In Vitro Th Differentiation Protocol

2016 ◽  
pp. 183-191 ◽  
Author(s):  
Takashi Sekiya ◽  
Akihiko Yoshimura
Keyword(s):  
Differentiation Protocol ◽  
Th Differentiation

scholarly journals An In Vitro Differentiation Protocol for Human Embryonic Bipotential Gonad and Testis Cell Development

2020 ◽  
Vol 15 (6) ◽  
pp. 1377-1391
Author(s):  
Ingrid M. Knarston ◽  
Svenja Pachernegg ◽  
Gorjana Robevska ◽  
Irene Ghobrial ◽  
Pei Xuan Er ◽  
...  
Keyword(s):  
Cell Development ◽  
In Vitro Differentiation ◽  
Differentiation Protocol ◽  
Bipotential Gonad

scholarly journals Do not keep it simple: recent advances in the generation of complex organoids

2020 ◽  
Vol 127 (11) ◽  
pp. 1569-1577 ◽  
Author(s):  
Philipp Wörsdörfer ◽  
Takashi I ◽  
Izumi Asahina ◽  
Yoshinori Sumita ◽  
Süleyman Ergün
Keyword(s):  
Stem Cell ◽  
3D Cell Culture ◽  
Adult Stem Cell ◽  
Inflammatory Cells ◽  
Stem Cell Population ◽  
Special Focus ◽  
Differentiation Protocol ◽  
Organ Specific ◽  
Culture Models

Abstract 3D cell culture models which closely resemble real human tissues are of high interest for disease modelling, drug screening as well as a deeper understanding of human developmental biology. Such structures are termed organoids. Within the last years, several human organoid models were described. These are usually stem cell derived, arise by self-organization, mimic mechanisms of normal tissue development, show typical organ morphogenesis and recapitulate at least some organ specific functions. Many tissues have been reproduced in vitro such as gut, liver, lung, kidney and brain. The resulting entities can be either derived from an adult stem cell population, or generated from pluripotent stem cells using a specific differentiation protocol. However, many organoid models only recapitulate the organs parenchyma but are devoid of stromal components such as blood vessels, connective tissue and inflammatory cells. Recent studies show that the incorporation of endothelial and mesenchymal cells into organoids improved their maturation and might be required to create fully functional micro-tissues, which will allow deeper insights into human embryogenesis as well as disease development and progression. In this review article, we will summarize and discuss recent works trying to incorporate stromal components into organoids, with a special focus on neural organoid models.

scholarly journals Improved Efficiency of Cardiomyocyte-Like Cell Differentiation from Rat Adipose Tissue-Derived Mesenchymal Stem Cells with a Directed Differentiation Protocol

2019 ◽  
Vol 2019 ◽  
pp. 1-16 ◽  
Author(s):  
Blanca Rebeca Ibarra-Ibarra ◽  
Martha Franco ◽  
Araceli Paez ◽  
Elvira Varela López ◽  
Felipe Massó
Keyword(s):  
Stem Cells ◽  
Mesenchymal Stem Cells ◽  
Adipose Tissue ◽  
Cell Differentiation ◽  
Cardiac Differentiation ◽  
Cardiomyocyte Differentiation ◽  
Cell Based Therapy ◽  
Differentiation Protocol ◽  
Differentiation Efficiency

