Methods of improving hematopoietic grafts

A technology of transplantation and hematopoietic cells, applied in the medical field, can solve the problems of poor implantation potential and the use of plasmids encoding oncogenes hindering clinical application, etc.

Pending Publication Date: 2020-04-10
ESTAB FR DU SANG +5
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In many cases, the introduction of plasmids encoding oncogenes in recipient cells or the use of non-GMP-grade feeder cells precludes their use in clinical applications
Finally, many of these protocols aim to produce cell populations with a surface phenotype similar to that of truly transplantable cord blood or adult HSCs, a strategy that has been shown to produce cells with poor engraftment potential

Method used

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  • Methods of improving hematopoietic grafts
  • Methods of improving hematopoietic grafts
  • Methods of improving hematopoietic grafts

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preparation example Construction

[0146] All embodiments described above for the method of preparing a hematopoietic cell graft of the invention are also encompassed in this aspect.

[0147] Expression of cell surface antigens CD135 and / or CD110 and / or expression of apelin receptor (APLNR) can be performed by any method known to the skilled person such as FACS, MACS, immunohistochemistry, Western blot, protein or antibody array, RT -PCR or by transcriptome approach to assess.

[0148] Depending on the method used to assess the expression of CD135, CD110 or APLNR, steps b) and c) may be sequential or simultaneous. Detection and selection of said expression can be simultaneous, for example, using FACS or MACS.

[0149] The identified and / or selected CD135+ and / or CD110+ and / or APLNR+ cells can be used as a hematopoietic graft, or can be included in or added to a hematopoietic graft (such as a bone marrow or cord blood graft) in order to enhance said transplantation effectiveness of the thing.

[0150] In anot...

Embodiment 1

[0272] Materials and methods

[0273] hiPSC expansion

[0274] The studies were performed using three different hiPSC lines: FD136-25, reprogrammed with retroviral vectors and Thomson's combination (endogenous expression of Oct4, Sox2, Nanog and Lin28); Pci-1426 and Pci-1432 Line (Phenocell), programmed with additional body weight (Sox2, Oct4, KLF, cMyc). hiPSCs were maintained on CellStart (Invitrogen, Carlsbad, USA) in TESR2 medium (Stem Cell Technologies, Bergisch Gladbach, Germany) and cells were plated every 5 days on freshly coated plates using standard TRYple select (Invitrogen). The mass passage method was passaged at 1:6.

[0275] EB differentiation

[0276] After 24 h, the cells were transferred to differentiation medium containing 24 ng / mL of SCF, 21 ng / mL of TPO, 21 ng / mL of FLT3L, 194 ng / mL of BMP4, 200 ng / mL of VEGF, 50 ng / mL of mL of IL3, 50 ng / mL of IL6, 5 ng / mL of IL1, 100 ng / mL of GCSF, 5 ng / mL of IGF1 (PeproTech, Neuilly-sur-Seine, France). Medium w...

Embodiment 2

[0332] Materials and methods

[0333] hiPSC expansion, EB differentiation, assessment of human cell engraftment, T cell maturation and functional assays, quantitative PCR, were performed as described above.

[0334] cell sorting

[0335] Dissociated EB cells were stained with antibody CD110-PE (MPL) or CD135-PE (FLT3), then re-stained with PE-MicroBeads (Miltenyi), and finally used Cell separation device for sorting.

[0336] mouse transplantation

[0337] NOD.Cg-Prkdc scid Il2rg tm1Wjl / SzJ (NSG) (Charles River, L'Abresle, France) was housed at the IRSN animal care facility. All experiments and procedures were carried out in accordance with the regulations of the French Ministry of Agriculture on animal experiments and were approved by the local ethics committee.

[0338] Twenty-four hours before cell injection, mice aged 6-8 weeks and housed under sterile conditions were sublethally irradiated with 3.5 Gy from a 137Cs source (2.115 Gy / min). To ensure consistency ...

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Abstract

The present invention relates to a method of preparing hematopoietic cell graft or enriching a population of cells for hematopoietic stem cells that are capable of long-term multilineage engraftment and self-renewal. It also relates to hematopoietic grafts comprising said hematopoietic stem cells as well as their uses in therapy.

Description

technical field [0001] The invention relates to the field of medicine, in particular to human hematopoietic grafts. In particular, the present invention relates to the identification and selection of hematopoietic stem cells capable of long-term multi-lineage engraftment and self-renewal, ie, hematopoietic stem cells suitable for hematopoietic transplantation. Background technique [0002] Hematopoietic stem cells (HSCs) are rare cells in human bone marrow (BM) and blood that are responsible for the lifelong curative effects of allogeneic hematopoietic cell transplantation in hematological diseases or after radiotherapy / chemotherapy. These cells can be harvested from several sources including BM, mobilized peripheral blood, or human cord blood. Cord blood offers several advantages, namely a reduced need for HLA matching and a reduced risk of graft-versus-host disease. However, despite advances in the manipulation of HSCs, their numbers are often insufficient for allogeneic...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0789
CPCC12N5/0647A61K35/28C12N2501/105C12N2501/125C12N2501/145C12N2501/155C12N2501/165C12N2501/20C12N2501/22C12N2501/2301C12N2501/2303C12N2501/2306C12N2506/45
Inventor 劳伦斯·居约诺-哈尔曼德克里斯托夫·德斯特克蒂埃里·贾弗雷多阿兰·查佩尔卢瓦克·加伦卢克·杜艾
Owner ESTAB FR DU SANG
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