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Induced differentiation method for differentiating human induced pluripotent stem cells into leydig cells and application thereof

A technology of Leydig cells and pluripotent stem cells, which is applied in the field of induced differentiation of human induced pluripotent stem cells to Leydig cells, can solve the problems of unknown origin of LC development, difficulty in obtaining materials, and difficulty in expansion, and achieves the goal of improving serum The effect of testosterone levels

Inactive Publication Date: 2016-01-20
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the developmental origin of LC is unknown, and there are defects such as difficulty in obtaining materials, scarcity, and difficulty in amplification, which obviously restrict its clinical application prospects.

Method used

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  • Induced differentiation method for differentiating human induced pluripotent stem cells into leydig cells and application thereof
  • Induced differentiation method for differentiating human induced pluripotent stem cells into leydig cells and application thereof
  • Induced differentiation method for differentiating human induced pluripotent stem cells into leydig cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Example 1 Differentiation from iPS cell lines to hNCSCs

[0048] 1) Preparation of cell suspension: using the established human iPS cell line (hiPSCs, HumMolGenet.2013, 22(11):2221-33), hiPSCs cells grow in flat clones when expanded and cultured on Matrigel, and the cells are arranged close, such as figure 1 As shown in A. The hiPSCs cells were digested into small pieces with 0.5 mmol / L EDTA, and the cells were resuspended in mTeSR medium containing ROCK inhibitor. The ROCK inhibitor used therein was Y27632 (Calbiochem, San Diego, CA).

[0049] 2) Induce differentiation: collect suspension cells, inoculate in Petri dishes, use neural differentiation medium (80% Knockout TM DMEM, 18% Knockout TM SR, 1% (V / V) penicillin-streptomycin mixed solution, 1mML-glutamic acid, 0.1mM β-mercaptoethanol) suspension culture to form translucent spherical embryo bodies, such as figure 1 Shown in B. where Knockout TM DMEM and Knockout TM SRs were purchased from Invitrogen, Carlsb...

Embodiment 2

[0056] Example 2 Induction of differentiation from hNCSCs into Leydig cells (iPS-hNCSCs-LCs or hNCSCs-LCs)

[0057] Expand the obtained hNCSCs cells, and replace them with Leydig cells (LCs) differentiation medium (DMEM-F12 medium, add 2% FCS, 10ngPDGF-BB, 1ng / mlLH, 1nM thyroid hormone (T3 ), 70ng / mlIGF-I), induced for 14 days, cell differentiation was carried out, cell supernatant was collected, and cells were fixed. The expressions of mature LCs-related markers (LCs markers 3β-HSD, P450c17, steroidogenic acute regulatory protein (StAR), and steroidogenic factor 1 (SF-1)) were detected by immunofluorescence, such as Figure 5 As shown, immunostaining analysis indicated that hNCSCs-induced differentiation cells (hNCSCs-LCs) expressed 3β-HSD, P450C17, StAR, SF-1. The testosterone level in the culture medium was measured by testosterone ELISA detection, such as Image 6 As shown, it was found that hNCSCs-LCs gradually increased testosterone secretion after differentiation in v...

Embodiment 3

[0058] Example 3 The effect of iPS-hNCSCs-LCs (or called hNCSCs-LCs) in vivo

[0059] Previous studies have shown that the specific apoptosis inducer of Leydig cells in rats treated with ethylene dimethanesulfonate (EDS) for 4 days can deplete Leydig cells, so the EDS model was established by intraperitoneal injection of EDS into rats. Three groups of adult rats were selected, which were normal control group, EDS-control group and cell transplantation group. The normal control group was intraperitoneally injected with the same volume of normal saline on the 0th day and the 4th day. EDS-control group rats were intraperitoneally injected with EDS (75 mg / kg body weight) on day 0, and injected 20 μl of normal saline (10 μl / unilateral testis) into testes on day 4. The rats in the cell group were intraperitoneally injected with EDS (75 mg / kg body weight) on day 0, and on day 4, hNCSCs-LCs (1.5×10 6 resuspended in 10 μl PBS / single testis) and transplanted into rat testes. Serum te...

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Abstract

The invention provides an external directional induced differentiation method for differentiating human induced pluripotent stem (iPS) cells into leydig cells (LCs) through neural crest stem cells (NCSCs). By the utilization of an animal model, it is verified that the LCs from the iPS cells have the capacity of regenerating aged or damaged LCs, and a new testosterone-supplying scheme is provided for a patient with hypogonadism and particularly an LOH patient.

Description

technical field [0001] The invention relates to the technical field of stem cells and tissue engineering, in particular to a method for inducing differentiation of human induced pluripotent stem cells (iPS) cells into Leydig cells (LCs) and its application. Background technique [0002] Leydig cells (LCs) are distributed in the loose connective tissue between testicular germ cell tubes, and the testosterone secreted by them is the main source of testosterone in adult males. LCs transplantation is the best way to treat LOH and other diseases related to testosterone deficiency. It can better simulate the physiological characteristics of human testosterone secretion, so that the serum testosterone concentration and local testicular testosterone concentration can be effectively improved, and better treatment can be obtained. At the same time, try to avoid adverse consequences during the treatment process. However, the developmental origin of LC is unknown, and there are defects...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/077A61K35/52A61P5/26
CPCA61K45/00C12N5/0683C12N2500/32C12N2500/44C12N2500/99C12N2501/105C12N2501/11C12N2501/115C12N2501/135C12N2501/31C12N2501/395C12N2501/727C12N2506/45C12N2513/00C12N2533/32C12N2533/52C12N2533/54A61K35/52A61P5/26A61K38/1808A61K38/1825C12N2501/30C12N5/0606
Inventor 项鹏姜美花李伟强
Owner SUN YAT SEN UNIV
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