Cell-based therapy has become a resource for the treatment of cardiovascular diseases; however, there are some conundrums to achieve. In vitro cardiomyocyte generation could be a solution for scaling options in clinical applications. Variability on cardiac differentiation in previously reported studies from adipose tissue-derived mesenchymal stem cells (ASCs) and the lack of measuring of the cardiomyocyte differentiation efficiency motivate the present study. Here, we improved the ASC-derived cardiomyocyte-like cell differentiation efficiency with a directed cardiomyocyte differentiation protocol: BMP-4 + VEGF (days 0-4) followed by a methylcellulose-based medium with cytokines (IL-6 and IL-3) (days 5-21). Cultures treated with the directed cardiomyocyte differentiation protocol showed cardiac-like cells and “rosette-like structures” from day 7. The percentage of cardiac troponin T- (cTnT-) positive cells was evaluated by flow cytometry to assess the cardiomyocyte differentiation efficiency in a quantitative manner. ASCs treated with the directed cardiomyocyte differentiation protocol obtained a differentiation efficiency of up to 44.03% (39.96%±3.78) at day 15 without any enrichment step. Also, at day 21 we observed by immunofluorescence the positive expression of early, late, and cardiac maturation differentiation markers (Gata-4, cTnT, cardiac myosin heavy chain (MyH), and the sarcoplasmic/endoplasmic reticulum Ca2+ ATPase (SERCa2)) in cultures treated with the directed cardiomyocyte differentiation protocol. Unlike other protocols, the use of critical factors of embryonic cardiomyogenesis coupled with a methylcellulose-based medium containing previously reported cardiogenic cytokines (IL-6 and IL-3) seems to be favorable for in vitro cardiomyocyte generation. This novel efficient culture protocol makes ASC-derived cardiac differentiation more efficient. Further investigation is needed to identify an ASC-derived cardiomyocyte surface marker for cardiac enrichment.

scholarly journals CellSIUS provides sensitive and specific detection of rare cell populations from complex single cell RNA-seq data

2019 ◽  
Author(s):  
Rebekka Wegmann ◽  
Marilisa Neri ◽  
Sven Schuierer ◽  
Bilada Bilican ◽  
Huyen Hartkopf ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Populations ◽  
Rna Seq ◽  
Differentiation Protocol ◽  
Layer V ◽  
Retzius Cells ◽  
Rare Cells ◽  
Cell Cycle Status

AbstractComprehensive benchmarking of computational methods for single-cell RNA sequencing (scRNA-seq) analysis is scarce. Using a modular workflow and a large dataset with known cell composition, we benchmarked feature selection and clustering methodologies for scRNA-seq data. Results highlighted a methodology gap for rare cell population identification for which we developed CellSIUS (Cell Subtype Identification from Upregulated gene Sets). CellSIUS outperformed existing approaches, enabled the identification of rare cell populations and, in contrast to other methods, simultaneously revealed transcriptomic signatures indicative of the rare cells’ function. We exemplified the use of our workflow and CellSIUS for the characterization of a human pluripotent cell 3D spheroid differentiation protocol recapitulating deep-layer corticogenesis in vitro. Results revealed lineage bifurcation between Cajal-Retzius cells and layer V/VI neurons as well as rare cell populations that differ by migratory, metabolic, or cell cycle status, including a choroid plexus neuroepithelial subgroup, revealing previously unrecognized complexity in human stem cell-derived cellular populations.

scholarly journals In Vitro Differentiation of Human Skin-Derived Cells into Functional Sensory Neurons-Like

Cells ◽  
2020 ◽  
Vol 9 (4) ◽  
pp. 1000
Author(s):  
Adeline Bataille ◽  
Raphael Leschiera ◽  
Killian L’Hérondelle ◽  
Jean-Pierre Pennec ◽  
Nelig Le Goux ◽  
...  
Keyword(s):  
Stem Cells ◽  
Sensory Neurons ◽  
Calcium Imaging ◽  
Transcriptional Analysis ◽  
In Vitro Differentiation ◽  
Neural Crest Stem Cells ◽  
Differentiated Cells ◽  
Differentiation Protocol ◽  
Peripheral Neuron

