CA2219869A1 - Human cd-34 hematopoietic stem cells - Google Patents
Human cd-34 hematopoietic stem cellsInfo
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- CA2219869A1 CA2219869A1 CA002219869A CA2219869A CA2219869A1 CA 2219869 A1 CA2219869 A1 CA 2219869A1 CA 002219869 A CA002219869 A CA 002219869A CA 2219869 A CA2219869 A CA 2219869A CA 2219869 A1 CA2219869 A1 CA 2219869A1
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Description
Human CD34- Hematopoietic Stem Cells Field of the Invention The present invention relates to human hematopoietic stem cells and to a method of isolating and using such cells.
Background of the Invention The m~mm~ n hematopoietic system consists of a heterogeneous array of cells ranging from large numbers of dirrelellliated cells with defined function to rare pluripotent stem cells with extensive developmental and proliferative potential (1, 2, 3). The defining feature of a stem cell is its ability to repopulate the hematopoietic system of a recipient after transplantation. Stem cells are playing an increasingly important role in clinical and commercial applications, as the role of the stem cells in transplantation widens. Identification and purification of stem cells is essential both to determine the cellular and molecular factors that govern stem cell development and for the application of clinical procedures including stem cell transplant and gene therapy.
Cell surface expression of CD34 has become the distinguishing feature used to isolate stem cells because CD34 is downregulated as cells differentiate into more abundant mature cells (4). However, CD34 does not mark stem cells exclusively since only 1% of bone marrow (BM) cells are CD34+ and include clonogenic progenitors that are not able to repopulate after transplantation.
Therefore other markers such as Thy-l can be combined with CD34 to positively select for a more enriched cell fraction (5, 6, 7). Conversely, the CD34+ fraction can be enriched by elimin~ting cells that express markers that are expressed on non-repopulating cells (e.g.lineage antigens). For these reasons, all current clinical and experimental protocols ~tili~ing human stem cells including ex vivo culture, gene therapy, and bone marrow transplantation, focus on CD34+ cells.
There have been reports (8, 9, 10) of murine CD34- hematopoietic stem cells which are capable of long term repopulation. For human hematopoietic stem cells, however, CD34+ cells have been regarded as a stem cell marker without exception.
Brief Description of the Drawings The present invention will be further understood from the following description with reference to the Figures, in which:
Figure lA demonstrates cell surface expression of CD34 on cord blood cells depleted for lineage markers (Lin-). (Panel I) Lin- cells were purified as in (11, 12) and stained with a class III monoclonal antibody for CD34 (581) conjugated to FITC (Becken Dickenson, BD). Cells residing in R1 were considered CD34 negative (CD34-). (Panel II) CD34- cells were purified using standard cell sorting techniques and re-analyzed using the same CD34-581 antibody. (Panel III and Panel IV) Purified R1 cells were stained and re-analyzed using a class I monoclonal antibody for CD34 (Immun-133) (Coulter) and a class II monoclonal antibody for CD34 (Q-Bend-10) (Becton Dickinson, BD).
Figure lB demonstrates the immunostaining of purified populations for CD34 expression. Representative cells are shown from a total of 25-75 cells examined for each treatment (n=2).
Figure lC demonstrates a comparison of cell surface markers between Lin-CD34- versus Lin-CD34+ cord blood cells commonly used to further subdivide p~ ive cells.
Figure 2 demonstrates the level of human cell engraftment in NOD/SCID mice transplanted with highly purified Lin-CD34- cells at various doses.
Figure 3 demonstrates the multilineage differentiation of human Lin-CD34- cells in NOD/SCID mice. Bone marrow from a highly engrafted mouse transplanted with 120,000 Lin-CD34- cord blood cells was stained with various human-specific monoclonal antibodies and analyzed by flow cytometry.
Approximately 106 mononuclear cells collected from mouse BM was prepared as shown previously for multilineage analysis (12). (A) Cells with medium to high forward scatter (region Rl) were gated and further analyzed. (B) Histogram of CD45 (pan-leukocyte marker) expression indicating that 2.5% of the cells present in the murine bone marrow are h~ n, gated R2. All further lineage markers was done on cells within gate R2 (CD45+). (C) Isotype control for non-specific IgG staining of PE and FITC fluorescence. (D) Expression of myeloid marker CD33 and granulocyte marker CD15; (E) pan-B cell markers CDl9 and CD20; (F) CD38 and the imm~h1re hematopoietic marker CD34; (G
and H) T-cell markers CD2, CD3, CD4 and CD8.
Figure 4 is a frequency analysis of Lin-CD34- cells found in human hematopoietic tissue. (A) Column I; Fetal liver collected from 8 week old human fetus, n=3 Column II; Fetal blood aspirated from 19 week old fetus, n=l Column III; Cord blood collected from placenta at time of birth, n=3. (B) Column I; Normal Adult Bone marrow, n=2 Column II; Bone marrow from a normal adult donor after 5 days of G-CSF ~lmini~tration, n=2 Column III;
Peripherial blood collected from a normal adult donor after 5 days of G-CSF
a-lministration, n=2.
Figure 5 demonstrates the percentage of the input cells after in-vitro culture of lin- CD34- cells. Purified cells were counted and seeded (250-2000) in wells cont~ining serum free media (SF) ( see examples) (solid bar) or SF
supplemented with 25% HWEC- conditioned media ( shaded bar). Cells were counted each day and the percentage of the input cells was calculated ( n=3).
Figure 6 is an analysis of CD34 and CD38 expression of highly purified populations after in vitro culture. A representative experiment (n=4) of CD34 and CD38 cell surface expression performed on initially purified lin-CD34-CD38- cells, and purified cells after 2 and 4 days of culture in SF, SF
supplemented with 5% FCS or 25% HWEC-CM. The entire contents of individual wells was collected at 2 and 4 days (5000-10, 000 cells), stained with monoclonal antibodies CD34 and CD38 directly conjugated to FITC and PE
respectively (Beckton Dickinson, BD). Stained populations were then washed and analyzed using standard flow cytometric techniques as done previously (11,12) followed by the display of histograms using the Cell Quest software program (BD) Figure 7 demonstrates the capacity of expanded lin-CD34-, lin-CD38-CD38- or lin-CD34-CD38+ cell fractions to engraft NOD/SCID mice.
Figure 8 demonstrates the multilineage differentiation of human lin-CD34- cells in NOD/SCID mice after ex vivo culture. (A) Histogram of CD45 (human-specific pan-leukocyte marker) expression indicating that 7% of the cells present in the murine BM are hllm~n (B) Forward and Side scatter of the CD45 human cells. Subsequent analysis of lineage markers was done on CD45+ cells within gate Rl (lymphoid and blast cells) or R2 (myeloid cells) gates. (C) Analysis for the presence of imm~tllre cells using the CD34 and CD38 markers. ( D) Analysis for the presence of human B cell lineage cells using CDl9 and CD20 markers. (E-F) Analysis for the presence of human T
lymphocytes using the panel of T cell markers: CD2, CD3, CD4 and CD8. (G-H) Analysis for the presence of myeloid cells using CD33, CD14, CD15 and CD13 markers.
Description of the Invention The present inventors have identified and isolated a population of human hematopoietic stem cells which do not express CD34 (CD34-), CD38 (CD38-) or lineage specific markers (Lin-) and which are able to generate by proliferation and dirrelellLiation multiple lineages of the human hematopoietic system, as evidenced by their ability to produce multilineage human engraftment of immune-deficient NOD/SCID mice after transplantation.
Moreover, the repopulative capacity and the dirrerellliative capacity of the lin-CD34-CD38- cells can be stimulated by in vitro culture of these cells.
In initial studies, a population of CD34-Lin- cells were isolated from human fetal liver, cord blood, bone marrow and mobilized peripheral blood and bone marrow. These cells do not express any lineage specific markers and are also devoid of HLA-DR and Thy-l, two other markers associated with primitive cells, but are heterogeneous for CD38 and c-kit expression. The NOD/SCID transplantation studies show that the repopulating cells are exclusively present in the CD38- fraction and not the CD38+ fraction and these cells are characterized as Lin-CD34-CD38-. These novel Lin-CD34- cells are most abundant in fetal tissues, with progressive reductions in cord blood (CB) and adult bone marrow (BM) or mobilized peripheral blood (M-PB).
Transplantation of the Lin- CD34- cells in NOD/SCID mice engrafted myeloid, lymphoid and erythroid lineages, which demonstrates that Lin- CD34- cells repopulate cells of the hematopoietic system. The results also show that Lin-CD34-CD38- cells are developmentally earlier than CD34+ cells and can produce CD34+ cells following in vitro culture or after repopulation. Thus, these cells appear to be at the top of the heirarchical structure of human stem cells.
The identification of this novel stem cell within the hierarchy of human hematopoiesis has important implications for understanding the origin of hematopoietic diseases such as leukemia and for clinical procedures such as stem cell transplantation and gene therapy. This novel stem cell can be important for the treatment of infection, for the reconstitution of deficient ormissing cell populations as for example for cancer patients after myeloablative therapy and for the treatment of genetic abnormalities and defects.
The present invention provides a therapeutic composition and a method for the treatment of hematopoietic disorders. In particular, the composition andmethod can be used to treat hematopoietic disorders such as leukemia and for several clinical procedures such as stem cell transplantation, therapy, gene therapy, for combating infection and for cell reconstitution.
In accordance with an aspect of the present invention there is provided an essentially pure population of human hematopoietic cells characterized as Lin- CD34-.
In accordance with another aspect of the present invention there is provided an essentially pure population of human hematopoietic cells characterized as Lin-CD34-CD38-.
In accordance with another aspect of the present invention there is provided a method for providing an essentially pure population of human hematopoietic cells characterized as Lin- CD34-.
In accordance with another aspect of the invention there is provided a method for the treatment of hematopoietic disorders such as leukemia comprising the use of human hematopoietic cells characterized as Lin- CD34-.
In accordance with another aspect of the present invention is a method for the ex vivo generation of human hematopoietic cells using Lin-CD34- cells.
In accordance with another aspect of the present invention is a gene therapy method for providing genetically altered human hematopoietic cells characterized as Lin- CD34- to a patient.
