In vitro generation of mouse polarized embryo-like structures from embryonic and trophoblast stem cells

AbstractNonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our objective was to develop macaque trophoblast stem cells (TSC) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first trimester placental villous cytotrophoblasts followed by culture in TSC medium to “reprogram” the cells to a proliferative state. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was >4,000-fold higher in ST culture media compared to TSC media. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and macaque placental nonclassical MHC class I molecule, Mamu-AG. TSC differentiation to extravillous trophoblasts (EVTs) with or without the ALK-5 inhibitor A83-01 resulted in differing morphologies but similar expression of Mamu-AG and CD56 as assessed by flow cytometry, hence further refinement of relevant EVT markers is needed. Our preliminary characterization of macaque TSCs suggests that these cells represent a proliferative, self-renewing TSC population capable of differentiating to STs in vitro thereby establishing an experimental model of primate placentation.

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Derivation and Culture of Mouse Trophoblast Stem Cells In Vitro

Embryonic Stem Cell Protocols ◽

10.1385/1-59745-037-5:35 ◽

2006 ◽

pp. 35-44 ◽

Cited By ~ 1

Author(s):

Satoshi Tanaka

Keyword(s):

Stem Cells ◽

Trophoblast Stem Cells ◽

Trophoblast Stem

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Innate immunity in an in vitro murine blastocyst model using embryonic and trophoblast stem cells

Journal of Bioscience and Bioengineering ◽

10.1016/j.jbiosc.2013.09.001 ◽

2014 ◽

Vol 117 (3) ◽

pp. 358-365 ◽

Cited By ~ 8

Author(s):

Hiroaki Aikawa ◽

Miho Tamai ◽

Keisuke Mitamura ◽

Fakhria Itmainati ◽

Glen N. Barber ◽

...

Keyword(s):

Stem Cells ◽

Innate Immunity ◽

Trophoblast Stem Cells ◽

Trophoblast Stem

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Derivation of Haploid Trophoblast Stem Cells Via Conversion In Vitro

SSRN Electronic Journal ◽

10.2139/ssrn.3244533 ◽

2018 ◽

Author(s):

Keli Peng ◽

Xu Li ◽

Congyu Wu ◽

Yuna Wang ◽

Jian Yu ◽

...

Keyword(s):

Stem Cells ◽

Trophoblast Stem Cells ◽

Trophoblast Stem

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Murine Trophoblast Stem Cells and Their Differentiated Cells Attenuate Zika Virus In Vitro by Reducing Glycosylation of the Viral Envelope Protein

Cells ◽

10.3390/cells10113085 ◽

2021 ◽

Vol 10 (11) ◽

pp. 3085

Author(s):

Biswas Neupane ◽

Mona Fendereski ◽

Farzana Nazneen ◽

Yan-Lin Guo ◽

Fengwei Bai

Keyword(s):

Stem Cells ◽

Zika Virus ◽

Viral Envelope ◽

Viral Envelope Protein ◽

Zikv Infection ◽

Trophoblast Stem Cells ◽

Differentiated Cells ◽

Trophoblast Stem ◽

Stage Embryo

Zika virus (ZIKV) infection during pregnancy can cause devastating fetal neuropathological abnormalities, including microcephaly. Most studies of ZIKV infection in pregnancy have focused on post-implantation stage embryos. Currently, we have limited knowledge about how a pre-implantation stage embryo deals with a viral infection. This study investigates ZIKV infection on mouse trophoblast stem cells (TSCs) and their in vitro differentiated TSCs (DTSCs), which resemble the cellular components of the trophectoderm layer of the blastocyst that later develops into the placenta. We demonstrate that TSCs and DTSCs are permissive to ZIKV infection; however, ZIKV propagated in TSCs and DTSCs exhibit substantially lower infectivity, as shown in vitro and in a mouse model compared to ZIKV that was generated in Vero cells or mouse embryonic fibroblasts (MEFs). We further show that the low infectivity of ZIKV propagated in TSCs and DTSCs is associated with a reduced level of glycosylation on the viral envelope (E) proteins, which are essential for ZIKV to establish initial attachment by binding to cell surface glycosaminoglycans (GAGs). The decreased level of glycosylation on ZIKV E is, at least, partially due to the low-level expression of a glycosylation-related gene, Hexa, in TSCs and DTSCs. Furthermore, this finding is not limited to ZIKV since similar observations have been made as to the chikungunya virus (CHIKV) and West Nile virus (WNV) propagated in TSCs and DTSCs. In conclusion, our results reveal a novel phenomenon suggesting that murine TSCs and their differentiated cells may have adapted a cellular glycosylation system that can limit viral infectivity by altering the glycosylation of viral envelope proteins, therefore serving as a unique, innate anti-viral mechanism in the pre-implantation stage embryo.