Skin-derived precursor cells (SKPs) are neural crest stem cells that persist in certain adult tissues, particularly in the skin. They can generate a large type of cell in vitro, including neurons. SKPs were induced to differentiate into sensory neurons (SNs) by molecules that were previously shown to be important for the generation of SNs: purmorphamine, CHIR99021, BMP4, GDNF, BDNF, and NGF. We showed that the differentiation of SKPs induced the upregulation of neurogenins. At the end of the differentiation protocol, transcriptional analysis was performed on BRN3A and a marker of pain-sensing nerve cell PRDM12 genes: 1000 times higher for PRDM12 and 2500 times higher for BRN3A in differentiated cells than they were in undifferentiated SKPs. Using immunostaining, we showed that 65% and 80% of cells expressed peripheral neuron markers BRN3A and PERIPHERIN, respectively. Furthermore, differentiated cells expressed TRPV1, PAR2, TRPA1, substance P, CGRP, HR1. Using calcium imaging, we observed that a proportion of cells responded to histamine, SLIGKV (a specific agonist of PAR2), polygodial (a specific agonist of TRPA1), and capsaicin (a specific agonist of TRPV1). In conclusion, SKPs are able to differentiate directly into functional SNs. These differentiated cells will be very useful for further in vitro studies.

scholarly journals 12-O-Tetradecanoylphorbol-13-acetate increases cardiomyogenesis through PKC/ERK signaling

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Katarzyna Anna Radaszkiewicz ◽  
Deborah Beckerová ◽  
Lucie Woloszczuková ◽  
Tomasz Witold Radaszkiewicz ◽  
Petra Lesáková ◽  
...  
Keyword(s):  
Cellular Response ◽  
Embryonic Stem ◽  
Kinase C ◽  
Differentiation Protocol ◽  
Extracellular Signal ◽  
Cell Differentiation Factor ◽  
Canonical Wnt ◽  
Erk Activity ◽  
Mes Cells

Abstract 12-O-Tetradecanoylphorbol-13-acetate (TPA) is the most widely used diacylglycerol (DAG) mimetic agent and inducer of protein kinase C (PKC)-mediated cellular response in biomedical studies. TPA has been proposed as a pluripotent cell differentiation factor, but results obtained have been inconsistent. In the present study we show that TPA can be applied as a cardiomyogenesis-promoting factor for the differentiation of mouse embryonic stem (mES) cells in vitro. The mechanism of TPA action is mediated by the induction of extracellular signal-regulated kinase (ERK) activity and the subsequent phosphorylation of GATA4 transcription factor. Interestingly, general mitogens (FGF, EGF, VEGF and serum) or canonical WNT signalling did not mimic the effect of TPA. Moreover, on the basis of our results, we postulate that a TPA-sensitive population of cardiac progenitor cells exists at a certain time point (after days 6–8 of the differentiation protocol) and that the proposed treatment can be used to increase the multiplication of ES cell-derived cardiomyocytes.

scholarly journals In Vitro Induction of Tendon-Specific Markers in Tendon Cells, Adipose- and Bone Marrow-Derived Stem Cells is Dependent on TGFβ3, BMP-12 and Ascorbic Acid Stimulation

2019 ◽  
Vol 20 (1) ◽  
pp. 149 ◽  
Author(s):  
Carlotta Perucca Orfei ◽  
Marco Viganò ◽  
John R. Pearson ◽  
Alessandra Colombini ◽  
Paola De Luca ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Ascorbic Acid ◽  
Growth Factors ◽  
Target Tissue ◽  
Tissue Specific ◽  
Cell Therapies ◽  
Differentiation Protocol ◽  
Tendon Cells

Mesenchymal Stem Cells (MSCs) and tissue-specific progenitors have been proposed as useful tools for regenerative medicine approaches in bone, cartilage and tendon-related pathologies. The differentiation of cells towards the desired, target tissue-specific lineage has demonstrated advantages in the application of cell therapies and tissue engineering. Unlike osteogenic and chondrogenic differentiation, there is no consensus on the best tenogenic induction protocol. Many growth factors have been proposed for this purpose, including BMP-12, b-FGF, TGF-β3, CTGF, IGF-1 and ascorbic acid (AA). In this study, different combinations of these growth factors have been tested in the context of a two-step differentiation protocol, in order to define their contribution to the induction and maintenance of tendon marker expression in adipose tissue and bone marrow derived MSCs and tendon cells (TCs), respectively. Our results demonstrate that TGF-β3 is the main inducer of scleraxis, an early expressed tendon marker, while at the same time inhibiting tendon markers normally expressed later, such as decorin. In contrast, we find that decorin is induced by BMP-12, b-FGF and AA. Our results provide new insights into the effect of different factors on the tenogenic induction of MSCs and TCs, highlighting the importance of differential timing in TGF-β3 stimulation.