In accordance with another aspect of the present invention is a method for reconstituting deficient or missing human hematopoietic cell populations comprising the use of Lin- CD34- cells.
In accordance with another aspect of the present invention is a method for transplanting human hematopoietic cell populations to a patient comprising the use of Lin- CD34- cells.
In accordance with another aspect of the present invention is a method for combatting infection in a patient comprising ~dmini~tering an effective amount of human hematopoietic cells characterized as Lin- CD34-.
In accordance with yet a further aspect of the present invention there is provided a method utilizing human hematopoietic cells characterized as Lin-CD34- for the production of CD34+ human hematopoietic cells.
In accordance with a further aspect of the present invention is a method for increasing the repopulating capacity of human hematopoietic cells characterized as Lin-CD34- by culturing such cells in vitro for several days.
In accordance with a further aspect of the present invention is a method for screening candidate compounds affecting proliferation or dirr~relltiation ofstem cells characterized as Lin-CD34- and Lin-CD34-CD38-.
It was determined using CD34 class III fluorescent monoclonal antibodies that CD34- cells that do not express lineage markers exist in human hematopoietic tissues (Fig. lA). The possibility that CD34 protein was produced in the Lin-CD34- cells but not transported to the cell surface, was excluded using permeabilized, stained cytospins of purified Lin-CD34- that were further conjugated with fluourescent monoclonal antibodies. This confirmed that no CD34-FITC signal was detected in the Lin-CD34- cells (Fig.
lB) Heterogeneity within human Lin-CD34+ cells is well documented and further subdivision for the most p~ ive cells is typically based on the cell surface markers CD38, c-kit, Thy-1 and HLA-DR. The expression of these markers on both the Lin-CD34+ and Lin-CD34- cells was compared (Fig. lC).
Lin-CD34- cells displayed a bi-modal distribution of CD38 clearly dividing the population into two fractions in contrast to the high proportion of Lin-CD34+
cells that express CD38 (Fig. lC). Cell surface expression of c-kit was similar between that two populations, while the Lin-CD34- cells are almost exclusively Thy-1- and HLA-DR- (Fig. lC). Both the absence of HLA-DR expression and the presence of Thy-1 have been proposed to define more ~lhlliLive subfractions within the Lin-CD34+ population. Therefore, the Lin-CD34-population derived from CB is a distinct population which differs not only in CD34 expression from plilllilive Lin-CD34+ cells but also in phenotypic heterogeneity based on additional markers associated with stem cells.
Using clonogenic methylcellulose assays, it was determined whether Lin-CD34-, Lin-CD34-CD38- and Lin-CD34-CD38+ cells possessed any hematopoietic progenitor activity by comparing the CFC and LTC-IC content, respectively. Clonogenic capacity of Lin-CD34- cells was extremely low in comparison to Lin-CD34+CD38- cells (Table I). As many as 10,000 cells needed to be seeded on MS-5 stroma to detect a single LTC-IC within the Lin-CD34- cell fraction, while further purification demonstrated that detection of LTC-IC in the Lin-CD34-CD38- fraction required seeding of at least 2000 cells. By contrast, as few as 10 Lin-CD34+CD38- cells contain an LTC-IC. The Lin-CD34-CD38+ cells were devoid of LTC-IC activity (limit of detection at 10,000 cells) but contained a much higher capacity to form CFC specifically commilled to the erythroid lineage (Table I). The low efficiency to produce myeloid and erythroid colnlllilled progenitors as well as the more plilllilive LTC-IC is similar to observations made with murine Lin-CD34- cells in the same assay systems and suggest functional similarities may exist between the Lin-CD34- cells from these two species (8,10).
The only conclusive method to detect stem cells is to determine their ability to repopulate recipient hosts (1, 2,3). A repopulation assay was developed for primitive human cells based on their ability to initiate multilineage human engraftment in immune-deficient NOD/SCID mice (13, 14). Based on cell purification and gene marking, the cell capable of repopulating NOD/SCID mice (termed the SCID-Repopulating Cell, SRC) was established as distinct from and more plilnilive than the majority of progenitors detected in in vitro assays (15). Only the Lin-CD34+CD38- cells and not the Lin-CD34+CD38+ cells could give rise to multilineage engraftment (15, 12).
Moreover, transplantation of as many as 10-6 Lin+CD34- cells also did not engraft (12). To determine whether highly purified Lin-CD34- cells had SRC
activity and to determine the frequency of any repopulating cells, Lin-CD34-cells were transplanted at varying cell doses into NOD/SCID mice using our standard protocols, and BM was analyzed for the presence of human cells after 8-12 weeks. The level of human cell engraftment in 23 NOD/SCID mice was quantitated by FACS and DNA analysis for the presence of human cells and results are sllmm~ri~ed in Fig. 2. A large proportion of transplanted mice were engrafted with human cells indicating that the Lin-CD34- cells were able to repopulate NOD/SCID mice. We have termed this cell the CD34NEG-SCID
Repopulating Cell (CD34NEG-SRC). The frequency was 1 CD34NEG-SRC in 125,000 Lin-CD34- cells. The differentiative and proliferative capacity of the CD34NEG-SRC cell was assessed by flow cytometric analysis. A
representative engrafted NOD/SCID mouse 10 weeks after the transplant of 120,000 Lin-CD34- cells is shown in Fig.3. Cells with medium to high forward scatter (region R1, Fig. 3A) were gated and further analyzed based on CD45 expression, a human specific pan-leukocyte marker (Fig.3B). The isotype control is shown in Fig.3C. The BM of this mouse contained 2.5% CD45+
human cells (Fig.3B) or at least 106 total human cells indicating that the Lin-CD34- cells have extensive proliferative capacity. Granulocytes (CD15+) were present among myeloid cells (CD33+) (Fig.3D). B-lymphoid cells were also present in the murine BM as shown by staining for CD19 and CD20 (Fig. 3E).
Interestingly, human T-cells expressing both CD2 and CD3 (Fig. 3G), along with CD4 and CD8 positive cells (Fig.3H) were also identified. NOD/SCID
mice transplanted with highly purified p~ ilive Lin-CD34+CD38- cells never gave rise to engraftment contailling T-cells demonstrating the unique in vivo repopulation behavior of the Lin-CD34- cells. In addition to multilineage engraftment, imm~ re CD34+ and CD34+CD38- cells were detected (Fig.3F).
It was concluded that the Lin-CD34- cells have the ability to repopulate NOD/SCID mice and differentiate in vivo into multiple lineages of myeloid and lymphoid cells. The production of CD34+CD38- and CD34+CD38+ cells in vivo suggests that Lin-CD34- cells are developmentally earlier than CD34+
cells in the hierarchy of human hematopoiesis.
There is evidence that the frequency of plilllilive cells changes during ontogeny with the highest proportions seen in the fetus. A variety of fetal, neonatal, and adult sources of human hematopoietic tissue were analyzed in an attempt to identify and quantify the Lin-CD34- population. The results indicate (Fig.4A,4B) that Lin-CD34- cells are produced early in human ontogeny and can persist throughout adult life and that the mechanisms that operate during the mobilization of CD34+ cells with G-CSF also affect Lin-CD34- cells.
This data provides the first identification of a novel human hematopoietic stem cell that does not express CD34 or lineage-specific markers. As d~te~ ed by all available monoclonal antibodies, this population is not only distinct by the absence of CD34, but also by the lack of HLA-DR
and Thy- 1 markers. In addition to these phentotypic differences, several lines of evidence functionally distinguish these two stem cell populations. While the Lin-CD34- cells have limited hematopoietic activity in vitro, Lin-CD34+CD38-cells are highly clonogenic based on their ability to produce CFC and LTC-IC.
Although both stem cell fractions are capable of repopulation, the presence of T-cells within the multilineage engraftment is a unique characteristic of Lin-CD34- transplantation since we have never detected human T cells in mice transplanted with Lin-CD34+CD38- cells (n=25). Therefore it is unlikely that NOD/SCID repopulation is derived from the co~ "~ tion of Lin-CD34+CD38-cells. Furthermore, based on LTC-IC frequency a millill~Ulll of 1 LTC-IC resides within 10 highly purified Lin-CD34+CD38- cells. In contrast to flow cytometry, in which we have determined that the Lin-CD34- population is 99% (or in some cases 100%) pure, the LTC-IC assay allows us to detect a smaller number of col~ ting Lin-CD34+CD38- cells. Using this assay, only a single LTC-IC could be detected in as many as 10 000 CD34-Lin- cells.
If this LTC-IC activity came from a Lin-CD34+CD38- cell, a maximum of 10 Lin-CD34+CD38- cells could be contained in the Lin-CD34- purified fraction (0.1% cont~min~tion). In addition, repopulated mice have not been observed when only 10 Lin-CD34+CD38- cells were transplanted (12). Since the frequency of SRC derived from Lin-CD34- cells is 1 in 125 000, a maximum of 125 Lin-CD34+CD38- cells could have been transplanted, again less than the number needed to repopulate CD34+CD38- cells.
The in vivo differentiation of human Lin-CD34- cells into CD34+ and lineage positive cells suggests that the Lin-CD34- cells preceed CD34+ cells in the hierarchy of human hematopoiesis.
The identification of these novel repopulating cells, termed CD34neg-SCID repopulating cells (CD34neg-SRC), provides an OppOl lUllily to examine the differentiation and proliferation potential of these cells and to establish their relationship to other cells within the human stem cell hierarchy. However, in vivo repopulation is a complex system m~king it difficult to establish hierarchical relationships. Moreover, it was found that there is 1 CD34neg-SRC
in 105 Lin-CD34- cells. While this may reflect the true frequency of primitive cells capable of repopulation, it it also possible that other plilllilive cells exist within this cell fraction. The inability to demonstrate activity of these plilllliive cells could be due to limitations in currently available hematopoietic assays.
