  
Fibroblast Growth Factor 8 ◽  
Brown University

AbstractDopamine (DA) neuron subtypes modulate specific physiological functions and are involved in distinct neurological disorders. Embryonic stem cell (ESC) derived DA neurons have the potential to aid in the study of disease mechanisms, drug discovery, and possibly cell replacement therapies. DA neurons can be generated from ESCs in vitro, but the subtypes of ESC-derived DA neurons have not been investigated in detail despite the diversity of DA neurons observed in vivo. Due to cell culture heterogeneity, sampling methods applied to ESC-derived cultures can be ambiguous and potentially biased. Therefore, we developed a quantification method to capture the depth of DA neuron production in vitro by estimating the error associated with systematic random sampling. Using this method, we quantified calbindin+ and calretinin+ subtypes of DA neurons generated from mouse ESCs. We found a higher production of the calbindin+ subtype (11−27%) compared to the calretinin+ subtype (2-13%) of DA neuron; in addition, DA neurons expressing neither subtype marker were also generated. We then examined whether exogenous sonic hedgehog (SHH) and fibroblast growth factor 8 (FGF8) affected subtype generation. Our results demonstrate that exogenous SHH and FGF8 did not alter DA neuron subtype generation in vitro. These findings suggest that a deeper understanding DA neuron derivation inclusive of mechanisms that govern the in vitro subtype specification of ESC-derived DA neurons is required.NoteAll research was planned and conducted while members were at Brown UniversityResearch fundingNIH/NCRR/NIGMS RI Hospital COBRE Center for Stem Cell Biology (8P20GM103468-04) (MZ) Brown Institute for Brain Science Pilot Grant (4-63662) (MZ/DHK)

scholarly journals EGFR confers exquisite specificity of Wnt9a-Fzd9b signaling in hematopoietic stem cell development

2018 ◽  
Author(s):  
Stephanie Grainger ◽  
Nicole Nguyen ◽  
Jenna Richter ◽  
Jordan Setayesh ◽  
Brianna Lonquich ◽  
...  
Keyword(s):  
Stem Cell ◽  
Wnt Signaling ◽  
Hematopoietic Stem Cell ◽  
Cell Biology ◽  
Embryonic Stem ◽  
Cell Development ◽  
List Type ◽  
Hematopoietic Stem

SummaryThe mechanisms of Wnt-Frizzled (Fzd) signaling selectivity and their biological implications remain unclear. We demonstrate for the first time that the epidermal growth factor receptor (EGFR) is required as a co-factor for Wnt signaling. Using genetic studies in zebrafish, paired within vitrocell biology and biochemistry, we have determined that Fzd9b signals specifically with Wnt9ain vivoandin vitroto elicit β-catenin dependent Wnt signals that regulate hematopoietic stem and progenitor cell (HSPC) development in the dorsal aorta. This requirement is conserved in the derivation of HSPCs from human embryonic stem cells. Wnt9a-Fzd9b specificity requires two intracellular domains in Fzd9b, which interact with EGFR as a required co-factor to promote signal transduction. EGFR phosphorylates one tyrosine residue on Fzd9b, a requirement for the Wnt signal. These findings indicate that Wnt signaling interactions can be exquisitely specific and inform protocols for derivation of HSPCsin vitro.HighlightsAnin vitrosignaling screen identifies Fzd9b as a Wnt9a-specific receptor.Fzd9b and Wnt9a regulate hematopoietic stem cell development as a cognate pair.WNT9A and FZD9 are required for HSPC derivation from human pluripotent cellsin vitro.EGFR confers specificity to Wnt9a-Fzd9b signaling in zebrafish and human cells.

scholarly journals In vitro and in vivo analyses of human embryonic stem cell-derived dopamine neurons

