scholarly journals Differentiation and Establishment of Dental Epithelial-Like Stem Cells Derived from Human ESCs and iPSCs

2020 ◽  
Vol 21 (12) ◽  
pp. 4384 ◽  
Author(s):  
Gee-Hye Kim ◽  
Jihye Yang ◽  
Dae-Hyun Jeon ◽  
Ji-Hye Kim ◽  
Geun Young Chae ◽  
...  
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Cell Line ◽  
Pluripotent Stem Cells ◽  
Tooth Development ◽  
Embryonic Stem ◽  
T Antigen ◽  
Nodule Formation ◽  
Epithelial Stem Cells ◽  
Human Escs

Tooth development and regeneration occur through reciprocal interactions between epithelial and ectodermal mesenchymal stem cells. However, the current studies on tooth development are limited, since epithelial stem cells are relatively difficult to obtain and maintain. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) may be alternative options for epithelial cell sources. To differentiate hESCs/hiPSCs into dental epithelial-like stem cells, this study investigated the hypothesis that direct interactions between pluripotent stem cells, such as hESCs or hiPSCs, and Hertwig’s epithelial root sheath/epithelial rests of Malassez (HERS/ERM) cell line may induce epithelial differentiation. Epithelial-like stem cells derived from hES (EPI-ES) and hiPSC (EPI-iPSC) had morphological and immunophenotypic characteristics of HERS/ERM cells, as well as similar gene expression. To overcome a rare population and insufficient expansion of primary cells, EPI-iPSC was immortalized with the SV40 large T antigen. The immortalized EPI-iPSC cell line had a normal karyotype, and a short tandem repeat (STR) analysis verified that it was derived from hiPSCs. The EPI-iPSC cell line co-cultured with dental pulp stem cells displayed increased amelogenic and odontogenic gene expression, exhibited higher dentin sialoprotein (DSPP) protein expression, and promoted mineralized nodule formation. These results indicated that the direct co-culture of hESCs/hiPSCs with HERS/ERM successfully established dental epithelial-like stem cells. Moreover, this differentiation protocol could help with understanding the functional roles of cell-to-cell communication and tissue engineering of teeth.

scholarly journals Hair Follicle Dermal Cells Support Expansion of Murine and Human Embryonic and Induced Pluripotent Stem Cells and Promote Haematopoiesis in Mouse Cultures

2018 ◽  
Vol 2018 ◽  
pp. 1-14
Author(s):  
Jun Liu ◽  
Claire A. Higgins ◽  
Jenna C. Whitehouse ◽  
Susan J. Harris ◽  
Heather Crawford ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Hair Follicle ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Dermal Papilla ◽  
Epithelial Stem Cells ◽  
Stem Cell Maintenance ◽  
Induced Pluripotent ◽  
Dermal Cells

In the hair follicle, the dermal papilla (DP) and dermal sheath (DS) support and maintain proliferation and differentiation of the epithelial stem cells that produce the hair fibre. In view of their regulatory properties, in this study, we investigated the interaction between hair follicle dermal cells (DP and DS) and embryonic stem cells (ESCs); induced pluripotent stem cells (iPSCs); and haematopoietic stem cells. We found that coculture of follicular dermal cells with ESCs or iPSCs supported their prolonged maintenance in an apparently undifferentiated state as established by differentiation assays, immunocytochemistry, and RT-PCR for markers of undifferentiated ESCs. We further showed that cytokines that are involved in ESC support are also expressed by cultured follicle dermal cells, providing a possible explanation for maintenance of ES cell stemness in cocultures. The same cytokines were expressed within folliclesin situin a pattern more consistent with a role in follicle growth activities than stem cell maintenance. Finally, we show that cultured mouse follicle dermal cells provide good stromal support for haematopoiesis in an established coculture model. Human follicular dermal cells represent an accessible and readily propagated source of feeder cells for pluripotent and haematopoietic cells and have potential for use in clinical applications.

