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Growth inhibitor

A technology of inhibitors and manufacturing methods, applied in the direction of cell culture active agents, medical preparations containing active ingredients, bone/connective tissue cells, etc., can solve problems such as cells that have not been explored

Pending Publication Date: 2021-02-02
ORIZURU THERAPEUTICS INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] On the other hand, it has not been recognized that insulin-producing cell populations or pancreatic β-cell populations induced by further differentiation from pluripotent stem cell-derived endocrine precursor cell populations or cell populations at a later stage of differentiation are mixed with proliferating cells, and the removal of such cells has not been explored

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0351] Example 1: Reduction of Non-Target Cells (Ki67 Positive Cells) in Populations of Endocrine Precursor Cells Treated with FGF Receptor 1 Inhibitors

[0352] 1. Method

[0353] (1) Preparation of insulin-producing cell populations

[0354] Differentiation induction from endocrine precursor cell populations to insulin-producing cell populations was performed according to the above-mentioned step 6) or previous reports (Nature Biotechnology 2014; 32:1121-1133).

[0355] The endocrine precursor cell population induced by iPS cell differentiation was treated with differentiation factors (ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, ascorbic acid) and FGF receptor 1 inhibitor (CAS192705-79-6) Insulin-producing cell populations were obtained by culturing in the differentiation induction medium (Improved MEM / 1% B27 / Penisilin Streptomycin) for 12 days. The number of Ki67-positive cells in the obtained cell population was evaluated by flow cytometry.

[0356] (2) In viv...

Embodiment 2

[0373] Example 2: Reduction of non-target cells (Ki67-positive cells) and maintenance of target cells (insulin-positive (and NKX6.1-positive)) in populations of insulin-producing cells treated with FGF receptor 1 inhibitors cell)

[0374] 1. Method

[0375] 1) The endocrine precursor cell population induced by iPS cell differentiation was treated with differentiation factors (ALK5 inhibitor II, T3, LDN, γ-secretase inhibitor RO, ascorbic acid) and FGF receptor 1 inhibitor (CAS192705-79 -6) was cultured in a differentiation induction medium (Improved MEM / 1% B27 / Penisilin Streptomycin) for 11 days to obtain an insulin-producing cell population.

[0376] 2) The endocrine precursor cell population obtained by iPS cell differentiation induction was placed in the differentiation induction medium (Improved MEM / 1% B27 / Penisilin Streptomycin) were cultured for 4 days, and differentiated into insulin-producing cell populations. Next, FGF receptor 1 inhibitor ( CAS192705-79-6, 1 μM) ...

Embodiment 3

[0387] Example 3: Reduction of non-target cells (Ki67-positive cells) in cell populations treated with FGFR1 inhibitors other than CAS192705-79-6

[0388] 1. Method

[0389] The endocrine precursor cell population obtained by iPS cell differentiation induction was cultured in a differentiation induction medium (Improved MEM / 1% B27 / Penisilin Streptomycin) was cultured for 8 days, and then, in the cells containing differentiation factors (T3, ALK5 inhibitor II, ZnSO 4 , heparin, N-acetylcysteine, Trolox, R428) differentiation induction medium (MCDB131 / 20mM Glucose / NaHCO 3 / FAF-BSA / ITS-X / Glutamax / ascorbic acid / Penisilin Streptomycin) were cultured for 4 days in the medium supplemented with CAS192705-79-6, E-3810 or PD173074 at three concentrations of low, medium and high respectively. The number of Ki67-positive cells in the obtained cell population was evaluated by flow cytometry.

[0390] 2. Results

[0391] Table 4 shows the rate of Ki67-positive cells in the cell populat...

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Abstract

The present invention relates to a method for producing an insulin-producing cell population or a pancreas beta cell population in which Ki67 positive cells are reduced, the method comprising treatinga population of endocrine progenitor cells or further differentiated cells with an FGFR1 inhibitor.

Description

technical field [0001] The invention relates to a method for removing highly proliferative Ki67 positive cells present in insulin-producing cell groups or pancreatic beta cell groups induced by differentiation of pluripotent stem cells. Background technique [0002] Studies on the differentiation and induction of pluripotent stem cells such as iPS cells and ES cells into insulin-secreting cells (insulin-producing cells, pancreatic β cells) and their application to the treatment of diabetes are advancing. [0003] So far, various methods have been developed and reported for inducing the differentiation of pluripotent stem cells into insulin-secreting cells. Non-Patent Document 1 describes that by using CAS192705-79-6 (1-[2-amino-6-(3,5-dimethoxy-benzene) as an inhibitor of fibroblast growth factor receptor (FGFR) 1 Base)-pyrido(2,3-d)pyrimidin-7-yl]-3-tert-butyl-urea) can promote the differentiation of endocrine precursor cells derived from human iPS cells. [0004] In addi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077
CPCC12N5/0676C12N2501/113A61K35/39C12N5/0678C12N2500/38C12N2501/727C12N2501/70C12N2506/02C12N2506/45C12N2501/155C12N2501/119
Inventor 山添则子日吉秀行持田泰佑伊藤亮豊田太郎木村东
Owner ORIZURU THERAPEUTICS INC