Method for producing erythroid progenitor cells

A technology of erythroid progenitor cells and red blood cells, which is applied in the medical field and can solve problems such as complex implementation, limited production output, and high cost

Pending Publication Date: 2020-07-03
ESTAB FR DU SANG +3
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, current methods have serious drawbacks for large-scale production, such as a too rapid orientation towards erythrocyte maturation, which significantly limits production yield, and differentiation into cells that cannot properly perform the enucleation step (Akimov S et al., 2005, " Stem Cells, 23(9):1423-1433; Hirose SI et al., 2013, Stem Cell Reports, 1(6):499-508; Huang X, 2013, Molecular Therapy (Mol. Ther.), 22(2):451-463)
Finally, other methods use co-culture with feeder cells (Kurita R et al., 2013, PloS One, 8(3):e59890), which is complex and expensive to implement

Method used

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  • Method for producing erythroid progenitor cells
  • Method for producing erythroid progenitor cells
  • Method for producing erythroid progenitor cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0272] Example 1: Expansion of erythroid progenitor cells from HSC derived from cord blood and cell apheresis

[0273] Materials and Method

[0274] Medium used: IMDM (Biochrom), supplemented with transferrin (330μg / mL), insulin (10μg / mL), 5% AB serum pool (EFS) and heparin (3U / mL). From day 0 to day 7, supplement the medium with IL-3 (5ng / mL), SCF (100ng / mL) and EPO (2IU / mL). From day 7 to the end of erythroblast progenitor cell expansion, SCF (100ng / mL) and EPO (2IU / mL) were added to the medium.

[0275] HSC:

[0276] According to the protocol provided by Milenyi (see Human CD34 MicroBead Kit, from Miltenyi Biotec), HSCs were obtained after magnetic separation using CD34+ beads.

[0277] Cell maintenance:

[0278] Cells were seeded at a concentration of 10,000 cells / ml on day 0, cells were diluted 1 / 5 in new medium on day 4, cells were washed on day 7 and cultured at 100,000 cells / ml in new medium . On the 11th day, the cells were diluted to 100,000 cells / ml and seeded in new medi...

Embodiment 2

[0299] Example 2: Expansion of erythroid progenitor cells from engineered HSCs derived from cell apheresis that overexpress HTERT, BMI1 and constitutively overexpress BCL-XL or inducible overexpress HTERT, BMI1 and LMO2

[0300] Materials and Method

[0301] Medium used: IMDM (Biochrom), supplemented with transferrin (330μg / mL), insulin (10μg / mL), 5% AB serum pool (EFS) and heparin (3U / mL). From day 0 to day 7, supplement the medium with IL-3 (5ng / mL), SCF (100ng / mL) and EPO (2IU / mL). From day 7 to the end of erythroblast progenitor cell expansion, SCF (100ng / mL) and EPO (2IU / mL) were added to the medium.

[0302] HSC:

[0303] HSC was obtained from apheresis after magnetic separation using Miltenyi CD34+ beads.

[0304] Cell maintenance:

[0305] Inoculate cells at a concentration of 100,000 cells / ml on day 0 for two consecutive infections of HTERT BMI1 lentiviral supernatant and BCL-XL retrovirus supernatant or HTERT BMI1 lentiviral supernatant and LMO2 lentivirus Supernatant: On t...

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Abstract

The invention relates to a method for the in vitro production of erythroid progenitor cells, involving bringing hematopoietic stem cells, which may or may not be genetically modified, into contact with a defined cell culture medium that comprises a glucocorticoid hormone and an autophagy inducer.

Description

[0001] The invention belongs to the medical field. More specifically, the present invention relates to novel methods for generating erythroid progenitor cells and red blood cells. Background technique [0002] Maintaining a constant supply of oxygen to tissues is essential for the survival of many organisms, especially humans. Red blood cells transport oxygen through the bloodstream. This transport is provided by hemoglobin, a protein unique to red blood cells that can bind oxygen. When red blood cells reach the tissue, oxygen diffuses through the capillary walls. Therefore, the role of red blood cells is crucial. [0003] In case of emergency (bleeding) and pathological conditions (blood disease, cancer, etc.), red blood cell transfusion is required. In 2016, more than 100 million blood bags were collected and distributed worldwide to meet blood transfusion needs. Fifty percent of these products are distributed in rich countries, accounting for only 15% of the global populati...

Claims

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Application Information

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Patent Type & AuthorityApplications(China)
IPC IPC(8): C12N5/078
CPCC12N5/0641C12N2501/39C12N2501/999C12N2502/45C12N2506/02C12N2506/45C12N2510/04
Inventor劳伦斯·居约诺-哈尔曼德卢瓦克·加伦弗雷德里克·奥雷德尼古拉斯·瑞贝格弗雷德里克·雷莱克斯卢克·杜艾
OwnerESTAB FR DU SANG