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Non-tumorigenic expansion of pluripotent stem cells

A technology of pluripotent stem cells and stem cells, applied in the field of non-tumorigenic proliferation, can solve the problems of insufficient maintenance of cell stability and inability to demonstrate long-term guarantee

Inactive Publication Date: 2012-10-24
BUDDHIST TZU CHI GEN HOSPITAL
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Problems solved by technology

This substitution did not show long-term guaranteed results, and such attempts have proven insufficient to maintain cell robustness and continued propagation
Furthermore, this substitution also demonstrated that hES cells grown in the medium replacement did form differentiated cells around hES cell colonies in trophoblast-free culture, a sign that the optimal situation was not achieved

Method used

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  • Non-tumorigenic expansion of pluripotent stem cells
  • Non-tumorigenic expansion of pluripotent stem cells
  • Non-tumorigenic expansion of pluripotent stem cells

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Embodiment

[0022] RT-PCR and quantitative RT-PCR of this gene and other differentiation marker genes

[0023]Undifferentiated or differentiated hES cells cultured on HUCMSCs or MEF feeder layers were mechanically removed and treated with RLT lysis buffer (Qiagen). The first cDNA was synthesized using the SuperScript III One-Step RT-PCR kit (invitrogen) according to the manufacturer's instructions. Table 1 shows the sequence, binding temperature and product size of each pair of primers. All PCR samples were analyzed by electrophoresis on a 2% agarose gel containing 0.5 μg / ml ethidium bromide (Sigma). For quantitative RT-PCR (qRT-PCR), the FastStart universal SYBR green master (ROX, Roche, USA) gene expression assay was used in the ABI StepOne Plus system (Applied Biosystems) with GAPDH as an internal control. The sequences and binding temperatures of the primers are shown in Table 1.

[0024] Table 1

[0025]

[0026] Isolation and proliferation of HUCMSCs

[0027] Human umbilical...

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Abstract

A method for expansion of human embryonic stem (hES) cells in a medium including human umbilical cord-derived mesenchymal stem cells (HUCMSCs) as a feeder is provided. The human embryonic stem cells (hES) maintain the features of embryonic stem cells in the medium, such as pluripotency, unlimited undifferentiated proliferation and normal karyotypes. Also provided is a method for non-tumorigenic expansion of the human embryonic stem cells (hES) that is free from forming teratoma.

Description

technical field [0001] The present invention relates to the proliferation of human pluripotent stem cells, in particular to the non-tumorigenic proliferation of human embryonic stem (hES) cells. Background technique [0002] Human embryonic stem (hES) cells are pluripotent and have a strong ability to differentiate into most cell types of adults. Therefore, this cell is a strong guarantee for regenerative medicine. Desirable specific properties of the hES cells include immortality, pluripotency, and unrestricted undifferentiated growth. Traditionally, pluripotent embryonic stem cells have been cultured on feeder layer cells such as mouse embryonic fibroblasts (MEFs) to maintain the cells in an undifferentiated state. The feeder layer is necessary to maintain the cells; and hES cells are typically cultured on the feeder layer until differentiation is desired. Unfortunately, the mouse trophoblast maintenance cells are often contaminated by contact with hES cells, which does...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0775C12N5/0735
CPCC12N2502/11C12N5/0606C12N2502/1388
Inventor 丁大清朱堂元
Owner BUDDHIST TZU CHI GEN HOSPITAL
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