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Method for obtaining ngn3-expressing cells and insulin producing-beta cells

a technology of ngn3 and beta cells, which is applied in the field of obtaining ngn3expressing cells and insulin producingbeta cells, can solve the problems of poor reproducibility of bone marrow, lack of organ donors, and methods that are really satisfying

Inactive Publication Date: 2011-02-10
INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

The present invention provides methods for obtaining insulin-producing beta cells using a specific type of enzyme called a histone deacetylase inhibitor (HDACi). These methods involve contacting pancreatic explants or Ngn3-expressing cells with an HDACi, such as trichostatin A (TSA) or valproic acid (VPA). The invention also provides a pharmaceutical composition for the treatment of diabetes that includes an HDACi, such as TSA. The technical effect of this invention is the ability to efficiently and effectively produce insulin-producing beta cells for the treatment of diabetes.

Problems solved by technology

Thus, one of the major problems limiting islet transplantation therapy is the lack of organ donors.
Yet, despite of the intensive efforts which have been devoted to define an effective method for obtaining beta cells, none of these methods really are satisfying.
Indeed, even if some of these strategies may be useful, each of these approaches has shown limitations (e.g., raised problems of rejection and potential transfer of viruses form animal to human with xenograft; important risk of carcinogenesis and difficulties to control in vitro some steps of development with embryonic stem cells; early stage of approaches based on adult stem cells, liver and intestine cells, and poor reproducibility encountered with bone marrow.)
With regard to this serious public health problem, well understanding the pancreatic development and processes controlling it is an essential and critical challenge.
However, the mechanisms regulating cell fate choices during pancreatic development are still unclear.
However, to date, pancreatic phenotypes have not been reported in specific HDAC-deficient mice.

Method used

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  • Method for obtaining ngn3-expressing cells and insulin producing-beta cells
  • Method for obtaining ngn3-expressing cells and insulin producing-beta cells
  • Method for obtaining ngn3-expressing cells and insulin producing-beta cells

Examples

Experimental program
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example 1

Material & Methods

[0117]Animals and dissection of dorsal pancreatic rudiments: Pregnant Wistar rats were purchased from the CERJ (Le Genest, France). The first day post-coitum was taken as embryonic day 0.5 (E0.5). Pregnant female rats at 13.5 days of gestation were killed by CO2asphyxiation, according to the French Animal Care Committee's guidelines. Dorsal pancreatic buds from E13.5 rat embryos were dissected as described previously (2).

[0118]Organ culture, HDACi treatments, BrdU incorporation: Pancreases were laid on 0.45 μm filters (Millipore) at the air-medium interface in Petri dishes, containing RPMI 1640

[0119](Invitrogen) supplemented with penicillin (100 U / mL), streptomycin (100 μg / mL), HEPES (10 mmol / L), L-glutamine (2mmol / L), non-essential amino acids (1x, Invitrogen) and 10% heat-inactivated calf serum (HyClone). Cultures were maintained at 37° C. in humidified 95% air / 5% CO2. Medium was changed every other day. Explants were cultured in the presence of VPA or TSA (Sigma...

example 2

[0126]In vivo HDACi injections in rat: Experiments were submitted for ethical evaluation to the “Comité Régional d′Ethique pour l′Experimentation Animale Ile-de-France-Paris Descartes” and approved (register number P2.CHS.081.09). Pregnant rat wistar females are injected intraperitoneally daily with HDACi between E13.5 and E20.5. The NaB treated group received 1000 mg / kg whereas the VPA treated group received 300 mg / kg, in Phosphate Buffer Saline solution (PBS). After 7 days of treatment, the animals are killed by CO2 asphyxiation, according to the French Animal Care Committee's guidelines. Embryos are dissected and each pancreas is fixed or frozen for subsequent analysis by immunohistochemistry or western-blot respectively.

example 3

[0127]In vitro HDACi treatment of human pancreas: Human pancreases are extracted from foetal tissue fragments obtained immediately after voluntary abortions performed around 9 weeks of development, in compliance with the current French legislation. Human pancreases are cultured in the same conditions described above used for rat pancreases, and treated until 14 days with 800nM TSA.

Results

[0128]HDACs are down-regulated during pancreas development: The inventors first analyzed the expression of different HDACs during rat pancreas development. Using Western blotting, we found that both class I (HDAC1-3) and class II (HDAC4-7) HDACs were expressed at E13.5, E17.5 and in the adult pancreas. The expression levels of most HDACs (with the exception of HDAC3) decreased during development. The inventors next measured total HDAC activity and observed a 86.1% ±6.5% decrease at E17.5 compared with E13.5. To determine whether decreased HDAC activity correlated with increased histone acetylation, ...

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Abstract

The present invention relates to a method for obtaining Ngn3-expressing cells and insulin producing-beta cells by contacting a Pdx1-expressing pancreas explant with an amount of at least one histone deacetylase inhibitor (HDACi). The inventive methods have the advantage of being simple and quick, and of providing large amounts of Ngn3-expressing cells and insulin producing-beta cells, that are useful therapeutic tools. The invention also relates to a pharmaceutical composition for the treatment of diabetes which comprises an amount of at least one HDACi.

Description

FIELD OF THE INVENTION[0001]The invention relates to a method for obtaining a population of Ngn3-expressing cells or a population of insulin producing-beta cells by contacting a Pdx1-expressing pancreatic explant with an amount of at least one histone deacetylase inhibitor (HDACi). The invention also relates to a pharmaceutical composition for the treatment of diabetes which comprises an amount of at least one HDACi.BACKGROUND OF THE INVENTION[0002]Nowadays, more than 150 million people suffer from diabetes in the world. This disease is rising heavily and it is estimated than in the next 20 years, 300 million people could be affected. Insulinotherapy is the large-scale diabetes mellitus treatment. It consists in recurrent injections of insulin every day. The hope is to replace this heavy treatment, which is associated with side effects, by a definitive cure. In this goal, islets transplantation was tested according to the Edmonton protocol (66). However, 5 to 10 organ donors are req...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): C12N5/071C07C237/20
CPCF02C1/05F01K25/08Y02E20/14
Inventor SCHARFMANN, RAPHAELHAUMAITRE-SARRON, CECILELENOIR, OLIVIA
Owner INST NAT DE LA SANTE & DE LA RECHERCHE MEDICALE (INSERM)
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