2005 ◽  
Vol 92 (5) ◽  
pp. 1265-1276 ◽  
Author(s):  
Chang-Hwan Park ◽  
Yang-Ki Minn ◽  
Ji-Yeon Lee ◽  
Dong Ho Choi ◽  
Mi-Yoon Chang ◽  
...  
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Human Embryonic Stem Cell ◽  
Embryonic Stem ◽  
Dopamine Neurons

scholarly journals Znanstvena istraživanja u 21. stoljeću - etički aspekti uporabe laboratorijskih životinja i alternativni modeli

2022 ◽  
Vol 53 (5) ◽  
Author(s):  
Ivana Kmetič ◽  
Monika Roller ◽  
Marina Miletić ◽  
Teuta Murati
Keyword(s):  
Stem Cell ◽  
Embryonic Stem Cell ◽  
Embryo Culture ◽  
Embryonic Stem ◽  
Whole Embryo Culture ◽  
Cell Test

U toksikološkim istraživanjima uz uporabu klasičnih (in vivo) istraživanja, primjenjuju se alternativni test sustavi. Korištenje laboratorijskih životinja, embrija, humanog i animalnog tkiva, kultura stanica i fetalnog seruma u istraživanjima smatra se etički problematičnim te se ograničava zakonima, pravilnicima i praksom. Razmatranjem načina kojima bi se neetičnost mogla izbjeći, došlo je do razvoja “3R” načela (akronim za tri pristupa koja bi se trebala provoditi pri istraživanjima na laboratorijskim životinjama), a to su: smanjenje/racionalizacija uporabe laboratorijskih životinja (engl. Reduction), načelo njihove zamjene (engl. Replacement) i poboljšanje uvjeta uzgoja, smještaja i skrbi za životinje (engl. Refinement). Većina je alternativnih testova toksičnosti još uvijek u postupku validacije. Pojedini in vitro testovi za istraživanja embriotoksičnosti (etički posebno osjetljivo područje) koja su priznala nadležna regulatorna tijela, su EST (engl. Embryonic Stem cell Test), WEC (engl. Whole- Embryo Culture) i MM (engl. MicroMass) test. Standardizacija protokola i uvođenje novih in vitro modela predstavlja važan segment napretka u toksikološkim istraživanjima. Znanstvena budućnost tu vidi mogućnost razvoja i implementacije načela etičnosti u istraživanja primjenjujući sustave koji će promišljeno i bez korištenja živih organizama dijelom nadomjestiti metode u biomedicini, veterinarskoj medicini, biotehnologiji i užem smislu - toksikologiji i farmakologiji.

Accept or Reject: The Role of Immune Tolerance in the Development of Stem Cell Therapies and Possible Future Approaches

2020 ◽  
pp. 019262332091824
Author(s):  
Richard Haworth ◽  
Michaela Sharpe
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Embryonic Stem ◽  
Immune Recognition ◽  
Cell Of Origin ◽  
In Vivo Models ◽  
Cell Product ◽  
A Cell

In 2011, Goldring and colleagues published a review article describing the potential safety issues of novel stem cell-derived treatments. Immunogenicity and immunotoxicity of the administered cell product were considered risks in the light of clinical experience of transplantation. The relative immunogenicity of mesenchymal stem cells, embryonic stem cells (ESCs), and induced pluripotent stem cells (iPSCs) was being addressed through in vitro and in vivo models. But the question arose as to whether the implanted cells needed to be identical to the recipient in every respect, including epigenetically, to evade immune recognition? If so, this set a high bar which may preclude use of many cells derived from iPSCs which have vestiges of a fetal phenotype and epigenetic memory of their cell of origin. However, for autologous iPSCs, the immunogenicity reduces once the surface antigen expression profile becomes close to that of the parent somatic cells. Therefore, a cell product containing incompletely differentiated cells could be more immunogenic. The properties of the administered cells, the immune privilege of the administration site, and the host immune status influence graft success or failure. In addition, the various approaches available to characterize potential immunogenicity of a cell therapy will be discussed.