For example, these cells could possess the developmental potential to repopulate NOD/SCID mice but are unable to under the current transplant conditions (e.g. some repopulating cells have much slower kinetics, or may lack expression of a particular adhesion molecule, etc) and therefore are never detected. In the murine system, some stem cells will only repopulate long-term but not short-term and vice versa (1,2,3). Therefore, ex vivo cultures are better suited for studies that examine the proliferative and differentiative potential of primitive cells, particularly in response to cytokine stimulation, because the conditions are well defined and it is straightforward to (measure developmental changes). Many studies based on ex vivo culture have demonstrated that there is heterogeneity within the CD34+ cell fraction (4). Subfractionation of the CD34+ cells on the basis of Thyl, CD38, and HLA-DR expression together with in vitro clonogenic and LTC-IC assays have demonstrated the progenitor-progeny relationship of the various cell types that make up the stem cell hierarchy (4, 14). The availability of the SRC assay to detect even earlier celltypes has added more information about the org~ni7~tion of cells within this hierarchy (14). Moreover, it was demonstrated that the SRC (derived from CD34+CD38- cells) can be expanded for 4 days in serum-free cultures without inducing their differentiation. However, all SRC are lost within an additional 4days of culture conco-ll-llilallt to the appearance of more differentiated CD38+cells (11). At the same time, both colony-forming cells (CFC) and long-term culture initiating cells (LTC-IC) could be greatly expanded during 8 days of culture demonstrating that the majority of the SRC are a distinct population (11), but may be closely related to ELTC-IC (18). Thus, in vitro culture systemscan be used to identify very fine transitions in the developmental program by combining both flow cytometry and functional CFC, LTC-IC and SRC assays.
It is now demonstrated hel~wilh that ex vivo culture of Lin-CD34- cells can induce the appearance of CD34+ cells and can increase the proportion of Lin-CD34- cells that have SRC activity. These studies provide new insight into the developmental program of human hematopoietic stem cells.
Our earlier studies demonstrated that the Lin-CD34- cell fraction expressed a bimodal distribution of CD38 allowing for further purification into CD38- and CD38+ subpopulations (12). To determine whether the Lin-CD34-, Lin-CD34-CD38-, Lin-CD34-CD38+ cells could be induced to proliferate andlor differentiate, these cells were cultured in defined serum-free (SF) conditions that have been previously shown could expand Lin34+38- cells and maintain and modestly increase POS CD34 POS-SRC(ll, 17). Cells were plated in methlycellulose assays at day 0, and after day 4 of liquid culture (Table II) to determine the effect of cytokine stimulation on the clonogenic progenitors present in lin-CD34-, lin-CD34-CD38- and lin-CD34-CD38+ cell fractions. Both the lin-CD34- and more purified lin-CD34-CD38- cells have a low plating efficiency (PE), l in 89 and 1 in 297 CFC respectively, whereas a higher PE of lin-CD34-CD38+ cells 1 in 10.4 cells was seen. Interestingly, the clonogenic capacity of the lin-CD34-CD38+ cells is restricted to the erythroid lineage. After 4 days of culture in SF media or SF media supplemented with the addition of 25% conditioned medium obtained from primary human umbilical vein endothelial cells (HWEC-CM), the PE of all the sub-populations examined had decreased, whereas the addition of 5% of FCS increased the clonogenicity of lin-CD34- cells and to a greater extent lin-CD34-CD38+
(Table II). This difference in clonogenicity may reflect heterogenity within Lin-CD34- cells and demonstrates that the CD38+ subfraction is already co~ llilled to the erythroid lineage suggesting that the CD34neg-SRC resides in the Lin-CD34-CD38- subfraction.
To determine whether the lin-CD34-CD38- cells could be stimulated to proliferate, changes in cell number were recorded between day 0 and 4 of culture with SF or 25% HWEC-CM (Fig.5). The total number of cells decreased by 2 fold at day 4 in SF media. Supplementation of 5% fetal calf serum (FCS) showed no increase in the viability of these cells (data not shown).However, the addition of HWEC-CM to SF media maintained or slightly increased the total cellularity (Fig.5). These results demonstrate that culture conditions that are optimal for Lin-CD34+CD38- cells (11), are unable to support lin-CD34-CD38- cells, while soluble components present in primary HWEC-CM seem to permit their survival.
The effect of culture on the differentiatiation program of lin-CD34-CD38- cells, from individual wells was analyzed by flow cytometry after 2 and 4 days of culture in various conditions (Fig.6). Surprisingly, Lin-CD34-CD38-cells seeded in SF media began to express CD34 which could be enhanced with the addition of 5% serum (Fig.6). In contrast, the majority of cells obtained after 2 or 4 days of culture in the presence of 25% HWEC conditioned medium still maintained the original Lin-CD34-CD38- phenotype. The stimulation of Lin-CD34-CD38- cells to differentiate and produce CD34+CD38- cells suggests that precede the CD34+CD38- population in the hierarchy of human hematopoiesis. Moreover, these results indicate that the lin-CD34-CD38- cells respond to signals present in SF or 5% serum conditions and that HWEC-CM can inhibit this stimulation.
To confirm which fraction contained CD34neg-SRC, both Lin-CD34-CD38- and Lin-CD34-CD38+ cells were transplanted into NOD/SCID mice.
Transplantation with as few as 10,000 or 4,000 Lin-CD34-CD38- cells derived from CB or BM, respectively resulted in engraft (Fig. 7), whereas as many as 180,000 Lin-CD34-CD38+ cells were not capable of repopulation (Fig 7).
These data indicate that the CD34neg-SRC present in the Lin-CD34- fraction are restricted to the CD38- subfraction. However, since an entire CB sample contains only 1 or 2 CD34neg-SRC (e.g. frequency is 1 CD34neg-SRC in 125,000 Lin-CD34- cells and one CB sample contains up to 250,000 Lin-CD34- cells) the losses associated with subselecting based on CD38 expression result in only 9% of samples which repopulate NOD/SCID mice.
As in vitro culture of lin-CD34-CD38- cells caused developmental changes recognized by the appearance of CD34+ cells, we evaluated whether the repopulating activity ofthe Lin-CD34-, lin-CD34-CD38-, and lin-CD34-CD38+ cell fractions was affected by ex vivo culture (Fig 7, panel II). Purifiedfractions from 43 CB and 3 BM samples were cultured for 2 and 4 days in SF, SF supplemented with 5%FCS or 25% HWEC-CM and transplanted at various doses into 136 recipient NOD/SCID mice (Fig. 7, Panel II). A total of 13 out 29 mice transplanted with less than 125,000 to as few as 8,700 Lin-CD34- cells that had been cultured for 4 days were engrafted. A large increase in the number of CD34neg-SRC must have occurred since 125,000 uncultured Lin-CD34- cells were required to repopulate NOD/SCID mice. Similarly, 35% of the mice transplanted with cultured lin-CD34-CD38- cells were engrafted as compared to 9% of mice transplanted with similar doses of uncultured lin-CD34-CD38- ( Fig. 7, Panel I vs II). By contrast, 5,000 and 10,000 lin-CD34+CD38- CB cells, known to contain 10-20 SRC, cultured for 4 days in the presence of serum or the addition of 25% HWEC-CM were unable to engraft NOD/SCID mice, confirming the absence of collt~ ting SRC from this fraction in Lin-CD34-CD38- repopulating cells. HWEC-CM provided significant increases in the proportion of repopulating cells. Consistent with the inability of lin-CD34-CD38+ cells to engraft at day 0, mice transplanted with cultured cells were not engrafted (limit of detection < 0.05%) confinning the absence of repopulating cells within this fraction.
The BM of engrafted mice was analyzed by multiparameter flow cytometry to determine whether cultured Lin-CD34- repopulating cells possessed the same in vivo proliferative and differentiative capacity as uncultured cells. A representative analysis of the BM of a NOD/SCID mouse transplanted with an initial population of 40,000 lin-CD34- cells after 4 days of culture is shown (Fig 8). The BM of this mouse contained 7% human cells as detected by CD45 expression, a human specific pan-leukocyte marker (Fig. 8).
Both B and T-lymphoid cells were present in the murine BM as shown by staining for CDl9, CD20 and CD4, CD3 antigens (Fig. 8D-F). The presence of CD33+, CD14+, CD15+ and CD13+ cells indicated the differentiation potential of lin-CD38-CD38- cells to the myeloid lineages (Fig. 8G-H). The engraftment pattern of mice transplanted with expanded lin-CD34-CD38- cells is similar to that observed with unstimulated purified lin-CD34- cells. The presence of human T-cells is a unique feature of Lin-CD34- engraftment since T-cells have neither been detected in mice transplanted with purified Lin-CD34+CD38- cells before or after ex vivo culture (11, 17).
It is believed that the repopulating cells of the human hematopoietic system are the lin-CD34-CD38- sub-population and short term ex vivo culture of this fraction has been observed to increase the proportion of repopulating cells. The ability of Lin-CD34-CD38- cells to produce CD34+CD38- cells in vitro and in vivo, demonstrates the developmental capacity of these cells and further suggests that this population of cells is more primitive than the CD34 positive fraction. In addition, conditions evaluated here provide the foundationfor future gene transfer and ex-vivo expansion of this novel population and for the identification of factors that stimulate their proliferation and dirreretlliation.
Furthermore, the knowledge that Lin-CD34- cells exist and can repopulate human hematopoietic cells provides a novel therapeutic composition and a method for the treatment of hematopoietic disorders. In particular, the composition and method can be used to treat hematopoietic disorders such as leukemia and for several clinical procedures such as stem cell transplantation, therapy, for combating infection and for cell reconstitution. These cells can also be used to generate CD34+ cells.
Examples The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.
Methods of synthetic chemistry, protein and peptide biochemistry and immunology referred to but not explicitly described in this disclosure and examples are reported in the scientific literature and are well known to those skilled in the art.
Example 1 - Analysis of Lin-CD34- cells found in human hematopoietic tissue Mononuclear cells were isolated from various human hematopoietic cell sources and stained with monoclonal antibodies for CD2, CD3, CD4, CD7, CD13, CD14, CD15, CD16, CDl9, CD20, and glycophorin conjugated to FITC, CD38 conjugated to PE and CD34 conjugated to Cy-5 . Cells gated Rl did not express lineage associated markers (Lin-) and were further analyzed for the expression of CD34 and CD38.
