Method to distinguish hematopoietic progenitor cells

Details Method to distinguish hematopoietic progenitor cells Method to distinguish hematopoietic progenitor cells

1993-11-04

2003-04-29

Kunz, Gary L. (Department: 1647)

Chemistry: molecular biology and microbiology

Measuring or testing process involving enzymes or...

Involving antigen-antibody binding, specific binding protein...

C435S069520, C530S351000

Reexamination Certificate

active

06555324

ABSTRACT:

FIELD OF THE INVENTION
This invention relates to the field of flow cytometry and the use of fluorescently labelled markers to distinguish between various cells types, and more particularly, this invention relates to a method for distinguishing among hematopoietic progenitor cells preferably by means of flow cytometry-wherein said cells are labelled with fluorescently labelled monoclonal antibodies.
BACKGROUND OF THE INVENTION
In U.S. Pat. No. 4,714,680, Civin described a population of pluripotent lympho-hematopoietic cells which were substantially free of mature lymphoid and myeloid cells. Civin also described an antigen, MY-10 and a monoclonal antibody (of the same name) thereto, which was present on these cells. These cells make up to about 1% of all cells in normal adult bone marrow, and generally comprise a mixture of totipotent, pluripotent stem cells and lineage committed progenitor cells with the latter cells predominating.
Since that time, MY-10 has been classified by the International Workshop on Human Leukocyte Antigens as falling with the cluster designated as “CD34. ” Anti-CD34 monoclonal antibodies are commercially available from a number of sources including Becton Dickinson Immunocytometry Systems, San Jose, Calif. (“BDIS”).
Given that most of the cells within a CD34
+
population of cells are lineage committed, a number of researchers have used various means in an attempt to distinguish lineage committed from non-lineage committed cells. For example, Loken et al., Blood, 70:1316 (1987), used flow cytometry and a combination of light scatter and immunofluorescence staining to identify the various stages of B lymphocyte maturation. Working backward from the most mature cell to the most immature cell within a cell type, Loken, Terstappen and their colleagues showed that certain antigens are expressed on mature stages of cells but not on immature stages while different antigens may be expressed on more immature cells the expression of which then is lost as the cells mature. They also showed that cells at different levels of maturity had different light scatter properties.
In Blood, 77:1218 (1991), Terstappen et al. showed that a cell population that was CD34
+
and CD38
−
was enriched for human hematopoietic stem cells (“HSC”). HSC are defined as those cells that are capable of both limited self-renewal and differentiation into the two principle progenitor components (i.e., myeloid and lymphoid progenitors). (As used herein, “myeloid” progenitors include cells that give rise to monocytes, granulocytes and megakaryocytes but exclude cells that can-give rise to cells in the erythroid lineage. Among the “erythroid” progenitors, therefore, are only those progenitors that give rise to cells within the erythroid lineage.) Terstappen et al. demonstrated that cells within the population of cells having this phenotype were capable of limited self-renewal and differentiation into the various hematopoietic cell types. Like other researchers in the field, Terstappen et al. reported that cells of this phenotype did not express CD3, CD10, CD33 or CD71.
Following this, in co-pending and commonly assigned patent application U.S. Ser. No. 07/895,491, filed Jun. 8 , 1992, Terstappen and his co-workers described a more enriched population of human cells having the phenotype CD34
+
/CD38
−
/HLA-DR
+
. They showed that this population of cells was enriched for HSC.
In addition to this work, others have examined the effects of cell adhesion molecules on HSC enriched populations. For example, Simmons et al. described the expression of VCAM-1 on certain bone marrow stromal cells as they relate to progenitor cells. See Blood, 80:388 (1992). Similarly, Ogawa, Blood, 81:2844 (1993), has described the suspected effects of different growth factors on progenitor cells.