Exploring early differentiation and pluripotency in domestic animals

2017 ◽  
Vol 29 (1) ◽  
pp. 101 ◽  
Author(s):  
R. Michael Roberts ◽  
Ye Yuan ◽  
Toshihiko Ezashi
Keyword(s):  
Stem Cells ◽  
Pluripotent Stem Cells ◽  
Inner Cell Mass ◽  
Cell Mass ◽  
Embryonic Stem ◽  
Short Review ◽  
Potential Usefulness ◽  
Meat Production ◽  
Human Escs ◽  
Domestic Species

This short review describes some general features of the origins of the pluripotent inner cell mass and epiblast during the early development of eutherian mammals and the two kinds of embryonic stem cell (ESC), naïve and primed type, that have been produced from these structures. We point out that the derivation of pluripotent stem cells from domesticated species continues to be fraught with difficulties, most likely because the culture requirements of these cells are distinct from those of mouse and human ESCs. Generation of induced pluripotent stem cells (iPSCs) from the domesticated species has been more straightforward, although the majority of the iPSC lines remain dependent on the continued expression of one or more integrated reprogramming genes. Although hope for the potential usefulness of these cells in genetic modification of livestock and other domestic species has dimmed, ESCs and iPSCs remain our best source of self-renewing populations of pluripotent cells, with potential usefulness in preserving and propagating valuable animal breeds and making contributions to fields such as regenerative medicine, toxicology and even laboratory meat production.

scholarly journals Quantitative Transcription Factor Analysis of Undifferentiated Single Human Embryonic Stem Cells

2009 ◽  
Vol 55 (12) ◽  
pp. 2162-2170 ◽  
Author(s):  
Anders Ståhlberg ◽  
Martin Bengtsson ◽  
Martin Hemberg ◽  
Henrik Semb
Keyword(s):  
Gene Expression ◽  
Stem Cells ◽  
Transcription Factor ◽  
Embryonic Stem Cells ◽  
Cell Line ◽  
Human Embryonic Stem Cells ◽  
Single Cells ◽  
Embryonic Stem ◽  
Measurement Noise ◽  
Sox2 Expression

Abstract Background: Human embryonic stem cells (hESCs) require expression of transcription factor genes POU5F1 (POU class 5 homeobox 1), NANOG (Nanog homeobox), and SOX2 [SRY (sex determining region Y)-box 2] to maintain their capacity for self-renewal and pluripotency. Because of the heterogeneous nature of cell populations, it is desirable to study the gene regulation in single cells. Large and potentially important fluctuations in a few cells cannot be detected at the population scale with microarrays or sequencing technologies. We used single-cell gene expression profiling to study cell heterogeneity in hESCs. Methods: We collected 47 single hESCs from cell line SA121 manually by glass capillaries and 57 single hESCs from cell line HUES3 by flow cytometry. Single hESCs were lysed and reverse-transcribed. Reverse-transcription quantitative real-time PCR was then used to measure the expression POU5F1, NANOG, SOX2, and the inhibitor of DNA binding genes ID1, ID2, and ID3. A quantitative noise model was used to remove measurement noise when pairwise correlations were estimated. Results: The numbers of transcripts per cell varied >100-fold between cells and showed lognormal features. POU5F1 expression positively correlated with ID1 and ID3 expression (P < 0.05) but not with NANOG or SOX2 expression. When we accounted for measurement noise, SOX2 expression was also correlated with ID1, ID2, and NANOG expression (P < 0.05). Conclusions: We demonstrate an accurate method for transcription profiling of individual hESCs. Cell-to-cell variability is large and is at least partly nonrandom because we observed correlations between core transcription factors. High fluctuations in gene expression may explain why individual cells in a seemingly undifferentiated cell population have different susceptibilities for inductive cues.

scholarly journals Generation of Sheep Induced Pluripotent Stem Cells With Defined DOX-Inducible Transcription Factors via piggyBac Transposition

2021 ◽  
Vol 9 ◽  
Author(s):  
Moning Liu ◽  
Lixia Zhao ◽  
Zixin Wang ◽  
Hong Su ◽  
Tong Wang ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Cell Types ◽  
T Antigen ◽  
Sv40 Large T Antigen ◽  
Adult Individual ◽  
Induced Pluripotent