Identification of CD34- cells with no lineage markers in human hematopoietic tissues To determine whether CD34- cells that do not express lineage markers exist in human hematopoietic tissues, human cord blood (CB) cells were first depleted of mononuclear cells that express 15 different lineage-specific antigens from human cord blood. This Lin- population was 99% pure (data not shown). The Lin- cells were then stained with the most widely used CD34 class III monoclonal antibody conjugated to FITC. Flow cytometric analysis showed two distinct populations of CD34+ and CD34- cells (Fig lA panel 1). The CD34- cells (gated Rl, Fig. lA, I) were collected by flow sorting, reanalysis demonstrated their high purity (99%; Fig. lA, II). To confirm that these cells did not express any surface CD34 antigen, the sorted cells were re-stained with two other CD34 Class I and II antibodies that recognize different epitopes of the CD34 molecule (Fig. lA, III and IV). No CD34+ cells were detected, attesting to the high purity and lack of CD34 cell surface expression of this Lin-CD34- population.
To exclude the possibility that CD34 protein was produced in the Lin-CD34- cells but not transported to the cell surface, cytospins of purified Lin-CD34- were permeabilized, stained with CD34 monoclonal antibodies conjugated to FITC and counter stained with DAPI (Fig. lB). No CD34-FITC
signal was detected in the Lin-CD34- cells. The specificity of the procedure is shown by the detection of cell surface and intracellular expression of CD34 on a population of purified Lin-CD34+ cells under similar conditions. Background fluorescence was indicated by staining cells with IgG conjugated to FITC as isotype control (Fig. lB). These results indicate that a population of Lin- cells exist in human CB that do not produce intracellular or cell surface CD34.
Heterogeneity within human Lin-CD34+ cells is well documented and further subdivision for the most p~ ilive cells is typically based on the cell surface markers CD38, c-kit, Thy- 1 and HLA-DR. The expression of these markers on both the Lin-CD34+ and Lin-CD34- cells was compared (Fig. lC).
Lin-CD34- cells displayed a bi-modal distribution of CD38 clearly dividing the population into two fractions in contrast to the high proportion of Lin-CD34+
cells that express CD38 (Fig. lC). Cell surface expression of c-kit was similar between that two populations, while the Lin-CD34- cells are almost exclusively Thy-l- and HLA-DR- (Fig. lC). Both the absence of HLA-DR expression and the presence of Thy-l have been proposed to define more plilllilive subfractions within the Lin-CD34+ population. Therefore, the Lin-CD34-population derived from CB is a distinct population which differs not only in CD34 expression from plilllilive Lin-CD34+ cells but also in phenotypicheterogeneity based on additional markers associated with stem cells.
Example 2- Cell Immunostaining Cord blood cells purified by flow cytometry as done previously (11,12) were cytospun onto slides, permeabilized, and incubated in a BSA solution. The results are shown in Figure lB. (Column I) Lin-CD34+CD38- cells were stained with isotype control antibody conjugated to FITC (Becton Dickinson) and countered stained with DNA binding DAPI as a control for non-specific background flourescence. (Column II) Lin-CD34+CD38- cells and (Column III) Lin-CD34- cells were stained with CD34 monoclonal antibodies followed by DAPI counter stain. All slides were examined using a fluorescent microscope ~ltili~ing the al~propliate filters for DAPI to detect the nucleus ofcells and FITC for the presence of CD34 protein.
Both Lin-CD34- and Lin-CD34+ purified cells were stained with monoclonal antibodies conjugated to flourochromes for CD38 (Becton Dickinson, BD), c-kit (BD), Thy-l (Coulter) and HLA-DR (BD). Stained populations were then washed and analyzed using standard flow cytometric techniques (J. Exp Med, PNAS) followed by the display of histograms using the Cell Quest software program (BD) (n=3).
Example 3 - Cell Engraftment Purified cell populations at the indicated dose were transplanted by tail vein injection into sublethally irradiated mice (375 cGy using a 137Cs g-irradiator) according to a standard protocol as previously described (16,17).
Mice were sacrificed 8 to 12 weeks post transplant and the BM from the femurs, tibiae and iliac crests of each mouse were flushed into IMDM
contaillillg 10% FCS. Mouse BM cells was analyzed using FACS analysis and by southern analysis using genomic DNA extracted by standard protocols in which the level of human cell engraftment was determined by comparing the characteristic 2.7 kb band with those of hl-m~n:mouse DNA mixtures as controls (limit of detection 0.05% human DNA) (16, 17). The results are shown in Figures 2 and 7.
Example 4 - Determin~tion of Hematopoietic Progenitor Activity of Lin-CD34-Lin-CD34+CD38- and Lin-CD34-CD38+
Highly purified cells were plated in clonogenic methlycellulose assays and seeded on MS-5 stroma in order to qua~ ale the CFC and LTC-IC content, respectively. Clonogenic capacity of Lin-CD34- cells was extremely low in comparison to Lin-CD34+CD38- cells (250 CFC vs. 8.9 CFC per 800 cells) (Table I). As many as 10,000 cells needed to be seeded on MS-5 stroma to detect a single LTC-IC within the Lin-CD34- cell fraction, while further purification demonstrated that detection of LTC-IC in the Lin-CD34-CD38-fraction required seeding of at least 2000 cells. By contrast, as few as 10 Lin-CD34+CD38- cells contain an LTC-IC. The Lin-CD34-CD38+ cells were devoid of LTC-IC activity (limit of detection at 10,000 cells) but contained a much higher capacity to form CFC specifically col-llllilled to the erythroid lineage (Table I). The low efficiency to produce myeloid and erythroid colllll-illed progenitors as well as the more ~l;lllilive LTC-IC is similar to observations made with murine Lin-CD34- cells in the same assay systems and suggest functional similarities may exist between the Lin-CD34- cells from these two species (8, 10).
Example 5 - Multilineage Differentiation of Human lin-CD34- cells in NOD/SCID mice after ex vivo Culture A representative mouse was transplanted with 50,000 expanded lin-CD34-CB cells after 2 days of ex-vivo culture in the presence of SF medium supplemented with 5% FCS. Mouse BM was extracted 10 weeks after transplant and analyzed by multiparameter flow cytometry (11, 12). The results are shown in Figure 8.
CD34- Cell Culture with Growth Factors Lin-CD34- cells were incubated in 50 ml of SF condition consisting of IMDM supplemented with 1% BSA (Stem Cell Technologies),5 mg/ml of human insulin (Humulin R from Eli Lilly and Co.),100 mg/ml of human transferrin (Gibco, BRL),10 mg/ml of low density lipoproteins (Sigma Chemical Co.), 10-4 M Beta-mercaptoethanol and growth factors (GF). GF
cocktail was used at final concentrations of 300 ng/ml of SCF (Amgen) and Flt-3 (Immunex), 50 ng/ml of G-CSF (Amgen),10 ng/ml of IL-3 (Amgen) and IL-6 (Amgen). 25% of condition media obtained from a fresh umbilical vein endothelial cell culture in a low percentage of serum ( 10%) and passaged four times, was added in some wells. Cells were cultured in flat bottomed suspension wells of 96-well plates (Nunc), incubated for 2 and 4 days at 37~C
and 5% CO2 and 50 ml of fresh GF cocktail was added to each well every other day.
Example 6 - Effect of ex vivo culture on the number of clonogenic pro~enitors present in the lin- CD34-~ lin-CD34-CD38- and lin-CD34-CD38+ cell fractions An aliquot of 800 to 2,500 lin-CD34-, lin- CD34-CD38- or lin-CD34-CD38+ cells were plated in clonogenic progenitor assays under standard conditions at the initiation of ex vivo cultures (day 0). Cells present after 4 days of culture in the presence of SF or SF supplemented with 5% FCS or 25% of HWEC-CM were plated in the same conditions. The number of CFC/800 input cells were estimated (mean ~ SEM; n-number of experiment). The results are seen in Table II.
Example 7 - The effect of liquid culture on the development and potential differentiation of lin-CD34-CD38- cells Individual wells were analyzed by flow cytometry after 2 and 4 days of culture in various conditions (Fig.6). Lin-CD34-CD38- cells seeded in SF
media began to express CD34 which could be enhanced with the addition of serum (Fig.6). In contrast, the majority of cells obtained after 2 or 4 days of culture in the presence of 25% HWEC conditioned medium still maintained the same phenotype. The acquisition of CD34 demonstrates the differentiation capacity of Lin-CD34-CD38- these cells in-vitro. The production of CD34+CD38- cells suggests that lin-CD34-CD38- cells precede CD34+CD38-population in the hierarchy of human hematopoiesis.
Example 8- Cytokine stimulation of Lin-CD34-CD38- Repopulating ActivityThe percentage of engrafted mice were compared before and after ex-vivo culture of Lin- CD34-, lin- CD34-CD38- and lin-CD34-CD38+ fractions.
Purified fractions from 43 CB and 3 BM samples were cultured for 2 and 4 days in SF, SF supplemented with 5%FCS or 25% HWEC-CM and transplanted at various doses into 136 recipient NOD/SCID mice (Fig. 7). Mice transplanted with cultured lin-CD34-CD38+ cells were not engrafted consistent with the inability of this fraction to engraft at day 0 (limit of detection <
0.05%). Transplanted Lin-CD34- cells we capable of engrafting mice with as few as 8, 700 Lin-CD34- CB cells cultured for 4 days in SF conditions. A total of 13 out 29 mice transplanted with cultured Lin-CD34- cells (cell dose ranging from 8, 700 to 55, 000) were engrafted, indicating that ex-vivo culture significantly increases the frequency of SRC (1 in 30,000 versus 1 in 125,000 atday 0). The increase in SRC activity could be explained by the acquisition of the CD34 antigen due to a differentiation response of a subfraction of lin-CD34-CD38- cells (Fig.6). However, similar increases in SRC was observed after stimulation of Lin-CD34-CD38- cells for 4 days in the presence of SF+25% HWEC-CM which does not cause differentiation into CD34+CD38-cells. As many as 35 % of the mice transplanted with cultured lin-CD34-CD38-cells engraft compared to 9% at day 0 ( Fig. l). In contrast,5,000 and 10,000 lin-CD34+CD38- CB cells CO~ g 10-20 SRC cultured for 4 days in the presence of serum or the addition of 25% HWEC-CM were unable to engraft NOD/SCID confirming the absence of collt~ ting SRC from this fraction in Lin-CD34-CD38- repopulating cells (data not shown). The fact that repopulating cells within lin-CD34+CD38- and lin-CD34-CD38- fractions respond differently to the presence of 25%HWEC-CM, and that no phenotypic change of lin-CD34-CD38- cells occurred after 4 days of culture suggests that elements other than the acquisition of CD34 antigen must play a role in the increase of repopulating activity of Lin-CD34-CD38- cells.