Global transcriptional profiling reveals similarities and differences between human stem cell-derived cardiomyocyte clusters and heart tissue

2012 ◽  
Vol 44 (4) ◽  
pp. 245-258 ◽  
Author(s):  
Jane Synnergren ◽  
Caroline Améen ◽  
Andreas Jansson ◽  
Peter Sartipy
Keyword(s):  
Stem Cell ◽  
Ion Channels ◽  
Human Heart ◽  
Embryonic Stem ◽  
Heart Tissue ◽  
Phenotypic Characterization ◽  
Adult Heart ◽  
Promising Source

It is now well documented that human embryonic stem cells (hESCs) can differentiate into functional cardiomyocytes. These cells constitute a promising source of material for use in drug development, toxicity testing, and regenerative medicine. To assess their utility as replacement or complement to existing models, extensive phenotypic characterization of the cells is required. In the present study, we used microarrays and analyzed the global transcription of hESC-derived cardiomyocyte clusters (CMCs) and determined similarities as well as differences compared with reference samples from fetal and adult heart tissue. In addition, we performed a focused analysis of the expression of cardiac ion channels and genes involved in the Ca2+-handling machinery, which in previous studies have been shown to be immature in stem cell-derived cardiomyocytes. Our results show that hESC-derived CMCs, on a global level, have a highly similar gene expression profile compared with human heart tissue, and their transcriptional phenotype was more similar to fetal than to adult heart. Despite the high similarity to heart tissue, a number of significantly differentially expressed genes were identified, providing some clues toward understanding the molecular difference between in vivo sourced tissue and stem cell derivatives generated in vitro. Interestingly, some of the cardiac-related ion channels and Ca2+-handling genes showed differential expression between the CMCs and heart tissues. These genes may represent candidates for future genetic engineering to create hESC-derived CMCs that better mimic the phenotype of the cardiomyocytes present in the adult human heart.

The Role of Nucleophosmin In Hematopoietic Stem Cells and the Pathogenesis of Myelodysplastic Syndrome

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 95-95 ◽  
Author(s):  
Keisuke Ito ◽  
Paolo Sportoletti ◽  
John G Clohessy ◽  
Grisendi Silvia ◽  
Pier Paolo Pandolfi
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Myelodysplastic Syndrome ◽  
Cell Biology ◽  
De Novo ◽  
Stem Cell Biology ◽  
Hematopoietic Stem

Abstract Abstract 95 Myelodysplastic syndrome (MDS) is an incurable stem cell disorder characterized by ineffective hematopoiesis and an increased risk of leukemia transformation. Nucleophosmin (NPM) is directly implicated in primitive hematopoiesis, the pathogenesis of hematopoietic malignancies and more recently of MDS. However, little is known regarding the molecular role and function of NPM in MDS pathogenesis and in stem cell biology. Here we present data demonstrating that NPM plays a critical role in the maintenance of hematopoietic stem cells (HSCs) and the transformation of MDS into leukemia. NPM is located on chromosome 5q and is frequently lost in therapy-related and de novo MDS. We have previously shown that Npm1 acts as a haploinsufficient tumor suppressor in the hematopoietic compartment and Npm1+/− mice develop a hematologic syndrome with features of human MDS, including increased susceptibility to leukemogenesis. As HSCs have been demonstrated to be the target of the primary neoplastic event in MDS, a functional analysis of the HSC compartment is essential to understand the molecular mechanisms in MDS pathogenesis. However, the role of NPM in adult hematopoiesis remains largely unknown as Npm1-deficiency leads to embryonic lethality. To investigate NPM function in adult hematopoiesis, we have generated conditional knockout mice of Npm1, using the Cre-loxP system. Analysis of Npm1 conditional mutants crossed with Mx1-Cre transgenic mice reveals that Npm1 plays a crucial role in adult hematopoiesis and ablation of Npm1 in adult HSCs leads to aberrant cycling and followed by apoptosis. Analysis of cell cycle status revealed that HSCs are impaired in their ability to maintain quiescence after Npm1-deletion and are rapidly depleted in vivo as well as in vitro. Competitive reconstitution assay revealed that Npm1 acts cell-autonomously to maintain HSCs. Conditional inactivation of Npm1 leads to an MDS phenotype including a profoundly impaired ability to differentiate into cells of the erythroid lineage, megakaryocyte dyspoiesis and centrosome amplification. Furthermore, Npm1 loss evokes a p53-dependent response and Npm1-deleted HSCs undergo apoptosis in vivo and in vitro. Strikingly, transfer of the Npm1 mutation into a p53-null background rescued the apoptosis of Npm1-ablated HSCs and resulted in accelerated transformation to an aggressive and lethal form of acute myeloid leukemia. Our findings highlight the crucial role of NPM in stem cell biology and identify a new mechanism by which MDS can progress to leukemia. This has important therapeutic implications for de novo MDS as well as therapy-related MDS, which is known to rapidly evolve to leukemia with frequent loss or mutation of TRP53. Disclosures: No relevant conflicts of interest to declare.