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Laminin is the ECM niche for trophoblast stem cells

Life Science Alliance ◽

10.26508/lsa.201900515 ◽

2020 ◽

Vol 3 (2) ◽

pp. e201900515 ◽

Cited By ~ 3

Author(s):

Daiji Kiyozumi ◽

Itsuko Nakano ◽

Ryoko Sato-Nishiuchi ◽

Satoshi Tanaka ◽

Kiyotoshi Sekiguchi

Keyword(s):

Stem Cells ◽

Dependent Manner ◽

Trophoblast Stem Cells ◽

Stem Cell Maintenance ◽

Trophoblast Stem ◽

Integrin Ligands ◽

Integrin Ligand ◽

Proliferation In Vitro

The niche is a specialized microenvironment for tissue stem cells in vivo. It has long been emphasized that niche ECM molecules act on tissue stem cells to regulate their behavior, but the molecular entities of these interactions remain to be fully elucidated. Here, we report that laminin forms the in vivo ECM niche for trophoblast stem cells (TSCs), the tissue stem cells of the placenta. TSCs expressed fibronectin-binding, vitronectin-binding, and laminin-binding integrins, whereas the integrin ligands present in the TSC niche were collagen and laminin. Therefore, the only niche integrin ligand available for TSCs in vivo was laminin. Laminin promoted TSC adhesion and proliferation in vitro in an integrin binding–dependent manner. Importantly, when the integrin-binding ability of laminin was genetically ablated in mice, the size of the TSC population was significantly reduced compared with that in control mice. The present findings underscore an ECM niche function of laminin to support tissue stem cell maintenance in vivo.

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Trophoblast stem cells differentiate in vitro into invasive trophoblast giant cells

Developmental Biology ◽

10.1016/j.ydbio.2004.03.040 ◽

2004 ◽

Vol 271 (2) ◽

pp. 362-371 ◽

Cited By ~ 64

Author(s):

Myriam Hemberger ◽

Martha Hughes ◽

James C Cross

Keyword(s):

Stem Cells ◽

Giant Cells ◽

Trophoblast Stem Cells ◽

Trophoblast Stem ◽

Trophoblast Giant Cells

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Cellular Dynamics of Mouse Trophoblast Stem Cells: Identification of a Persistent Stem Cell Type1

Biology of Reproduction ◽

10.1095/biolreprod.115.137125 ◽

2016 ◽

Vol 94 (6) ◽

Cited By ~ 5

Author(s):

Kaori Motomura ◽

Mami Oikawa ◽

Michiko Hirose ◽

Arata Honda ◽

Sumie Togayachi ◽

...

Keyword(s):

Stem Cells ◽

Cell Biology ◽

Expression Profiles ◽

Single Type ◽

Trophoblast Stem Cells ◽

Trophoblast Stem ◽

Differential Expression Pattern

Abstract Mouse trophoblast stem cells (TSCs) proliferate indefinitely in vitro, despite their highly heterogeneous nature. In this study, we sought to characterize TSC colony types by using methods based on cell biology and biochemistry for a better understanding of how TSCs are maintained over multiple passages. Colonies of TSCs could be classified into four major types: type 1 is compact and dome-shaped, type 4 is flattened but with a large multilayered cell cluster, and types 2 and 3 are their intermediates. A time-lapse analysis indicated that type 1 colonies predominantly appeared after passaging, and a single type 1 colony gave rise to all other types. These colony transitions were irreversible, but at least some type 1 colonies persisted throughout culture. The typical cells comprising type 1 colonies were small and highly motile, and they aggregated together to form primary colonies. A hierarchical clustering based on global gene expression profiles suggested that a TSC line containing more type 1 colony cells was similar to in vivo extraembryonic tissues. Among the known TSC genes examined, Elf5 showed a differential expression pattern according to colony type, indicating that this gene might be a reliable marker of undifferentiated TSCs. When aggregated with fertilized embryos, cells from types 1 and 2, but not from type 4, distributed to the polar trophectoderm in blastocysts. These findings indicate that cells typically found in type 1 colonies can persist indefinitely as stem cells and are responsible for the maintenance of TSC lines. They may provide key information for future improvements in the quality of TSC lines.

