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Detection of haematopoietic stem cells and progeny and uses thereof

a technology applied in the field of haematopoietic stem cells and progeny detection, can solve the problems of not being able to define the spatial distribution of hemopoietic stem cells (hsc) within the bm, and may take many weeks or months for natural recovery of such hpc cells to their normal levels

Inactive Publication Date: 2005-01-06
PETER MACCALLUM CANCER INST
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Benefits of technology

[0201] In gene therapy applications, genes are introduced into cells in order to achieve in vivo synthesis of a therapeutically effective genetic product, for example for replacement of a defective gene. “Gene therapy” includes both conventional gene therapy where a lasting effect is achieved by a single treatment, and the administration of gene therapeutic agents, which involves the one time or repeated administration of a therapeutically effective DNA or mRNA. Antisense RNAs and DNAs can be used as therapeutic agents for blocking the expression of certain genes in vivo.
[0202] Other indications of gene therapy include introduction of drug resistance genes to enable normal stem cells to have an advantage and be subject to selective pressure during chemotherapy. Suitable drug resistance genes include, but are not limited to, the gene encoding the multi-drug resistance (MDR) protein.

Problems solved by technology

The populations of such HPC cells may take many weeks or months to recover naturally to their normal levels.
Until recently, it has not been possible to define the spatial distribution of hemopoietic stem cells (HSC) within the BM.
This is due to the rarity of HSC and the lack of a single, unique antigenic marker allowing their unambiguous identification in situ.

Method used

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  • Detection of haematopoietic stem cells and progeny and uses thereof
  • Detection of haematopoietic stem cells and progeny and uses thereof
  • Detection of haematopoietic stem cells and progeny and uses thereof

Examples

Experimental program
Comparison scheme
Effect test

example 1

HA Expression on Murine Hemopoietic Cells

[0242] Expression of HA was demonstrated by flow cytometric analysis through the binding of a biotinylated form of hyaluronic acid binding protein (HABP), which demonstrates absolute specificity for HA (4). Using this approach, HABP binding was detected on two murine sub-populations enriched for HSC: Lin−Sca+Kit+(FIG. 1a) and Lin−Rhdull and was completely eliminated by prior treatment of the cells with the enzyme hyaluronidase (HY) (FIG. 1b). In addition, a similar proportion of Lin− Rh123dull cells isolated from CD44− / − mice (2) exhibited binding of HABP (25.0% compared to 26.5%), demonstrating that HA detected on the cell surface is not due to the binding of exogenous HA to its major receptor CD44 (19) but due to de-novo synthesis by primitive hemopoietic cells themselves. Importantly, RT-PCR analysis demonstrated that Lin−Sca+Kit+, Lin−HABP+ but not HABP− cells express Has-1, Has-2 and Has-3 (FIG. 2a+b).

example 2

HA Expression of Human HPC

[0243] The expression of HA by primitive BM cells is not a unique feature of the mouse but is also a characteristic of human hemopoietic progenitors. FACS analysis of human umbilical cord blood (CB) (FIG. 3) using CD34, CD38 and HABP showed putative Human HSC synthesise HA. Interestingly, there was a significant decrease in the level of HA expression in correlation with a maturing cell phenotype, with no HA expression detected on CD34−CD38+ cells. In accord with data in the mouse, isolated human CD34+ and Lin−HA+ cells but not Lin−HA− also express HAS1, HAS2 and HAS3 (FIG. 4). Collectively these data demonstrate that HA is synthesized by and expressed on murine and human hemopoietic populations enriched for HSC.

[0244] However, repopulating HSC represent only a minor proportion of Lin−Rh123dull cells (5). In order to examine whether HSC were contained within the HABP+ subpopulation, FACS was used to isolate Lin−HABP+ and Lin−HABP−, and Lin− Sca+HABP+ and L...

example 3

The Potential Role of HA as a Negative Regulator of HSC Proliferation

[0248] In vitro stroma-free, cytokine-dependent cultures demonstrated that the ligation of HA by a the surrogate soluble ligand HABP results in a profound suppression of both murine and human HSC proliferation while stimulated by a potent combination of early acting hemopoietic growth factors. Both murine and putative human HSC (Lin−Sca+Kit+ and Lin−CD34+CD38− cells respectively) were cultured in serum free conditions in the presence of multiple cytokines consisting of either SCF (75ng / ml), IL-11, FLT3 Ligand and IL-6 (all 100 ng / ml) or G-CSF, SCF, FLT3 Ligand, MGDF (all 100 ng / ml), Il-6 and IL-3 (both 10 ng / ml) for murine and human HSC respectively, and various concentrations of HABP. As shown in FIG. 5 this resulted in a significant inhibition of cell proliferation corresponding to the presence of increasing concentrations of HABP. This unexpected data shows a marked growth inhibitory effect of HA on hemopoiesis...

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Abstract

The present invention relates to a method of identifying a haematopoietic stem cell (HSC) or progeny thereof comprising the steps of: obtaining a cell sample including HSC or progeny thereof; detecting the presence of at least one carbohydrate sequence having a sequence of at least one disaccharide repeat of glucuronic acid and N-acetylglucosamine or an equivalent thereof; and identifying a HSC or progeny thereof having the sequence or equivalent thereof. The invention also relates to methods of enriching cell populations for HSC or progeny thereof, for isolating HSC or progeny thereof and cell preparations obtained using the methods of their invention and their uses.

Description

CROSS-REFERENCES [0001] This application is a continuation-in-part of published PCT Application No. PCT / AU02 / 01443 filed Oct. 24, 2002 which claims priority to Australian Application No. PR 8565 filed Oct. 30, 2001 both of which applications are incorporated herein in their entirety and to which applications is claimed priority. FIELD OF THE INVENTION [0002] The present invention relates to the identification of a specific population of cell types, in particular, haematopoietic stem cells (HSC) and cells deriving from HSC. The invention also provides for methods of isolation and uses of the stem cells and their progeny derived from the methods. Methods of regulating development of the stem cells and their progeny are also provided. BACKGROUND [0003] There exist a strong interest in identifying specific cell types in an effort to gain enriched populations of the cells. Having possession of an enriched population may allow for a better understanding of the specific cell types or even ...

Claims

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Application Information

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Patent Type & Authority Applications(United States)
IPC IPC(8): A61K35/12A61P7/00A61P7/06A61P9/00A61P35/02C12N5/0789C12N15/09C12Q1/04G01N33/53G01N33/566G01N33/569
CPCC12N5/0647G01N33/5308G01N2400/40G01N33/56972G01N2333/91097G01N33/56966A61P35/02A61P7/00A61P7/06A61P9/00
Inventor NILSSON, SUSANSIMMONS, PAULHAYLOCK, DAVID
Owner PETER MACCALLUM CANCER INST
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