Table OneHematopoietic Progenitor Activity of Lin-CD34-~ Lin-CD34-CD38-and Lin-CD34-CD38+
Phenotype of purified CFC/800 cellsFrequency of population LTC-IC
CD34-Lin- 8.9+3 <1/10,000 (n=2) (n=4) 5,501_1640(n=2) CD34-Lin-CD38- 2.7_2 ~1/2,000 (n=36) (n=4) CD34-Lin-CD38+ 77+31 <1/10,000 (n=10) (n=4) CD34+CD38- 250_46 >1/10 (n=4) (n=4) Table Two Effect of ex vivo culture on the number of clonogenic progenitors present in the lin- CD34-, lin-CD34-CD38- and lin-CD34-CD38+ cell fractions.
Phenotype of purified Da~l 0 After in vitro stimulation population CD34-Lin- 8.9 _3 Serum Free 10 _0.9 (n=4) (n=4) 5% Serum 76 _21 (n=2) CD34-Lin-CD38- 2.7_ 2 5% Serum 6.4 _4.5 (n=7) (n=36) Serum Free 1.2 _1 (n=9) 25% HWEC 0.5% _0.5 (n=7) CD34-Lin-CD38+ 77 _31 Serum Free 20 _10 (n=4) (n=10) 5% Serum 134_ 41 (n=4) *CFC/800 Input Cells References l.Till, J.E., McCulloch, E.A. 1980. Hemopoietic stem cell differentiation. Biochim.Biophys. Acta. 605(4):431-59.
Background of the Invention The m~mm~ n hematopoietic system consists of a heterogeneous array of cells ranging from large numbers of dirrelellliated cells with defined function to rare pluripotent stem cells with extensive developmental and proliferative potential (1, 2, 3). The defining feature of a stem cell is its ability to repopulate the hematopoietic system of a recipient after transplantation. Stem cells are playing an increasingly important role in clinical and commercial applications, as the role of the stem cells in transplantation widens. Identification and purification of stem cells is essential both to determine the cellular and molecular factors that govern stem cell development and for the application of clinical procedures including stem cell transplant and gene therapy.
Cell surface expression of CD34 has become the distinguishing feature used to isolate stem cells because CD34 is downregulated as cells differentiate into more abundant mature cells (4). However, CD34 does not mark stem cells exclusively since only 1% of bone marrow (BM) cells are CD34+ and include clonogenic progenitors that are not able to repopulate after transplantation.
Therefore other markers such as Thy-l can be combined with CD34 to positively select for a more enriched cell fraction (5, 6, 7). Conversely, the CD34+ fraction can be enriched by elimin~ting cells that express markers that are expressed on non-repopulating cells (e.g.lineage antigens). For these reasons, all current clinical and experimental protocols ~tili~ing human stem cells including ex vivo culture, gene therapy, and bone marrow transplantation, focus on CD34+ cells.
There have been reports (8, 9, 10) of murine CD34- hematopoietic stem cells which are capable of long term repopulation. For human hematopoietic stem cells, however, CD34+ cells have been regarded as a stem cell marker without exception.
Brief Description of the Drawings The present invention will be further understood from the following description with reference to the Figures, in which:
Figure lA demonstrates cell surface expression of CD34 on cord blood cells depleted for lineage markers (Lin-). (Panel I) Lin- cells were purified as in (11, 12) and stained with a class III monoclonal antibody for CD34 (581) conjugated to FITC (Becken Dickenson, BD). Cells residing in R1 were considered CD34 negative (CD34-). (Panel II) CD34- cells were purified using standard cell sorting techniques and re-analyzed using the same CD34-581 antibody. (Panel III and Panel IV) Purified R1 cells were stained and re-analyzed using a class I monoclonal antibody for CD34 (Immun-133) (Coulter) and a class II monoclonal antibody for CD34 (Q-Bend-10) (Becton Dickinson, BD).
Figure lB demonstrates the immunostaining of purified populations for CD34 expression. Representative cells are shown from a total of 25-75 cells examined for each treatment (n=2).
Figure lC demonstrates a comparison of cell surface markers between Lin-CD34- versus Lin-CD34+ cord blood cells commonly used to further subdivide p~ ive cells.
Figure 2 demonstrates the level of human cell engraftment in NOD/SCID mice transplanted with highly purified Lin-CD34- cells at various doses.
Figure 3 demonstrates the multilineage differentiation of human Lin-CD34- cells in NOD/SCID mice. Bone marrow from a highly engrafted mouse transplanted with 120,000 Lin-CD34- cord blood cells was stained with various human-specific monoclonal antibodies and analyzed by flow cytometry.
Approximately 106 mononuclear cells collected from mouse BM was prepared as shown previously for multilineage analysis (12). (A) Cells with medium to high forward scatter (region Rl) were gated and further analyzed. (B) Histogram of CD45 (pan-leukocyte marker) expression indicating that 2.5% of the cells present in the murine bone marrow are h~ n, gated R2. All further lineage markers was done on cells within gate R2 (CD45+). (C) Isotype control for non-specific IgG staining of PE and FITC fluorescence. (D) Expression of myeloid marker CD33 and granulocyte marker CD15; (E) pan-B cell markers CDl9 and CD20; (F) CD38 and the imm~h1re hematopoietic marker CD34; (G
and H) T-cell markers CD2, CD3, CD4 and CD8.
Figure 4 is a frequency analysis of Lin-CD34- cells found in human hematopoietic tissue. (A) Column I; Fetal liver collected from 8 week old human fetus, n=3 Column II; Fetal blood aspirated from 19 week old fetus, n=l Column III; Cord blood collected from placenta at time of birth, n=3. (B) Column I; Normal Adult Bone marrow, n=2 Column II; Bone marrow from a normal adult donor after 5 days of G-CSF ~lmini~tration, n=2 Column III;
Peripherial blood collected from a normal adult donor after 5 days of G-CSF
a-lministration, n=2.
Figure 5 demonstrates the percentage of the input cells after in-vitro culture of lin- CD34- cells. Purified cells were counted and seeded (250-2000) in wells cont~ining serum free media (SF) ( see examples) (solid bar) or SF
supplemented with 25% HWEC- conditioned media ( shaded bar). Cells were counted each day and the percentage of the input cells was calculated ( n=3).
Figure 6 is an analysis of CD34 and CD38 expression of highly purified populations after in vitro culture. A representative experiment (n=4) of CD34 and CD38 cell surface expression performed on initially purified lin-CD34-CD38- cells, and purified cells after 2 and 4 days of culture in SF, SF
supplemented with 5% FCS or 25% HWEC-CM. The entire contents of individual wells was collected at 2 and 4 days (5000-10, 000 cells), stained with monoclonal antibodies CD34 and CD38 directly conjugated to FITC and PE
respectively (Beckton Dickinson, BD). Stained populations were then washed and analyzed using standard flow cytometric techniques as done previously (11,12) followed by the display of histograms using the Cell Quest software program (BD) Figure 7 demonstrates the capacity of expanded lin-CD34-, lin-CD38-CD38- or lin-CD34-CD38+ cell fractions to engraft NOD/SCID mice.
Figure 8 demonstrates the multilineage differentiation of human lin-CD34- cells in NOD/SCID mice after ex vivo culture. (A) Histogram of CD45 (human-specific pan-leukocyte marker) expression indicating that 7% of the cells present in the murine BM are hllm~n (B) Forward and Side scatter of the CD45 human cells. Subsequent analysis of lineage markers was done on CD45+ cells within gate Rl (lymphoid and blast cells) or R2 (myeloid cells) gates. (C) Analysis for the presence of imm~tllre cells using the CD34 and CD38 markers. ( D) Analysis for the presence of human B cell lineage cells using CDl9 and CD20 markers. (E-F) Analysis for the presence of human T
lymphocytes using the panel of T cell markers: CD2, CD3, CD4 and CD8. (G-H) Analysis for the presence of myeloid cells using CD33, CD14, CD15 and CD13 markers.
Description of the Invention The present inventors have identified and isolated a population of human hematopoietic stem cells which do not express CD34 (CD34-), CD38 (CD38-) or lineage specific markers (Lin-) and which are able to generate by proliferation and dirrelellLiation multiple lineages of the human hematopoietic system, as evidenced by their ability to produce multilineage human engraftment of immune-deficient NOD/SCID mice after transplantation.
Moreover, the repopulative capacity and the dirrerellliative capacity of the lin-CD34-CD38- cells can be stimulated by in vitro culture of these cells.
In initial studies, a population of CD34-Lin- cells were isolated from human fetal liver, cord blood, bone marrow and mobilized peripheral blood and bone marrow. These cells do not express any lineage specific markers and are also devoid of HLA-DR and Thy-l, two other markers associated with primitive cells, but are heterogeneous for CD38 and c-kit expression. The NOD/SCID transplantation studies show that the repopulating cells are exclusively present in the CD38- fraction and not the CD38+ fraction and these cells are characterized as Lin-CD34-CD38-. These novel Lin-CD34- cells are most abundant in fetal tissues, with progressive reductions in cord blood (CB) and adult bone marrow (BM) or mobilized peripheral blood (M-PB).
Transplantation of the Lin- CD34- cells in NOD/SCID mice engrafted myeloid, lymphoid and erythroid lineages, which demonstrates that Lin- CD34- cells repopulate cells of the hematopoietic system. The results also show that Lin-CD34-CD38- cells are developmentally earlier than CD34+ cells and can produce CD34+ cells following in vitro culture or after repopulation. Thus, these cells appear to be at the top of the heirarchical structure of human stem cells.