Designing and Engineering Stem Cell Niches

MRS Bulletin ◽  
2010 ◽  
Vol 35 (8) ◽  
pp. 591-596 ◽  
Author(s):  
Ana I. Teixeira ◽  
Ola Hermanson ◽  
Carsten Werner
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Chemical Engineering ◽  
Polyglycolic Acid ◽  
Differentiated Cells ◽  
Extracellular Matrix Components ◽  
Stem Cell Niches

AbstractStem cells have received a lot of attention due to great promises in medical treatment, for example, by replacing lost and sick cells and re-constituting cell populations. There are several classes of stem cells, including embryonic, fetal, and adult tissue specific. More recently, the generation of so-called induced pluripotent stem (iPS) cells from differentiated cells has been established. Common criteria for all types of stem cells include their ability to self-renew and to retain their ability to differentiate in response to specific cues. These characteristics, as well as the instructive steering of the cells into differentiation, are largely dependent on the microenvironment surrounding the cells. Such “stem cell friendly” microenvironments, provided by structural and biochemical components, are often referred to as niches. Biomaterials offer attractive solutions to engineer functional stem cell niches and to steer stem cell state and fatein vitroas well asin vivo. Among materials used so far, promising results have been achieved with low-toxicity and biodegradable polymers, such as polyglycolic acid and related materials, as well as other polymers used as structural “scaffolds” for engineering of extracellular matrix components. To improve the efficiency of stem cell control and the design of the biomaterials, interfaces among stem cell research, developmental biology, regenerative medicine, chemical engineering, and materials research are rapidly developing. Here we provide an introduction to stem cell biology and principles of niche engineering and give an overview of recent advancements in stem cell niche engineering from two stem cell systems—blood and brain.

scholarly journals Platelet-Derived Growth Factor Receptor Alpha as a Marker of Mesenchymal Stem Cells in Development and Stem Cell Biology

2015 ◽  
Vol 2015 ◽  
pp. 1-8 ◽  
Author(s):  
Ramin M. Farahani ◽  
Munira Xaymardan
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Stem Cell ◽  
Cell Biology ◽  
Growth Factor Receptor ◽  
Stem Cell Marker ◽  
Factor Receptor Alpha ◽  
Lines Of Evidence

Three decades on, the mesenchymal stem cells (MSCs) have been intensively researched on the bench top and used clinically. However, ambiguity still exists in regard to their anatomical locations, identities, functions, and extent of their differentiative abilities. One of the major impediments in the quest of the MSC research has been lack of appropriatein vivomarkers. In recent years, this obstacle has been resolved to some degree as PDGFRαemerges as an important mesenchymal stem cell marker. Accumulating lines of evidence are showing that the PDGFRα+cells reside in the perivascular locations of many adult interstitium and fulfil the classic concepts of MSCsin vitroandin vivo. PDGFRαhas long been recognised for its roles in the mesoderm formation and connective tissue development during the embryogenesis. Current review describes the lines of evidence regarding the role of PDGFRαin morphogenesis and differentiation and its implications for MSC biology.