Pluripotent stem cells (PSCs) have the potential to differentiate to all cell types of an adult individual and are useful for studying mammalian development. Establishing induced pluripotent stem cells (iPSCs) capable of expressing pluripotent genes and differentiating to three germ layers will not only help to explain the mechanisms underlying somatic reprogramming but also lay the foundation for the establishment of sheep embryonic stem cells (ESCs) in vitro. In this study, sheep somatic cells were reprogrammed in vitro into sheep iPSCs with stable morphology, pluripotent marker expression, and differentiation ability, delivered by piggyBac transposon system with eight doxycycline (DOX)-inducible exogenous reprogramming factors: bovine OCT4, SOX2, KLF4, cMYC, porcine NANOG, human LIN28, SV40 large T antigen, and human TERT. Sheep iPSCs exhibited a chimeric contribution to the early blastocysts of sheep and mice and E6.5 mouse embryos in vitro. A transcriptome analysis revealed the pluripotent characteristics of somatic reprogramming and insights into sheep iPSCs. This study provides an ideal experimental material for further study of the construction of totipotent ESCs in sheep.

An extracellular matrix-based culture system for generation of human pluripotent stem cell derived-hepatocytes

2020 ◽  
Vol 16 ◽  
Author(s):  
Mehdi Forouzesh ◽  
Mojgan Hosseini ◽  
Mehran Ataei ◽  
Maryam Farzaneh ◽  
Seyed Esmaeil Khoshnam
Keyword(s):  
Stem Cells ◽  
Extracellular Matrix ◽  
Pluripotent Stem Cells ◽  
Liver Diseases ◽  
Therapeutic Potential ◽  
Embryonic Stem ◽  
3D Culture ◽  
Hepatic Differentiation ◽  
Human Escs ◽  
Culture Methods

: Liver disease (hepatic disease) adversely affects the normal function of the liver and causes liver problems. Druginduced liver injury (DILI) can be predicted by primary human hepatocytes. However, the sources of hepatocytes for largescale drug toxicity screening are limited. To solve this problem, pluripotent stem cells (PSCs), mesenchymal stem cells (MSCs), and hepatic stem cells (HSCs) have emerged as attractive cell sources for cell-based therapies. Human PSCs including embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) have the ability to undergo self-renewal and to differentiate into lineages of ectoderm, mesoderm, and endoderm. Human PSC can be used for generation of hepatocytes to facilitate the development of novel drugs for treatment of severe liver diseases. The therapeutic potential of PSC-derived hepatocytes for liver failure have been identified to enhance the development of chemically defined and xenogenic-free 3D culture methods. To date, several hepatic differentiation strategies and various extracellular matrix (ECM) components have been employed to produce hepatocytes or hepatic-like cells (HLCs) in vitro. In this review, we focused on the potential of Matrigel, collagen type 1, RoGel, and laminin as ECM on the differentiation and function of hESC- and hiPSC-derived hepatocytes. The hepatic differentiation of human ESCs and iPSCs would offer an ideal tool for cell therapy and liver diseases.

Platelet Production System Using An Immortalized Megakaryocyte Cell Line Derived From Human Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 2-2 ◽  
Author(s):  
Sou Nakamura ◽  
Naoya Takayama ◽  
Hiromitsu Nakauchi ◽  
Koji Eto
Keyword(s):  
Stem Cells ◽  
Cell Line ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Embryonic Stem ◽  
Negative Regulator ◽  
Vector System ◽  
Transfusion Therapy ◽  
Platelet Production ◽  
Promising Source