Abstract 12495: Fibrin-Thrombin Patch for Endothelial Cell Differentiation and Cardiac-Tissue Patch Manufacturing Using Human Induced-Pluripotent Stem Cells

Circulation ◽  
2014 ◽  
Vol 130 (suppl_2) ◽  
Author(s):  
Sophia Zhang ◽  
James Dutton ◽  
Liping Su ◽  
Jianyi Zhang ◽  
Lei Ye
Keyword(s):  
Stem Cells ◽  
Endothelial Cell ◽  
Cell Differentiation ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Cardiac Tissue ◽  
Differentiation Protocol ◽  
Endothelial Cell Differentiation ◽  
Induced Pluripotent

Aim: To explore the feasibility using fibrin-thrombin patch for endothelial cell differentiation and cardiac scaffold manufacturing using cardiac cells derived from human induced-pluripotent stem cells (hiPSCs). Method and Result: hiPSCs were dissociated into single cells and seeded into three-dimensional (3D) fibrin-thrombin patches and undergo a two-stage differentiation protocol. With this protocol, up to 45% of the differentiated hiPSCs assumed an EC phenotype, and after purification, greater than 95% of the cells displayed the EC phenotype (based on CD31 expression). The hiPSC-ECs continued to display EC characteristics for 4 weeks in vitro. Gene and protein expression levels of CD31, CD144 and von Willebrand factor-8 (vWF-8) were significantly up-regulated in differentiated hiPSC-ECs. hiPSC-ECs also have biological function to up-take Dil-conjugated acetylated LDL (Dil-ac-LDL) and form tubular structures on Matrigel. A human cardiac-tissue patch (hiCP) was developed by seeding the hiPSC-ECs with hiPSC-derived cardiomyocytes (hiPSC-CMs) and smooth muscle cells (hiPSC-SMCs) into a 3D fibrin scaffold. The hiCP began to contract 3 days after synthesis, 4 days earlier than patches that were created identically but without hiPSC-ECs, and continued to beat regularly (100-120 beats/min) for at least 4 weeks in vitro. Conclusion: These data demonstrate that this new 3D differentiation protocol can efficiently generate stable ECs from hiPSCs and, furthermore, that the differentiated hiPSC-ECs can be combined with hiPSC-CMs and -SMCs to construct an hiCP with improved contractile activity. Our observations also suggest that interactions between the cardiac endothelium and myocytes may contribute to the optimized beating of hiCPs.

scholarly journals CARATTERIZZAZIONE MOLECOLARE E FUNZIONALE DI NEURONI STRIATALI DERIVATI DA CELLULE STAMINALI EMBRIONALI UMANE

2012 ◽  
Author(s):  
Alessia Delli Carri
Keyword(s):  
Pluripotent Stem Cells ◽  
Neural Differentiation ◽  
Neurodegenerative Disorder ◽  
Striatal Neurons ◽  
Involuntary Muscle ◽  
Differentiation Protocol ◽  
Gradual Development ◽  
The Central Nervous System ◽  
Memory Control

The development of neural differentiation protocol aimed at obtaining functional neurons in vitro represents the future in medicine. The text will explain the protocol developed by Cattaneolab in order to obtain in vitro striatal neurons from human embryonic and pluripotent stem cells. The work has been published in 2013 on Development (Delli Carri A., Onorati M., et al.; 2013). Striatal neurons at ganglia level die in Huntington’s Disease, a neurodegenerative disorder of the central nervous system, characterized by a gradual development of involuntary muscle movements, progressive disorientation and confusion, personality disintegration, impairment of memory control.

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