While the work described above and in references such as Wognum et al., Blood, 76:697 (1990) and Udomsakdi et al., Blood, 80:2513 (1992), focused on the discrimination of non-lineage committed cells from lineage committed cells, on the discrimination of maturational stages within a lineage or on the use of a certain very select markers (e.g., EPO) to identify a specific progenitor lineage, there has been no careful systematic study using highly sensitive measuring means to discriminate among progenitor cells committed, specific lineage.
SUMMARY OF THE INVENTION
The present invention comprises a method to discriminate human lineage committed myeloid, lymphoid and erythroid progenitor cells. Cells that are enriched for HSC are identified using markers (e.g., monoclonal antibodies, fragments thereof, or recombinant binding proteins derived therefrom) that are specific for CD34, CD38 and HLA-DR antigens. These cells are CD34
+
/CD38
−
/HLA-DR
+
. These cells also express homogeneous levels of stem cell factor receptor (“SCFR”), Leu 8 (“L-selectin”), CD18, CD33, CD44, CD48, CD49e, CD50 and CD52. Low levels of granulocyte/monocyte-colony stimulating factor receptor (“GM-CSFR”), gp130/IL-6R (gp130 signal subunit of. the interleukin-6 receptor), IL-6R and Fas Ag are expressed on cells of this phenotype, while IL-7R is not expressed at all.
Progenitor cells committed to the erythroid, myeloid or lymphoid lineages can be identified using a marker for CD34, a marker for CD38 and one or more markers that are specific for cell adhesion molecules and/or cytokine receptors that are expressed on the surface of human cells. Progenitor cells are CD34
+
/CD38
+
. Progenitor cells that are committed to the lymphoid lineage show de novo expression of IL-7R, increased expression of Fas Ag, CD49e and CD52 and decreased expression of SCFR, GM-CSFR, IL-6R, gp130/IL-6R, Leu 8, CD18, CD33, CD44 and CD50. Progenitor cells that are committed to the erythroid lineage show a complete loss of L-selectin and decreased expression of IL-6R, CD18, CD48, CD50 and CD52. Progenitor cells committed to the myeloid lineage show increased expression of GM-CSFR, IL-6R, Fas Ag, CD18, CD33, CD44, CD48 and CD49e and decreased expression of SCFR, L-Selectin and CD52.
Using this information, if one were to isolate only those cells committed to the erythroid lineage, for example, one could combine CD34, CD38 and IL-7R antibodies and look for positive expression for each marker. To look for only myeloid committed progenitor cells, one could use CD34, CD38 and IL-6R antibodies and look for positive expression of these markers. To exclude those myeloid progenitors that may be erythroid, one could select for those progenitors that are IL-6R
+
and IL-7R
−
.
One means to identify and/or isolate cells of the desired lineage and/or phenotype comprises flow cytometry. Cell sorters and cells analyzers are available from a number of commercial sources, including Becton Dickinson Immunocytometry Systems (“BDIS”) under the name FACS®. The general principles of flow cytometry and the use of fluorescent markers therewith is described in Herzenberg et al., Sci. Amer., 234:108 (1976). See also U.S. Pat. No. 4,727,020. In this method, monoclonal antibodies conjugated to dyes having emission spectra that are distinguishable are used. Useful dyes include r-phycoerythrin (“PE”), allophycocyanin (“APC”), fluorescein isothiocyanate (“FITC”), Texas Red (Molecular Probes), CY-5 (Biological Detection Systems), peridinin chlorophyll protein complex (“PerCP”) and tandem conjugates thereof. While desired, it is not necessary that each dye have similar excitation maxima. See U.S. Pat. Nos. 4,520,110, 4,542,104, and 4,876,190.
Other means for isolating cells having the desired phenotype include columns containing a matrix of materials which will trap cells. The matrix can comprise a material which can be magnetized, such as steel wool, and used in combination with magnetic beads which are conjugated to antibodies. By serial passage of labelled cells through such a device, the desired phenotype can be achieved. Such a device is sold under the name “MACS” by Miltenyi Biotech GmbH. Alternatively, the matrix may comprise biotin and the antibo

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