Abstract Abstract 2 Human induced pluripotent stem cells (hiPSCs) are a promising source of blood cells, including platelets, for transfusion. However, there remains a need for: 1) a method to obtain large numbers of cells, 2) a system to provide cells of a predefined quality, and 3) a method to overcome the storage limitations of platelets caused by their short shelf life. To address these issues, we attempted to establish an immortalized megakaryocyte progenitor cell line derived from hiPSCs. We recently showed that the temporal profile of c-MYC activation during megakaryopoiesis is critical for normal platelet production from hiPSCs; that is, peak activation of c-MYC in megakaryocyte progenitors must be followed by a reduction of c-MYC expression for further maturation (Takayama et al. J Exp Med, 2010). Mechanistic analysis revealed that overexpression (O/E) of c-MYC increased megakaryocyte numbers but also induced apoptosis and senescence. Here we demonstrate that this phenomenon is primarily regulated by induction of the INK4A and ARF genes. When we examined the effects of a) c-MYC O/E and p53 knockdown, b) c-MYC O/E and BCL-XL (negative regulator of caspase family) O/E, and c) c-MYC O/E and BMI1 (negative regulator for both INK4A and ARF genes) O/E in hematopoietic progenitors derived from human embryonic stem cells (hESCs), we found that only c-MYC and BMI1 O/E (protocol b) increased numbers of CD41a+/CD42b+ non-polyploid megakaryocytes in an exponential manner for over 3 months. Neither c-MYC O/E and p53 knockdown (protocol a) nor c-MYC O/E and BCL-XL O/E (protocol c) were sufficient to maintain an increase in the megakaryocyte population, which suggests that down-regulation of INK4A and ARF contributes mainly to the prevention of excessive c-MYC-induced cell apoptosis and senescence. It thus appears that we were able to establish an immortalized megakaryocyte cell line (MKCL). As mentioned, a decline in c-MYC activation during hiPSC-derived megakaryocyte maturation is required for generation of functional CD41a+/CD42b+ platelets in vitro. Excessively sustained c-MYC expression in megakaryocytes was accompanied by increased ARF and INK4A expression and decreased GATA1 and NF-E2 expression, eventually leading to megakaryocyte senescence and apoptosis, and CD41a+/CD42blow/- platelet generation. By using an inducible expression vector system with c-MYC and BMI1 in this context, the MKCL was capable of generating polyploid megakaryocytes (>8N; 40%). The MKCL also subsequently showed proplatelet formation leading to the release of “functional” CD41a+/CD42b+ platelets. Furthermore, following transfusion of 6×108 platelets originally derived from the our immortalized MKCL into immunodeficient NOG mice, the platelets appeared to exhibit normal circulation a high degree of chimerism (human CD41a/ human CD41a + mouse CD41 was ∼67% at 2 hrs and 26% at 24 hrs post transfusion). We therefore propose that establishment of immortalized MKPCs through gene manipulation could potentially provide a stable supply of platelets at a predefined quality and quantity for transfusion therapy. Disclosures: No relevant conflicts of interest to declare.

Platelet Production System Using An Immortalized Megakaryocyte Cell Line Derived From Human Pluripotent Stem Cells

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. MRG-1-MRG-1
Author(s):  
Sou Nakamura ◽  
Naoya Takayama ◽  
Hiromitsu Nakauchi ◽  
Koji Eto
Keyword(s):  
Stem Cells ◽  
Cell Line ◽  
Pluripotent Stem Cells ◽  
Conflicts Of Interest ◽  
Embryonic Stem ◽  
Negative Regulator ◽  
Vector System ◽  
Transfusion Therapy ◽  
Platelet Production ◽  
Promising Source

