336 DIFFERENTIATION OF MOUSE EMBRYONIC STEM CELLS USING SF1 INTO THE STEROID-PRODUCING CELLS

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we obtained SF1 cDNA from mRNA of murine pituitary glands and constructed the SF1-expressing vector controlled by the CMV promoter. The SF1 transgenic mouse embryonic stem cells (SF1-mES cells) were established through transfection using the nucleofector (Lonza) and selection process using G418 at 250 μg mL–1. The SF1-mES cells were aggregated in hanging drops for 2 days and were transferred to suspension culture for an additional 1 day in mouse basal differentiation medium. Three-day-old SF1-mESC-derived EB were attached onto 6 well culture plates and differentiated into granulosa-like cells. Differentiated SF1-mES cells were analysed by expression of steroidogenesis-related genes and gonadal lineage-markers to the level of mRNA via real-time PCR method. To test the phenotype for granulosa-like cells, we confirmed transcripts of specific forkhead transcription factor FOXL2 and the follicle-stimulating hormone receptor (FSHR). On the other hand, we monitored some specific genes related to differentiation into testicular tissue. We observed the progress to primitive streak-mesendodermby gene expression analyses. In addition, we observed that differentiated SF1-mES cells express steroidogenic enzymes, such as 3β-hydroxysteroid dehydrogenase, cytochrome P450-containing enzyme (CYP)-11A1, and CYP19A1. Using the advanced approach, we explored culture conditions that optimize SF-1-mediated differentiation of ES cells into defined steroidogenic and gonadal lineages. We also induced granulosa-like cells. We established the effective protocol to generate ovarian cells. The derivation of these cells explores new avenues for the further study and potential application of these cells in steroidogenesis.

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64 EPITHELIAL MESENCHYMAL TRANSITION AND DIFFERENTIATION OF STEROIDOGENIC FACTOR 1 MOUSE EMBRYONIC STEM CELLS INTO THE STEROIDOGENIC CELLS

Reproduction Fertility and Development ◽

10.1071/rdv28n2ab64 ◽

2016 ◽

Vol 28 (2) ◽

pp. 162

Author(s):

H. Y. Kang ◽

Y.-K. Choi ◽

J.-U. Hwang ◽

E.-B. Jeung

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Epithelial Mesenchymal Transition ◽

Embryonic Stem ◽

Primitive Streak ◽

Mouse Embryonic Stem Cells ◽

Mrna Levels ◽

Forkhead Transcription Factor ◽

Steroidogenic Factor 1 ◽

Mesenchymal Transition

Steroidogenic factor 1 (SF-1) is essential for the development and function of steroidogenic tissues. Stable incorporation of SF-1 into embryonic stem cells has been reported to prime the cells for steroidogenesis. In this study, we transfected mouse embryonic stem cells (mESCs) with the mouse SF1 gene (SF1-mESCs) by using the nucleofector (Lonza), and selected SF1-mESCs by G418 250 μg mL–1. The selected cells were differentiated into granulosa-like cells through hanging-drops for 3 days, suspension culture for 1 day, then attachment onto 6-well plates. Expression of steroidogenesis-related genes and gonadal lineage-markers was analysed by real-time PCR. To test the phenotype for granulosa-like cells, transcripts of specific forkhead transcription factor (Foxl2) and follicle stimulating hormone receptor (Fshr) were measured. Also, expression of EMT-related genes, such as E-Cadherin (Cdh1), N-Cadherin (Cdh2), Snai1, Snai2 (Slug), Twist, and Vimentin, was monitored. SF1-mESCs were differentiated into the primitive streak‐mesendoderm and the steroidogenic enzymes such as 3β-hydroxysteroid dehydrogenase (Hsd3b1), cytochrome P450-containing enzyme (Cyp)-11a1, and Cyp19a1 were time-dependently changed. Next, the mRNA levels of Foxl2 and Fshr representing granulosa-like cell were increased during differentiation of SF1-mESCs. Especially, the level of oestradiol and Cdh2 was increased at a specific differentiation time. We induced differentiation of mESCs into the functional granulosa-like cells through transfection of the mouse SF1 gene. These cells will be useful for further study and potential application of these cells in steroidogenesis. This research was supported by a grant (15182MFDS460) from the Ministry of Food and Drug Safety in 2015.

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Angioblast Derived from ES Cells Construct Blood Vessels and Ameliorate Diabetic Polyneuropathy in Mice

Journal of Diabetes Research ◽

10.1155/2015/257230 ◽

2015 ◽

Vol 2015 ◽

pp. 1-17 ◽

Cited By ~ 8

Author(s):