scholarly journals Fezf2 transient expression via modRNA with concurrent SIRT1 inhibition enhances differentiation of cortical subcerebral / corticospinal neuron identity from mES cells

2020 ◽  
Author(s):  
Cameron Sadegh ◽  
Wataru Ebina ◽  
Anthony C. Arvanites ◽  
Lance S. Davidow ◽  
Lee L. Rubin ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cortical Neurons ◽  
Embryonic Stem ◽  
Induced Pluripotent Stem Cell ◽  
Sirtuin 1 ◽  
Projection Neurons ◽  
Specific Expression ◽  
Specific Differentiation

AbstractDuring late embryonic development of the cerebral cortex, the major class of cortical output neurons termed subcerebral projection neurons (SCPN; including the predominant population of corticospinal neurons, CSN) and the class of interhemispheric callosal projection neurons (CPN) initially express overlapping molecular controls that later undergo subtype-specific refinements. Such molecular refinements are largely absent in heterogeneous, maturation-stalled, neocortical-like neurons (termed “cortical” here) spontaneously generated by established embryonic stem cell (ES) and induced pluripotent stem cell (iPSC) differentiation. Building on recently identified central molecular controls over SCPN development, we used a combination of synthetic modified mRNA (modRNA) for Fezf2, the central transcription factor controlling SCPN specification, and small molecule screening to investigate whether distinct chromatin modifiers might complement Fezf2 functions to promote SCPN-specific differentiation by mouse ES (mES)-derived cortical-like neurons. We find that the inhibition of a specific histone deacetylase, Sirtuin 1 (SIRT1), enhances refinement of SCPN subtype molecular identity by both mES-derived cortical-like neurons and primary dissociated E12.5 mouse cortical neurons. In vivo, we identify that SIRT1 is specifically expressed by CPN, but not SCPN, during late embryonic and postnatal differentiation. Together, these data indicate that SIRT1 has neuronal subtype-specific expression in the mouse cortex in vivo, and its inhibition enhances subtype-specific differentiation of highly clinically relevant SCPN / CSN cortical neurons in vitro.

scholarly journals Conserved functional antagonism of CELF and MBNL proteins controls stem cell-specific alternative splicing in planarians

eLife ◽  
2016 ◽  
Vol 5 ◽  
Author(s):  
Jordi Solana ◽  
Manuel Irimia ◽  
Salah Ayoub ◽  
Marta Rodriguez Orejuela ◽  
Vera Zywitza ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Alternative Splicing ◽  
Cell Biology ◽  
Embryonic Stem ◽  
In Vivo Model ◽  
Stem Cell Regulation ◽  
Specific Alternative ◽  
Functional Antagonism

In contrast to transcriptional regulation, the function of alternative splicing (AS) in stem cells is poorly understood. In mammals, MBNL proteins negatively regulate an exon program specific of embryonic stem cells; however, little is known about the in vivo significance of this regulation. We studied AS in a powerful in vivo model for stem cell biology, the planarian Schmidtea mediterranea. We discover a conserved AS program comprising hundreds of alternative exons, microexons and introns that is differentially regulated in planarian stem cells, and comprehensively identify its regulators. We show that functional antagonism between CELF and MBNL factors directly controls stem cell-specific AS in planarians, placing the origin of this regulatory mechanism at the base of Bilaterians. Knockdown of CELF or MBNL factors lead to abnormal regenerative capacities by affecting self-renewal and differentiation sets of genes, respectively. These results highlight the importance of AS interactions in stem cell regulation across metazoans.

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