Chemistry: molecular biology and microbiology – Measuring or testing process involving enzymes or... – Involving nucleic acid
Reexamination Certificate
2000-11-03
2003-12-30
Le, Long V. (Department: 1641)
Chemistry: molecular biology and microbiology
Measuring or testing process involving enzymes or...
Involving nucleic acid
C435S002000, C435S343000, C435S372000, C530S350000, C530S388730, C530S827000
Reexamination Certificate
active
06670123
ABSTRACT:
BACKGROUND OF THE INVENTION
In vertebrates, most tissues are composed of differentiated cells that no longer divide. Nevertheless, there are tissues which retain an ‘embryonic’ cell population within themselves. The cellular composition of such embryonic populations is always changing, even in adult animals. This phenomenon is most evident in the mammalian hematopoietic system. This system, which is organized hierarchically, consists of a heterogeneous mixture of many different kinds of blood cells at all stages of differentiation—some morphologically recognizable and some not [23]. Mature, functional blood cells are divided into several lines, including erythroid, lymphoid, and myeloid, each possessing its own morphology, characteristics, and function. Each blood line derives from restricted progenitor cells, which become committed to a particular line of differentiation. However, despite this diversity, the various developing blood cells and progenitor cells derive from one discreet source: the embryonic cell population of multipotential, self-renewing hematopoietic stem cells [21, 22].
A stem cell is a cell capable of extensive proliferation: it generates more stem cells (through self-renewal) in addition to its differentiated progeny [20, 21]. In mammals and birds, a multipotential hematopoietic stem cell can give rise to red blood cells (erythrocytes), white blood cells (granulocytes), macrophages, platelets, and immunocompetent cells (lymphocytes) [22]. Thus, a single hematopoietic stem cell can generate a clone containing millions of differentiated cells, as well as a few stem cells. The continuous formation of new blood cells is accomplished in bone marrow by hematopoietic stem cells. Stem cells mature into progenitor cells, which then become lineage-committed, although not yet terminally-differentiated. Once committed, progenitor cells are no longer capable of maturing into all of the cell lineages which comprise the hematopoietic system [22].
Hematopoietic stem cells currently find use in a myriad of clinical settings. Indeed, with the recent remarkable progress in cell processing technology, there has been a rapid increase in the number of patients and types of diseases that are now treated with hematopoietic stem-cell transplantation, in both autologous and allogeneic cases. For example, autologous peripheral stem-cell support has largely replaced bone marrow transplantation as a means of regenerating the hematopoietic system of myeloma patients undergoing myeloablative chemotherapy. Stem cell transplantation is also used to treat patients with non-Hodgkin's lymphomas [25]. Moreover, peripheral blood autografting has been widely used in trials for the treatment of chemosensitive tumors [26].
Hematopoietic stem cells also may be used in vitro to enable the detection and assessment of growth factors associated with stem cell self-renewal and hematopoietic development. Furthermore, in vitro expansion of hematopoietic stem cells from various sources, including bone marrow and blood of the umbilical cord, is gaining importance, as it provides a clinically-potential graft in autologous or allogeneic transplant cases, and facilitates transduction of genes for the treatment of genetic diseases through gene therapy [24].
To be useful, however, hematopoietic stem cells first must be identified and isolated—a task made more difficult by the fact that hematopoietic stem cells comprise only a small proportion of the total cell population in bone marrow. Hematopoietic stem cells may be identified, for example, by detecting expression of specific cell-surface protein or carbohydrate antigen markers.
The CD34 antigen, which is a glycosylated transmembrane protein, has frequently been used as a marker for the identification of hematopoietic stem cells [26]. Detection of CD34 is also considered by some to be the first step in quality assessment of hematopoietic stem cell grafts [27]. The CD34 antigen was previously indicated to be present solely on stem cells, and not on lineage-committed progenitor cells. However, recent evidence suggests that expression of CD34 on the cell membrane does not always correlate with stem cell activity [28]. Indeed, in the mouse, there is a highly quiescent population of stem cells that lacks CD34 expression, but which has full reconstituting capacity. It also has been discovered that there is a similar population of dormant CD34-negative human hematopoietic stem cells. This information clearly casts some uncertainty on the benefits of using CD34 as a marker for isolating hematopoietic stem cells [28].
While it appears that lineage-committed hematopoietic cells may display epitopic characteristics associated with hematopoietic stem cells, it is not known how many of the antigen markers associated with differentiated cells are also present on stem cells. Accordingly, it is clear that significant problems exist in connection with the identification of hematopoietic stem cells, and new methods of detection of hematopoietic stem cells are needed.
SUMMARY OF THE INVENTION
The present invention is predicated on the discovery that the KIAA0918 gene is specifically expressed in the primitive hematopoietic Kg-1a cell line, which is close to the hematopoietic stem cell line, thereby providing a genetic marker for identifying hematopoietic stem cells. On the basis of this finding, the present invention provides an isolated nucleic acid sequence encoding KIAA0918, and an isolated nucleic acid sequence that hybridizes under high stringency conditions to a second nucleic acid that is complementary to a nucleic acid sequence encoding KIAA0918.
The present invention also discloses a purified KIAA0918 protein, and a purified protein encoded by a nucleic acid sequence that hybridizes under high stringency conditions to a second nucleic acid sequence that is complementary to a nucleic acid sequence encoding KIAA0918. Also provided is a method of making KIAA0918 protein.
The present invention is further directed to an antibody specific for KIAA0918, and a method for producing an antibody specific for KIAA0918 protein.
Additionally, the present invention discloses a vector comprising a nucleic acid sequence encoding KIAA0918, and a host cell transformed with a vector comprising a nucleic acid sequence encoding KIAA0918.
Also provided in the present invention is a method for detecting the presence of hematopoietic stem cells in a heterogeneous cell suspension that may contain hematopoietic stem cells, as well as a method for isolating hematopoietic stem cells from a heterogeneous cell suspension that may contain hematopoietic stem cells.
Finally, the present invention discloses a method for assessing gene expression in a tissue sample.
Additional objects of the present invention will be apparent in view of the description which follows.
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Berthou, C. et al., Granzyme B and Perforin Lytic Proteins are Expressed in CD34+ Peripheral Blood Progenitor Cells Mobilized by Chemotherapy and Granulocyte Colony-Stimulating Factor. Blood 86 (9): 3500-3506, 1995.
Ivanova, N.B. et al., Identification of Differentially Expressed Genes by Restri
Belyavsky Alexander
Shmelkov Sergey
Visser Jan
Amster Rothstein & Ebenstein LLP
Chen Changhwa J.
Le Long V.
New York Blood Center Inc.
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