Tatsuhito Himeno ◽

Hideki Kamiya ◽

Keiko Naruse ◽

Zhao Cheng ◽

Sachiko Ito ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Blood Vessels ◽

Progenitor Cell ◽

Therapeutic Strategy ◽

Es Cells ◽

Embryonic Stem ◽

Diabetic Polyneuropathy ◽

Mouse Embryonic Stem Cells ◽

Intraepidermal Nerve Fiber

Background. Although numerous reports addressing pathological involvements of diabetic polyneuropathy have been conducted, a universally effective treatment of diabetic polyneuropathy has not yet been established. Recently, regenerative medicine studies in diabetic polyneuropathy using somatic stem/progenitor cell have been reported. However, the effectiveness of these cell transplantations was restricted because of their functional and numerical impairment in diabetic objects. Here, we investigated the efficacy of treatment for diabetic polyneuropathy using angioblast-like cells derived from mouse embryonic stem cells.Methods and Results. Angioblast-like cells were obtained from mouse embryonic stem cells and transplantation of these cells improved several physiological impairments in diabetic polyneuropathy: hypoalgesia, delayed nerve conduction velocities, and reduced blood flow in sciatic nerve and plantar skin. Furthermore, pathologically, the capillary number to muscle fiber ratios were increased in skeletal muscles of transplanted hindlimbs, and intraepidermal nerve fiber densities were ameliorated in transplanted plantar skin. Transplanted cells maintained their viabilities and differentiated to endothelial cells and smooth muscle cells around the injection sites. Moreover, several transplanted cells constructed chimeric blood vessels with recipient cells.Conclusions. These results suggest that transplantation of angioblast like cells induced from embryonic stem cells appears to be a novel therapeutic strategy for diabetic polyneuropathy.

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Nat1 promotes translation of specific proteins that induce differentiation of mouse embryonic stem cells

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1617234114 ◽

2016 ◽

Vol 114 (2) ◽

pp. 340-345 ◽

Cited By ~ 34

Author(s):

Hayami Sugiyama ◽

Kazutoshi Takahashi ◽

Takuya Yamamoto ◽

Mio Iwasaki ◽

Megumi Narita ◽

...

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Translation Initiation ◽

Binding Proteins ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Translation Initiation Factor ◽

Eukaryotic Translation ◽

Eukaryotic Translation Initiation ◽

Mes Cells

Novel APOBEC1 target 1 (Nat1) (also known as “p97,” “Dap5,” and “Eif4g2”) is a ubiquitously expressed cytoplasmic protein that is homologous to the C-terminal two thirds of eukaryotic translation initiation factor 4G (Eif4g1). We previously showed that Nat1-null mouse embryonic stem cells (mES cells) are resistant to differentiation. In the current study, we found that NAT1 and eIF4G1 share many binding proteins, such as the eukaryotic translation initiation factors eIF3 and eIF4A and ribosomal proteins. However, NAT1 did not bind to eIF4E or poly(A)-binding proteins, which are critical for cap-dependent translation initiation. In contrast, compared with eIF4G1, NAT1 preferentially interacted with eIF2, fragile X mental retardation proteins (FMR), and related proteins and especially with members of the proline-rich and coiled-coil–containing protein 2 (PRRC2) family. We also found that Nat1-null mES cells possess a transcriptional profile similar, although not identical, to the ground state, which is established in wild-type mES cells when treated with inhibitors of the ERK and glycogen synthase kinase 3 (GSK3) signaling pathways. In Nat1-null mES cells, the ERK pathway is suppressed even without inhibitors. Ribosome profiling revealed that translation of mitogen-activated protein kinase kinase kinase 3 (Map3k3) and son of sevenless homolog 1 (Sos1) is suppressed in the absence of Nat1. Forced expression of Map3k3 induced differentiation of Nat1-null mES cells. These data collectively show that Nat1 is involved in the translation of proteins that are required for cell differentiation.

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Mouse embryonic stem cells switch migratory behaviour during early differentiation

10.1101/2020.12.07.415307 ◽

2020 ◽

Author(s):

Irene M. Aspalter ◽

Wolfram Pönisch ◽

Kevin J. Chalut ◽

Ewa K. Paluch

Keyword(s):

Stem Cells ◽

Embryonic Stem Cells ◽

Cell Fate ◽

Es Cells ◽

Embryonic Stem ◽

Mouse Embryonic Stem Cells ◽

Migratory Behaviour ◽

Es Cell ◽

Early Differentiation ◽

Cell State

AbstractDevelopment relies on a series of precisely orchestrated cell fate changes. While studies of fate transitions often focus on changes in gene regulatory networks, most transitions are also associated with changes in cell shape and cell behaviour. Here, we investigate changes in migratory behaviour in mouse embryonic stem (ES) cells during their first developmental fate transition, exit from ES cell state. We show that naïve pluripotent ES cells cannot efficiently migrate on 2-dimensional substrates but are able to migrate in an amoeboid fashion when placed in confinement. Exit from ES cell state, typically characterised by enhanced cell spreading, is associated with decreased migration in confinement and acquisition of mesenchymal-like migration on 2D substrates. Interestingly, confined, amoeboid-like migration of ES cells strongly depends on Myosin IIA, but not Myosin IIB. In contrast mesenchymal-like migration of cells exiting the ES cell state does not depend on Myosin motor activity but relies on the activity of the Arp2/3 complex. Together, our data suggest that during early differentiation, cells undergo a switch in the regulation of the actin cytoskeleton, leading to a transition from amoeboid-to mesenchymal-like migration.Summary statementNaïve mouse embryonic stem cells display amoeboid-like migration in confinement, but switch to mesenchymal-like migration as they exit the ES cell state.

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