Method of increasing differentiation of chondrogenic progenitor cells

A technology of progenitor cells and cartilage, applied in the field of peptides of chondrogenesis, can solve the problem of inconvenient interpretation of cartilage differentiation variability and other problems

Inactive Publication Date: 2011-12-07
AGENCY FOR SCI TECH & RES
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

In addition, the variability in chondrogenic differentiation of samples from different patients also gr

Method used

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  • Method of increasing differentiation of chondrogenic progenitor cells
  • Method of increasing differentiation of chondrogenic progenitor cells
  • Method of increasing differentiation of chondrogenic progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0459] Example 1. MSC cultures and differentiation of osteoblasts, chondrocytes and adipocytes

[0460] After informed consent, human bone marrow-derived mesenchymal stem cells (hBMSCs) were collected from the iliac crest as described (Sekiya et al., 2002) according to the guidelines of the National University Hospital of Singapore and cultured. to cultivate.

[0461] To prevent spontaneous differentiation, cells were maintained at subconfluent levels. MSCs were induced to differentiate into adipocytes and osteoblasts as described in (Liu et al., 2007), and 2×10 5 and 1.5×10 5 MSCs were induced to differentiate into adipocytes and osteoblasts in adipogenic and osteogenic media, respectively, for 14 days.

[0462] Adipogenic medium contained 0.5 mM isobutyl-methylxanthine (IBMX), 1 μM dexamethasone (Sigma), 10 μM insulin, 200 μM indomethacin and 1% antibiotic / antimycotic. Osteogenic medium contained 0.1 μΜ dexamethasone, 50 μΜ ascorbic acid-2-phosphate, 10 mM β-glycerophosp...

Embodiment 2

[0465] Example 2. Construction of expression plasmids and infection to MSCs

[0466] ZNF145 was amplified from cDNA of MSCs undergoing osteogenesis for 14 days and then cloned into pEntry3C (Invitrogen).

[0467] Sox9 ultimate ORF clone was from Invitrogen. The pLentiviral vectors for overexpression of ZNF145 or Sox9 were generated by LR recombination between pEntry3C and pLenti6 / V5 (Invitrogen). The lentivirus was produced by co-transfecting the pLentiviral vector and packaging mix (Invitrogen) used to overexpress ZNF145 or Sox9 into 293FT cells, and then infected MSCs with the virus supernatant to achieve ZNF145 or Sox9 overexpression, and the resulting The MSCs were selected with 5 μg / ml blasticidin for 7 days. Empty pLenti6 / V5 without insert was used as control (empty).

Embodiment 3

[0468] Example 3. Quantitative real-time PCR

[0469]To quantify the effect of ZNF145 overexpression or knockdown on MSC differentiation, quantitative real-time PCR was performed with a Taqman expression assay (as described by the manufacturer) and ABI 7700 Prism (Applied Biosystems).

[0470] Briefly, 0.3 μg of total RNA was converted into 30 μl of cDNA using the Large Capacity cDNA Library Kit, which was then diluted to 300 μl. Quantitative RT-PCR was performed as follows: initial denaturation at 50 °C for 2 min, 95 °C for 10 min, followed by 40 PCR cycles (95 15 seconds at 60°C, 1 minute at 60°C).

[0471] All probes were designed with 5' fluorescence generating 6-carboxyfluorescein and 3' quencher - tetramethyl-6-carboxyrhodamine. Expression of human GAPDH was used to normalize gene expression levels.

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Abstract

The present inventors disclose a method of promoting cartilage, bone or ligament repair or inducing cartilage tissue repair or regeneration, said method comprising increasing ZNF145 or a fragment, homologue, variant or derivative thereof in chondrogenic progenitor cells— For example, expression or activity in mesenchymal stem cells. The inventors also provide a chondrogenic progenitor cell, such as a mesenchymal stem cell (MSC), engineered to increase the expression or activity of ZNF145, or a fragment, homologue, variant or derivative thereof.

Description

technical field [0001] The present invention relates to the fields of development, cell biology, molecular biology and genetics. The present invention more particularly relates to a polypeptide that promotes chondrogenesis of chondrogenic progenitor cells, such as mesenchymal stem cells. Background technique [0002] A major area of ​​regenerative medicine is the use of stem cells in cartilage tissue engineering and reconstructive surgery. Stem cells differ from progenitor cells in that stem cells are capable of self-renewal and multilineage differentiation, whereas progenitor cells are capable of multilineage differentiation but not self-renewal (3). It is this ability to self-renew that makes stem cells especially useful in transplant medicine. Stem cells for cartilage repair can come from two main sources: ES cells from the inner cell mass of blastocyst-stage embryos and mesenchymal stem cells (MSCs). [0003] The most obvious advantage of using ES cells for cartilage ...

Claims

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Application Information

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IPC IPC(8): C12N5/10
CPCC12N2501/60C12N5/0655A61P19/02A61P19/08
Inventor 刘同明李永兴林濱
Owner AGENCY FOR SCI TECH & RES
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