The identification of this novel stem cell within the hierarchy of human hematopoiesis has important implications for understanding the origin of hematopoietic diseases such as leukemia and for clinical procedures such as stem cell transplantation and gene therapy. This novel stem cell can be important for the treatment of infection, for the reconstitution of deficient ormissing cell populations as for example for cancer patients after myeloablative therapy and for the treatment of genetic abnormalities and defects.
The present invention provides a therapeutic composition and a method for the treatment of hematopoietic disorders. In particular, the composition andmethod can be used to treat hematopoietic disorders such as leukemia and for several clinical procedures such as stem cell transplantation, therapy, gene therapy, for combating infection and for cell reconstitution.
In accordance with an aspect of the present invention there is provided an essentially pure population of human hematopoietic cells characterized as Lin- CD34-.
In accordance with another aspect of the present invention there is provided an essentially pure population of human hematopoietic cells characterized as Lin-CD34-CD38-.
In accordance with another aspect of the present invention there is provided a method for providing an essentially pure population of human hematopoietic cells characterized as Lin- CD34-.
In accordance with another aspect of the invention there is provided a method for the treatment of hematopoietic disorders such as leukemia comprising the use of human hematopoietic cells characterized as Lin- CD34-.
In accordance with another aspect of the present invention is a method for the ex vivo generation of human hematopoietic cells using Lin-CD34- cells.
In accordance with another aspect of the present invention is a gene therapy method for providing genetically altered human hematopoietic cells characterized as Lin- CD34- to a patient.
In accordance with another aspect of the present invention is a method for reconstituting deficient or missing human hematopoietic cell populations comprising the use of Lin- CD34- cells.
In accordance with another aspect of the present invention is a method for transplanting human hematopoietic cell populations to a patient comprising the use of Lin- CD34- cells.
In accordance with another aspect of the present invention is a method for combatting infection in a patient comprising ~dmini~tering an effective amount of human hematopoietic cells characterized as Lin- CD34-.
In accordance with yet a further aspect of the present invention there is provided a method utilizing human hematopoietic cells characterized as Lin-CD34- for the production of CD34+ human hematopoietic cells.
In accordance with a further aspect of the present invention is a method for increasing the repopulating capacity of human hematopoietic cells characterized as Lin-CD34- by culturing such cells in vitro for several days.
In accordance with a further aspect of the present invention is a method for screening candidate compounds affecting proliferation or dirr~relltiation ofstem cells characterized as Lin-CD34- and Lin-CD34-CD38-.
It was determined using CD34 class III fluorescent monoclonal antibodies that CD34- cells that do not express lineage markers exist in human hematopoietic tissues (Fig. lA). The possibility that CD34 protein was produced in the Lin-CD34- cells but not transported to the cell surface, was excluded using permeabilized, stained cytospins of purified Lin-CD34- that were further conjugated with fluourescent monoclonal antibodies. This confirmed that no CD34-FITC signal was detected in the Lin-CD34- cells (Fig.
lB) Heterogeneity within human Lin-CD34+ cells is well documented and further subdivision for the most p~ ive cells is typically based on the cell surface markers CD38, c-kit, Thy-1 and HLA-DR. The expression of these markers on both the Lin-CD34+ and Lin-CD34- cells was compared (Fig. lC).
Lin-CD34- cells displayed a bi-modal distribution of CD38 clearly dividing the population into two fractions in contrast to the high proportion of Lin-CD34+
cells that express CD38 (Fig. lC). Cell surface expression of c-kit was similar between that two populations, while the Lin-CD34- cells are almost exclusively Thy-1- and HLA-DR- (Fig. lC). Both the absence of HLA-DR expression and the presence of Thy-1 have been proposed to define more ~lhlliLive subfractions within the Lin-CD34+ population. Therefore, the Lin-CD34-population derived from CB is a distinct population which differs not only in CD34 expression from plilllilive Lin-CD34+ cells but also in phenotypic heterogeneity based on additional markers associated with stem cells.
Using clonogenic methylcellulose assays, it was determined whether Lin-CD34-, Lin-CD34-CD38- and Lin-CD34-CD38+ cells possessed any hematopoietic progenitor activity by comparing the CFC and LTC-IC content, respectively. Clonogenic capacity of Lin-CD34- cells was extremely low in comparison to Lin-CD34+CD38- cells (Table I). As many as 10,000 cells needed to be seeded on MS-5 stroma to detect a single LTC-IC within the Lin-CD34- cell fraction, while further purification demonstrated that detection of LTC-IC in the Lin-CD34-CD38- fraction required seeding of at least 2000 cells. By contrast, as few as 10 Lin-CD34+CD38- cells contain an LTC-IC. The Lin-CD34-CD38+ cells were devoid of LTC-IC activity (limit of detection at 10,000 cells) but contained a much higher capacity to form CFC specifically commilled to the erythroid lineage (Table I). The low efficiency to produce myeloid and erythroid colnlllilled progenitors as well as the more plilllilive LTC-IC is similar to observations made with murine Lin-CD34- cells in the same assay systems and suggest functional similarities may exist between the Lin-CD34- cells from these two species (8,10).
The only conclusive method to detect stem cells is to determine their ability to repopulate recipient hosts (1, 2,3). A repopulation assay was developed for primitive human cells based on their ability to initiate multilineage human engraftment in immune-deficient NOD/SCID mice (13, 14). Based on cell purification and gene marking, the cell capable of repopulating NOD/SCID mice (termed the SCID-Repopulating Cell, SRC) was established as distinct from and more plilnilive than the majority of progenitors detected in in vitro assays (15). Only the Lin-CD34+CD38- cells and not the Lin-CD34+CD38+ cells could give rise to multilineage engraftment (15, 12).
Moreover, transplantation of as many as 10-6 Lin+CD34- cells also did not engraft (12). To determine whether highly purified Lin-CD34- cells had SRC
activity and to determine the frequency of any repopulating cells, Lin-CD34-cells were transplanted at varying cell doses into NOD/SCID mice using our standard protocols, and BM was analyzed for the presence of human cells after 8-12 weeks. The level of human cell engraftment in 23 NOD/SCID mice was quantitated by FACS and DNA analysis for the presence of human cells and results are sllmm~ri~ed in Fig. 2. A large proportion of transplanted mice were engrafted with human cells indicating that the Lin-CD34- cells were able to repopulate NOD/SCID mice. We have termed this cell the CD34NEG-SCID
Repopulating Cell (CD34NEG-SRC). The frequency was 1 CD34NEG-SRC in 125,000 Lin-CD34- cells. The differentiative and proliferative capacity of the CD34NEG-SRC cell was assessed by flow cytometric analysis. A
representative engrafted NOD/SCID mouse 10 weeks after the transplant of 120,000 Lin-CD34- cells is shown in Fig.3. Cells with medium to high forward scatter (region R1, Fig. 3A) were gated and further analyzed based on CD45 expression, a human specific pan-leukocyte marker (Fig.3B). The isotype control is shown in Fig.3C. The BM of this mouse contained 2.5% CD45+
human cells (Fig.3B) or at least 106 total human cells indicating that the Lin-CD34- cells have extensive proliferative capacity. Granulocytes (CD15+) were present among myeloid cells (CD33+) (Fig.3D). B-lymphoid cells were also present in the murine BM as shown by staining for CD19 and CD20 (Fig. 3E).
Interestingly, human T-cells expressing both CD2 and CD3 (Fig. 3G), along with CD4 and CD8 positive cells (Fig.3H) were also identified. NOD/SCID
mice transplanted with highly purified p~ ilive Lin-CD34+CD38- cells never gave rise to engraftment contailling T-cells demonstrating the unique in vivo repopulation behavior of the Lin-CD34- cells. In addition to multilineage engraftment, imm~ re CD34+ and CD34+CD38- cells were detected (Fig.3F).
It was concluded that the Lin-CD34- cells have the ability to repopulate NOD/SCID mice and differentiate in vivo into multiple lineages of myeloid and lymphoid cells. The production of CD34+CD38- and CD34+CD38+ cells in vivo suggests that Lin-CD34- cells are developmentally earlier than CD34+
cells in the hierarchy of human hematopoiesis.
There is evidence that the frequency of plilllilive cells changes during ontogeny with the highest proportions seen in the fetus. A variety of fetal, neonatal, and adult sources of human hematopoietic tissue were analyzed in an attempt to identify and quantify the Lin-CD34- population. The results indicate (Fig.4A,4B) that Lin-CD34- cells are produced early in human ontogeny and can persist throughout adult life and that the mechanisms that operate during the mobilization of CD34+ cells with G-CSF also affect Lin-CD34- cells.
This data provides the first identification of a novel human hematopoietic stem cell that does not express CD34 or lineage-specific markers. As d~te~ ed by all available monoclonal antibodies, this population is not only distinct by the absence of CD34, but also by the lack of HLA-DR
and Thy- 1 markers. In addition to these phentotypic differences, several lines of evidence functionally distinguish these two stem cell populations. While the Lin-CD34- cells have limited hematopoietic activity in vitro, Lin-CD34+CD38-cells are highly clonogenic based on their ability to produce CFC and LTC-IC.
Although both stem cell fractions are capable of repopulation, the presence of T-cells within the multilineage engraftment is a unique characteristic of Lin-CD34- transplantation since we have never detected human T cells in mice transplanted with Lin-CD34+CD38- cells (n=25). Therefore it is unlikely that NOD/SCID repopulation is derived from the co~ "~ tion of Lin-CD34+CD38-cells. Furthermore, based on LTC-IC frequency a millill~Ulll of 1 LTC-IC resides within 10 highly purified Lin-CD34+CD38- cells. In contrast to flow cytometry, in which we have determined that the Lin-CD34- population is 99% (or in some cases 100%) pure, the LTC-IC assay allows us to detect a smaller number of col~ ting Lin-CD34+CD38- cells. Using this assay, only a single LTC-IC could be detected in as many as 10 000 CD34-Lin- cells.