Abstract Abstract MRG-1 Human induced pluripotent stem cells (hiPSCs) are a promising source of blood cells, including platelets, for transfusion. However, there remains a need for: 1) a method to obtain large numbers of cells, 2) a system to provide cells of a predefined quality, and 3) a method to overcome the storage limitations of platelets caused by their short shelf life. To address these issues, we attempted to establish an immortalized megakaryocyte progenitor cell line derived from hiPSCs. We recently showed that the temporal profile of c-MYC activation during megakaryopoiesis is critical for normal platelet production from hiPSCs; that is, peak activation of c-MYC in megakaryocyte progenitors must be followed by a reduction of c-MYC expression for further maturation (Takayama et al. J Exp Med, 2010). Mechanistic analysis revealed that overexpression (O/E) of c-MYC increased megakaryocyte numbers but also induced apoptosis and senescence. Here we demonstrate that this phenomenon is primarily regulated by induction of the INK4A and ARF genes. When we examined the effects of a) c-MYC O/E and p53 knockdown, b) c-MYC O/E and BCL-XL (negative regulator of caspase family) O/E, and c) c-MYC O/E and BMI1 (negative regulator for both INK4A and ARF genes) O/E in hematopoietic progenitors derived from human embryonic stem cells (hESCs), we found that only c-MYC and BMI1 O/E (protocol b) increased numbers of CD41a+/CD42b+ non-polyploid megakaryocytes in an exponential manner for over 3 months. Neither c-MYC O/E and p53 knockdown (protocol a) nor c-MYC O/E and BCL-XL O/E (protocol c) were sufficient to maintain an increase in the megakaryocyte population, which suggests that down-regulation of INK4A and ARF contributes mainly to the prevention of excessive c-MYC-induced cell apoptosis and senescence. It thus appears that we were able to establish an immortalized megakaryocyte cell line (MKCL). As mentioned, a decline in c-MYC activation during hiPSC-derived megakaryocyte maturation is required for generation of functional CD41a+/CD42b+ platelets in vitro. Excessively sustained c-MYC expression in megakaryocytes was accompanied by increased ARF and INK4A expression and decreased GATA1 and NF-E2 expression, eventually leading to megakaryocyte senescence and apoptosis, and CD41a+/CD42blow/− platelet generation. By using an inducible expression vector system with c-MYC and BMI1 in this context, the MKCL was capable of generating polyploid megakaryocytes (>8N; 40%). The MKCL also subsequently showed proplatelet formation leading to the release of “functional” CD41a+/CD42b+ platelets. Furthermore, following transfusion of 6×108 platelets originally derived from the our immortalized MKCL into immunodeficient NOG mice, the platelets appeared to exhibit normal circulation a high degree of chimerism (human CD41a/ human CD41a + mouse CD41 was ∼67% at 2 hrs and 26% at 24 hrs post transfusion). We therefore propose that establishment of immortalized MKPCs through gene manipulation could potentially provide a stable supply of platelets at a predefined quality and quantity for transfusion therapy. Disclosures: No relevant conflicts of interest to declare.

Angiopoietin-1 promotes endothelial differentiation from embryonic stem cells and induced pluripotent stem cells

Blood ◽  
2011 ◽  
Vol 118 (8) ◽  
pp. 2094-2104 ◽  
Author(s):  
Hyung Joon Joo ◽  
Honsoul Kim ◽  
Sang-Wook Park ◽  
Hyun-Jai Cho ◽  
Hyo-Soo Kim ◽  
...  
Keyword(s):  
Stem Cells ◽  
Induced Pluripotent Stem Cells ◽  
Pluripotent Stem Cells ◽  
Embryonic Stem ◽  
Human Escs ◽  
Induced Pluripotent ◽  
Precise Role ◽  
Angiopoietin 1

Abstract Angiopoietin-1 (Ang1) plays a crucial role in vascular and hematopoietic development, mainly through its cognate receptor Tie2. However, little is known about the precise role of Ang1 in embryonic stem cell (ESC) differentiation. In the present study, we used COMP-Ang1 (a soluble and potent variant of Ang1) to explore the effect of Ang1 on endothelial and hematopoietic differentiation of mouse ESCs in an OP9 coculture system and found that Ang1 promoted endothelial cell (EC) differentiation from Flk-1+ mesodermal precursors. This effect mainly occurred through Tie2 signaling and was altered in the presence of soluble Tie2-Fc. We accounted for this Ang1-induced expansion of ECs as enhanced proliferation and survival. Ang1 also had an effect on CD41+ cells, transient precursors that can differentiate into both endothelial and hematopoietic lineages. Intriguingly, Ang1 induced the preferential differentiation of CD41+ cells toward ECs instead of hematopoietic cells. This EC expansion promoted by Ang1 was also recapitulated in induced pluripotent stem cells (iPSCs) and human ESCs. We successfully achieved in vivo neovascularization in mice by transplantation of ECs obtained from Ang1-stimulated ESCs. We conclude that Ang1/Tie2 signaling has a pivotal role in ESC-EC differentiation and that this effect can be exploited to expand EC populations.

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