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Efficient derivation of stable pluripotent bovine embryonic stem cells

An embryonic stem cell and cell technology, which is applied in the field of efficient derivation of stable pluripotent bovine embryonic stem cells, can solve the problems of poor derivation efficiency, loss of proliferation ability and pluripotency markers, and failure to pass pluripotency tests.

Pending Publication Date: 2020-08-21
RGT UNIV OF CALIFORNIA +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0003] Despite years of research, the derivation of stable pluripotent embryonic stem cells (ESCs) in bovine species remains challenging
Most, if not all, reported bovine ESC lines fail the commonly used pluripotency assays, i.e. in vitro differentiation assays, in vivo teratoma formation and / or chimera formation
Also, they show poor derivation efficiency, limited proliferative capacity and loss of pluripotency markers after extensive passage

Method used

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  • Efficient derivation of stable pluripotent bovine embryonic stem cells
  • Efficient derivation of stable pluripotent bovine embryonic stem cells
  • Efficient derivation of stable pluripotent bovine embryonic stem cells

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Embodiment approach

[0089] Method for producing stable embryonic stem cells

[0090] Provided herein is a method for producing stable ungulate embryonic stem cells (ESCs), the method comprising, or consisting essentially of, or consisting of: culturing in a cell culture medium from an ungulate ungulate blastocyst cells or pluripotent cells isolated from animal-like embryos, the cell culture medium containing: (i) inactivated feeder cells; (ii) an effective amount of fibroblast growth factor 2 (FGF2) or its equivalents; and (iii) an effective amount of one or more Wnt signaling inhibitors. In one aspect, the ungulate blastocyst, pluripotent cell or ESC is bovine or ovine. In another aspect, the blastocyst cells, pluripotent cells or ESCs are detectably labeled.

[0091] The methods disclosed herein use the disclosed compositions and culture conditions to establish ungulate ESCs (e.g., bovine embryonic stem cells) that are multipotent, readily proliferate using single cell dissociation by trypsin...

Embodiment 1

[0126] CTFR medium supports derivation and long-term culture of pluripotent bESCs

[0127] In serum-free custom TeSR1 basal medium (without growth factors) supplemented with FGF2 and IWR1 ( C ustom T eSR1base medium supplemented with F GF2 and IW R 1) (referred to herein as "CTFR") conditions, bESCs were derivatized, propagated, cultured and subjected to several rounds of freezing and thawing. Cell lines can be established by the end of week 3 after ICM / embryo plating and remain unchanged for more than 50 passages (Figure 1). Unlike human ESCs and mouse EpiSCs, CTFR-bESCs did not display round colony morphology with clearly defined edges, but instead tightly adhered to each other, forming an interconnected honeycomb network that Clearly separated from the feeder layer ( Figure 1A ). CTFR-bESCs stained positive for alkaline phosphatase (AP) ( Figure 1A ). To assess the genetic stability of CTFR-bESCs after long-term culture, Applicants performed karyotype analysis in ...

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Abstract

This disclosure provides ungulate embryonic stem cells (ESCs) derived from the inner cell mass of pre-implantation blastocysts or pluripotent cells from embryos. From an agricultural and biomedical perspectives, the derivation of stable ESCs from domestic ungulates is important for genomic testing and selection, genetic engineering, and providing an experimental tool for studying human diseases. Cattle are one of the most important domestic ungulates that are commonly used for food and bioreactors.

Description

[0001] Cross References to Related Applications [0002] This application claims priority under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 62 / 616,975, filed January 12, 2018, the contents of which are incorporated herein by reference in their entirety. Background technique [0003] Despite years of research, the derivation of stable pluripotent embryonic stem cells (ESCs) in bovine species remains challenging. Most, if not all, of the reported bovine ESC lines fail the commonly used pluripotency assays, namely in vitro differentiation assays, in vivo teratoma formation and / or chimera formation. Also, they show poor derivation efficiency, limited proliferative capacity and loss of pluripotency markers after extensive passage. Therefore, there is a need in the art for methods of deriving stable bovine pluripotent ESCs. The present disclosure fulfills this need and provides related advantages as well. [0004] Summary of the invention [0005] Provided herein are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/07
CPCC12N5/0606C12N2500/99C12N2501/115C12N2501/415A01K67/0275A01K2207/12A01K2217/05C12N5/0609C12N5/061C12N15/01C12N15/8771C12N2501/15C12N2506/04
Inventor 帕布罗·胡安·罗斯亚宁娜·博格里奥蒂玛塞拉·维拉利诺胡安·卡洛斯·伊斯皮萨·贝尔蒙特吴军
Owner RGT UNIV OF CALIFORNIA
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