If this LTC-IC activity came from a Lin-CD34+CD38- cell, a maximum of 10 Lin-CD34+CD38- cells could be contained in the Lin-CD34- purified fraction (0.1% cont~min~tion). In addition, repopulated mice have not been observed when only 10 Lin-CD34+CD38- cells were transplanted (12). Since the frequency of SRC derived from Lin-CD34- cells is 1 in 125 000, a maximum of 125 Lin-CD34+CD38- cells could have been transplanted, again less than the number needed to repopulate CD34+CD38- cells.
The in vivo differentiation of human Lin-CD34- cells into CD34+ and lineage positive cells suggests that the Lin-CD34- cells preceed CD34+ cells in the hierarchy of human hematopoiesis.
The identification of these novel repopulating cells, termed CD34neg-SCID repopulating cells (CD34neg-SRC), provides an OppOl lUllily to examine the differentiation and proliferation potential of these cells and to establish their relationship to other cells within the human stem cell hierarchy. However, in vivo repopulation is a complex system m~king it difficult to establish hierarchical relationships. Moreover, it was found that there is 1 CD34neg-SRC
in 105 Lin-CD34- cells. While this may reflect the true frequency of primitive cells capable of repopulation, it it also possible that other plilllilive cells exist within this cell fraction. The inability to demonstrate activity of these plilllliive cells could be due to limitations in currently available hematopoietic assays.
For example, these cells could possess the developmental potential to repopulate NOD/SCID mice but are unable to under the current transplant conditions (e.g. some repopulating cells have much slower kinetics, or may lack expression of a particular adhesion molecule, etc) and therefore are never detected. In the murine system, some stem cells will only repopulate long-term but not short-term and vice versa (1,2,3). Therefore, ex vivo cultures are better suited for studies that examine the proliferative and differentiative potential of primitive cells, particularly in response to cytokine stimulation, because the conditions are well defined and it is straightforward to (measure developmental changes). Many studies based on ex vivo culture have demonstrated that there is heterogeneity within the CD34+ cell fraction (4). Subfractionation of the CD34+ cells on the basis of Thyl, CD38, and HLA-DR expression together with in vitro clonogenic and LTC-IC assays have demonstrated the progenitor-progeny relationship of the various cell types that make up the stem cell hierarchy (4, 14). The availability of the SRC assay to detect even earlier celltypes has added more information about the org~ni7~tion of cells within this hierarchy (14). Moreover, it was demonstrated that the SRC (derived from CD34+CD38- cells) can be expanded for 4 days in serum-free cultures without inducing their differentiation. However, all SRC are lost within an additional 4days of culture conco-ll-llilallt to the appearance of more differentiated CD38+cells (11). At the same time, both colony-forming cells (CFC) and long-term culture initiating cells (LTC-IC) could be greatly expanded during 8 days of culture demonstrating that the majority of the SRC are a distinct population (11), but may be closely related to ELTC-IC (18). Thus, in vitro culture systemscan be used to identify very fine transitions in the developmental program by combining both flow cytometry and functional CFC, LTC-IC and SRC assays.
It is now demonstrated hel~wilh that ex vivo culture of Lin-CD34- cells can induce the appearance of CD34+ cells and can increase the proportion of Lin-CD34- cells that have SRC activity. These studies provide new insight into the developmental program of human hematopoietic stem cells.
Our earlier studies demonstrated that the Lin-CD34- cell fraction expressed a bimodal distribution of CD38 allowing for further purification into CD38- and CD38+ subpopulations (12). To determine whether the Lin-CD34-, Lin-CD34-CD38-, Lin-CD34-CD38+ cells could be induced to proliferate andlor differentiate, these cells were cultured in defined serum-free (SF) conditions that have been previously shown could expand Lin34+38- cells and maintain and modestly increase POS CD34 POS-SRC(ll, 17). Cells were plated in methlycellulose assays at day 0, and after day 4 of liquid culture (Table II) to determine the effect of cytokine stimulation on the clonogenic progenitors present in lin-CD34-, lin-CD34-CD38- and lin-CD34-CD38+ cell fractions. Both the lin-CD34- and more purified lin-CD34-CD38- cells have a low plating efficiency (PE), l in 89 and 1 in 297 CFC respectively, whereas a higher PE of lin-CD34-CD38+ cells 1 in 10.4 cells was seen. Interestingly, the clonogenic capacity of the lin-CD34-CD38+ cells is restricted to the erythroid lineage. After 4 days of culture in SF media or SF media supplemented with the addition of 25% conditioned medium obtained from primary human umbilical vein endothelial cells (HWEC-CM), the PE of all the sub-populations examined had decreased, whereas the addition of 5% of FCS increased the clonogenicity of lin-CD34- cells and to a greater extent lin-CD34-CD38+
(Table II). This difference in clonogenicity may reflect heterogenity within Lin-CD34- cells and demonstrates that the CD38+ subfraction is already co~ llilled to the erythroid lineage suggesting that the CD34neg-SRC resides in the Lin-CD34-CD38- subfraction.
To determine whether the lin-CD34-CD38- cells could be stimulated to proliferate, changes in cell number were recorded between day 0 and 4 of culture with SF or 25% HWEC-CM (Fig.5). The total number of cells decreased by 2 fold at day 4 in SF media. Supplementation of 5% fetal calf serum (FCS) showed no increase in the viability of these cells (data not shown).However, the addition of HWEC-CM to SF media maintained or slightly increased the total cellularity (Fig.5). These results demonstrate that culture conditions that are optimal for Lin-CD34+CD38- cells (11), are unable to support lin-CD34-CD38- cells, while soluble components present in primary HWEC-CM seem to permit their survival.
The effect of culture on the differentiatiation program of lin-CD34-CD38- cells, from individual wells was analyzed by flow cytometry after 2 and 4 days of culture in various conditions (Fig.6). Surprisingly, Lin-CD34-CD38-cells seeded in SF media began to express CD34 which could be enhanced with the addition of 5% serum (Fig.6). In contrast, the majority of cells obtained after 2 or 4 days of culture in the presence of 25% HWEC conditioned medium still maintained the original Lin-CD34-CD38- phenotype. The stimulation of Lin-CD34-CD38- cells to differentiate and produce CD34+CD38- cells suggests that precede the CD34+CD38- population in the hierarchy of human hematopoiesis. Moreover, these results indicate that the lin-CD34-CD38- cells respond to signals present in SF or 5% serum conditions and that HWEC-CM can inhibit this stimulation.
To confirm which fraction contained CD34neg-SRC, both Lin-CD34-CD38- and Lin-CD34-CD38+ cells were transplanted into NOD/SCID mice.
Transplantation with as few as 10,000 or 4,000 Lin-CD34-CD38- cells derived from CB or BM, respectively resulted in engraft (Fig. 7), whereas as many as 180,000 Lin-CD34-CD38+ cells were not capable of repopulation (Fig 7).
These data indicate that the CD34neg-SRC present in the Lin-CD34- fraction are restricted to the CD38- subfraction. However, since an entire CB sample contains only 1 or 2 CD34neg-SRC (e.g. frequency is 1 CD34neg-SRC in 125,000 Lin-CD34- cells and one CB sample contains up to 250,000 Lin-CD34- cells) the losses associated with subselecting based on CD38 expression result in only 9% of samples which repopulate NOD/SCID mice.
As in vitro culture of lin-CD34-CD38- cells caused developmental changes recognized by the appearance of CD34+ cells, we evaluated whether the repopulating activity ofthe Lin-CD34-, lin-CD34-CD38-, and lin-CD34-CD38+ cell fractions was affected by ex vivo culture (Fig 7, panel II). Purifiedfractions from 43 CB and 3 BM samples were cultured for 2 and 4 days in SF, SF supplemented with 5%FCS or 25% HWEC-CM and transplanted at various doses into 136 recipient NOD/SCID mice (Fig. 7, Panel II). A total of 13 out 29 mice transplanted with less than 125,000 to as few as 8,700 Lin-CD34- cells that had been cultured for 4 days were engrafted. A large increase in the number of CD34neg-SRC must have occurred since 125,000 uncultured Lin-CD34- cells were required to repopulate NOD/SCID mice. Similarly, 35% of the mice transplanted with cultured lin-CD34-CD38- cells were engrafted as compared to 9% of mice transplanted with similar doses of uncultured lin-CD34-CD38- ( Fig. 7, Panel I vs II). By contrast, 5,000 and 10,000 lin-CD34+CD38- CB cells, known to contain 10-20 SRC, cultured for 4 days in the presence of serum or the addition of 25% HWEC-CM were unable to engraft NOD/SCID mice, confirming the absence of collt~ ting SRC from this fraction in Lin-CD34-CD38- repopulating cells. HWEC-CM provided significant increases in the proportion of repopulating cells. Consistent with the inability of lin-CD34-CD38+ cells to engraft at day 0, mice transplanted with cultured cells were not engrafted (limit of detection < 0.05%) confinning the absence of repopulating cells within this fraction.
The BM of engrafted mice was analyzed by multiparameter flow cytometry to determine whether cultured Lin-CD34- repopulating cells possessed the same in vivo proliferative and differentiative capacity as uncultured cells. A representative analysis of the BM of a NOD/SCID mouse transplanted with an initial population of 40,000 lin-CD34- cells after 4 days of culture is shown (Fig 8). The BM of this mouse contained 7% human cells as detected by CD45 expression, a human specific pan-leukocyte marker (Fig. 8).
Both B and T-lymphoid cells were present in the murine BM as shown by staining for CDl9, CD20 and CD4, CD3 antigens (Fig. 8D-F). The presence of CD33+, CD14+, CD15+ and CD13+ cells indicated the differentiation potential of lin-CD38-CD38- cells to the myeloid lineages (Fig. 8G-H). The engraftment pattern of mice transplanted with expanded lin-CD34-CD38- cells is similar to that observed with unstimulated purified lin-CD34- cells. The presence of human T-cells is a unique feature of Lin-CD34- engraftment since T-cells have neither been detected in mice transplanted with purified Lin-CD34+CD38- cells before or after ex vivo culture (11, 17).