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Placenta-derived macaque trophoblast stem cells: differentiation to syncytiotrophoblasts and extravillous trophoblasts reveals phenotypic reprogramming

Scientific Reports ◽

10.1038/s41598-020-76313-w ◽

2020 ◽

Vol 10 (1) ◽

Author(s):

Jenna Kropp Schmidt ◽

Logan T. Keding ◽

Lindsey N. Block ◽

Gregory J. Wiepz ◽

Michelle R. Koenig ◽

...

Keyword(s):

Stem Cells ◽

Chorionic Gonadotropin ◽

Cellular Proliferation ◽

Culture Media ◽

Gonadotropin Secretion ◽

Trophoblast Stem Cells ◽

Trophoblast Stem ◽

Mhc Class I Molecule ◽

Extravillous Trophoblasts

Abstract Nonhuman primates are excellent models for studying human placentation as experimental manipulations in vitro can be translated to in vivo pregnancy. Our objective was to develop macaque trophoblast stem cells (TSCs) as an in vitro platform for future assessment of primate trophoblast development and function. Macaque TSC lines were generated by isolating first and second trimester placental villous cytotrophoblasts followed by culture in TSC medium to maintain cellular proliferation. TSCs grew as mononuclear colonies, whereas upon induction of syncytiotrophoblast (ST) differentiation multinuclear structures appeared, indicative of syncytium formation. Chorionic gonadotropin secretion was > 4000-fold higher in ST culture media compared to TSC media. The secretion of chorionic gonadotropin by TSC-derived ST reflects a reprogramming of macaque TSCs to an earlier pregnancy phenotype. Characteristic trophoblast hallmarks were defined in TSCs and ST including expression of C19MC miRNAs and the macaque placental nonclassical MHC class I molecule, Mamu-AG. Extravillous trophoblasts (EVTs) were derived that express macaque EVT markers Mamu-AG and CD56, and also secrete high levels of MMP2. Our analyses of macaque TSCs suggests that these cells represent a proliferative, self-renewing population capable of differentiating to STs and EVTs in vitro thereby establishing an experimental model of primate placentation.

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Synergistic effect of basic fibroblast growth factor (bFGF) and epidermal growth factor on derivation of camel (Camelus dromedarius) trophoblast stem cells

Zygote ◽

10.1017/s0967199419000169 ◽

2019 ◽

Vol 27 (4) ◽

pp. 255-258

Author(s):

Faisal A. Alzahrani

Keyword(s):

Stem Cells ◽

Growth Factors ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Control Group ◽

Fibroblast Growth ◽

Trophoblast Stem Cells ◽

Trophoblast Stem ◽

Epidermal Growth

SummaryThis study aimed to optimize the derivation of trophectoderm from in vitro-produced camel embryos under feeder-free culture conditions using the basement membrane matrix Matrigel. Trophoblastic vesicles were obtained through mechanical microdissection of in vitro-produced camel (Camelus dromedarius) embryos. Supplementing the culture medium with 10 ng/ml of epidermal growth factor and 10 ng/ml fibroblast growth factor improved the attachment and subsequent outgrowths of cultured trophoblastic vesicles when compared with the control group and the groups supplemented individually with each growth factor. The expression levels of pluripotency genes octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), myelocytomatosis proto-oncogene (c-Myc) and anti-apoptotic gene B-cell lymphoma 2 (Bcl2) were increased in trophoblastic vesicles supplemented with both growth factors when compared with the control group. Conversely, both growth factors decreased the expression of apoptotic genes tumour protein p53 (p53) and Bcl-2-associated X protein (Bax). To the best of our knowledge, this may be the first report describing the derivation of trophoblast stem cells from in vitro-produced camel embryos.

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