It is believed that the repopulating cells of the human hematopoietic system are the lin-CD34-CD38- sub-population and short term ex vivo culture of this fraction has been observed to increase the proportion of repopulating cells. The ability of Lin-CD34-CD38- cells to produce CD34+CD38- cells in vitro and in vivo, demonstrates the developmental capacity of these cells and further suggests that this population of cells is more primitive than the CD34 positive fraction. In addition, conditions evaluated here provide the foundationfor future gene transfer and ex-vivo expansion of this novel population and for the identification of factors that stimulate their proliferation and dirreretlliation.
Furthermore, the knowledge that Lin-CD34- cells exist and can repopulate human hematopoietic cells provides a novel therapeutic composition and a method for the treatment of hematopoietic disorders. In particular, the composition and method can be used to treat hematopoietic disorders such as leukemia and for several clinical procedures such as stem cell transplantation, therapy, for combating infection and for cell reconstitution. These cells can also be used to generate CD34+ cells.
Examples The examples are described for the purposes of illustration and are not intended to limit the scope of the invention.
Methods of synthetic chemistry, protein and peptide biochemistry and immunology referred to but not explicitly described in this disclosure and examples are reported in the scientific literature and are well known to those skilled in the art.
Example 1 - Analysis of Lin-CD34- cells found in human hematopoietic tissue Mononuclear cells were isolated from various human hematopoietic cell sources and stained with monoclonal antibodies for CD2, CD3, CD4, CD7, CD13, CD14, CD15, CD16, CDl9, CD20, and glycophorin conjugated to FITC, CD38 conjugated to PE and CD34 conjugated to Cy-5 . Cells gated Rl did not express lineage associated markers (Lin-) and were further analyzed for the expression of CD34 and CD38.
Identification of CD34- cells with no lineage markers in human hematopoietic tissues To determine whether CD34- cells that do not express lineage markers exist in human hematopoietic tissues, human cord blood (CB) cells were first depleted of mononuclear cells that express 15 different lineage-specific antigens from human cord blood. This Lin- population was 99% pure (data not shown). The Lin- cells were then stained with the most widely used CD34 class III monoclonal antibody conjugated to FITC. Flow cytometric analysis showed two distinct populations of CD34+ and CD34- cells (Fig lA panel 1). The CD34- cells (gated Rl, Fig. lA, I) were collected by flow sorting, reanalysis demonstrated their high purity (99%; Fig. lA, II). To confirm that these cells did not express any surface CD34 antigen, the sorted cells were re-stained with two other CD34 Class I and II antibodies that recognize different epitopes of the CD34 molecule (Fig. lA, III and IV). No CD34+ cells were detected, attesting to the high purity and lack of CD34 cell surface expression of this Lin-CD34- population.
To exclude the possibility that CD34 protein was produced in the Lin-CD34- cells but not transported to the cell surface, cytospins of purified Lin-CD34- were permeabilized, stained with CD34 monoclonal antibodies conjugated to FITC and counter stained with DAPI (Fig. lB). No CD34-FITC
signal was detected in the Lin-CD34- cells. The specificity of the procedure is shown by the detection of cell surface and intracellular expression of CD34 on a population of purified Lin-CD34+ cells under similar conditions. Background fluorescence was indicated by staining cells with IgG conjugated to FITC as isotype control (Fig. lB). These results indicate that a population of Lin- cells exist in human CB that do not produce intracellular or cell surface CD34.
Heterogeneity within human Lin-CD34+ cells is well documented and further subdivision for the most p~ ilive cells is typically based on the cell surface markers CD38, c-kit, Thy- 1 and HLA-DR. The expression of these markers on both the Lin-CD34+ and Lin-CD34- cells was compared (Fig. lC).
Lin-CD34- cells displayed a bi-modal distribution of CD38 clearly dividing the population into two fractions in contrast to the high proportion of Lin-CD34+
cells that express CD38 (Fig. lC). Cell surface expression of c-kit was similar between that two populations, while the Lin-CD34- cells are almost exclusively Thy-l- and HLA-DR- (Fig. lC). Both the absence of HLA-DR expression and the presence of Thy-l have been proposed to define more plilllilive subfractions within the Lin-CD34+ population. Therefore, the Lin-CD34-population derived from CB is a distinct population which differs not only in CD34 expression from plilllilive Lin-CD34+ cells but also in phenotypicheterogeneity based on additional markers associated with stem cells.
Example 2- Cell Immunostaining Cord blood cells purified by flow cytometry as done previously (11,12) were cytospun onto slides, permeabilized, and incubated in a BSA solution. The results are shown in Figure lB. (Column I) Lin-CD34+CD38- cells were stained with isotype control antibody conjugated to FITC (Becton Dickinson) and countered stained with DNA binding DAPI as a control for non-specific background flourescence. (Column II) Lin-CD34+CD38- cells and (Column III) Lin-CD34- cells were stained with CD34 monoclonal antibodies followed by DAPI counter stain. All slides were examined using a fluorescent microscope ~ltili~ing the al~propliate filters for DAPI to detect the nucleus ofcells and FITC for the presence of CD34 protein.
Both Lin-CD34- and Lin-CD34+ purified cells were stained with monoclonal antibodies conjugated to flourochromes for CD38 (Becton Dickinson, BD), c-kit (BD), Thy-l (Coulter) and HLA-DR (BD). Stained populations were then washed and analyzed using standard flow cytometric techniques (J. Exp Med, PNAS) followed by the display of histograms using the Cell Quest software program (BD) (n=3).
Example 3 - Cell Engraftment Purified cell populations at the indicated dose were transplanted by tail vein injection into sublethally irradiated mice (375 cGy using a 137Cs g-irradiator) according to a standard protocol as previously described (16,17).
Mice were sacrificed 8 to 12 weeks post transplant and the BM from the femurs, tibiae and iliac crests of each mouse were flushed into IMDM
contaillillg 10% FCS. Mouse BM cells was analyzed using FACS analysis and by southern analysis using genomic DNA extracted by standard protocols in which the level of human cell engraftment was determined by comparing the characteristic 2.7 kb band with those of hl-m~n:mouse DNA mixtures as controls (limit of detection 0.05% human DNA) (16, 17). The results are shown in Figures 2 and 7.
Example 4 - Determin~tion of Hematopoietic Progenitor Activity of Lin-CD34-Lin-CD34+CD38- and Lin-CD34-CD38+
Highly purified cells were plated in clonogenic methlycellulose assays and seeded on MS-5 stroma in order to qua~ ale the CFC and LTC-IC content, respectively. Clonogenic capacity of Lin-CD34- cells was extremely low in comparison to Lin-CD34+CD38- cells (250 CFC vs. 8.9 CFC per 800 cells) (Table I). As many as 10,000 cells needed to be seeded on MS-5 stroma to detect a single LTC-IC within the Lin-CD34- cell fraction, while further purification demonstrated that detection of LTC-IC in the Lin-CD34-CD38-fraction required seeding of at least 2000 cells. By contrast, as few as 10 Lin-CD34+CD38- cells contain an LTC-IC. The Lin-CD34-CD38+ cells were devoid of LTC-IC activity (limit of detection at 10,000 cells) but contained a much higher capacity to form CFC specifically col-llllilled to the erythroid lineage (Table I). The low efficiency to produce myeloid and erythroid colllll-illed progenitors as well as the more ~l;lllilive LTC-IC is similar to observations made with murine Lin-CD34- cells in the same assay systems and suggest functional similarities may exist between the Lin-CD34- cells from these two species (8, 10).
Example 5 - Multilineage Differentiation of Human lin-CD34- cells in NOD/SCID mice after ex vivo Culture A representative mouse was transplanted with 50,000 expanded lin-CD34-CB cells after 2 days of ex-vivo culture in the presence of SF medium supplemented with 5% FCS. Mouse BM was extracted 10 weeks after transplant and analyzed by multiparameter flow cytometry (11, 12). The results are shown in Figure 8.
CD34- Cell Culture with Growth Factors Lin-CD34- cells were incubated in 50 ml of SF condition consisting of IMDM supplemented with 1% BSA (Stem Cell Technologies),5 mg/ml of human insulin (Humulin R from Eli Lilly and Co.),100 mg/ml of human transferrin (Gibco, BRL),10 mg/ml of low density lipoproteins (Sigma Chemical Co.), 10-4 M Beta-mercaptoethanol and growth factors (GF). GF
cocktail was used at final concentrations of 300 ng/ml of SCF (Amgen) and Flt-3 (Immunex), 50 ng/ml of G-CSF (Amgen),10 ng/ml of IL-3 (Amgen) and IL-6 (Amgen). 25% of condition media obtained from a fresh umbilical vein endothelial cell culture in a low percentage of serum ( 10%) and passaged four times, was added in some wells. Cells were cultured in flat bottomed suspension wells of 96-well plates (Nunc), incubated for 2 and 4 days at 37~C
and 5% CO2 and 50 ml of fresh GF cocktail was added to each well every other day.
Example 6 - Effect of ex vivo culture on the number of clonogenic pro~enitors present in the lin- CD34-~ lin-CD34-CD38- and lin-CD34-CD38+ cell fractions An aliquot of 800 to 2,500 lin-CD34-, lin- CD34-CD38- or lin-CD34-CD38+ cells were plated in clonogenic progenitor assays under standard conditions at the initiation of ex vivo cultures (day 0). Cells present after 4 days of culture in the presence of SF or SF supplemented with 5% FCS or 25% of HWEC-CM were plated in the same conditions. The number of CFC/800 input cells were estimated (mean ~ SEM; n-number of experiment). The results are seen in Table II.
Example 7 - The effect of liquid culture on the development and potential differentiation of lin-CD34-CD38- cells Individual wells were analyzed by flow cytometry after 2 and 4 days of culture in various conditions (Fig.6). Lin-CD34-CD38- cells seeded in SF
media began to express CD34 which could be enhanced with the addition of serum (Fig.6). In contrast, the majority of cells obtained after 2 or 4 days of culture in the presence of 25% HWEC conditioned medium still maintained the same phenotype. The acquisition of CD34 demonstrates the differentiation capacity of Lin-CD34-CD38- these cells in-vitro. The production of CD34+CD38- cells suggests that lin-CD34-CD38- cells precede CD34+CD38-population in the hierarchy of human hematopoiesis.
Example 8- Cytokine stimulation of Lin-CD34-CD38- Repopulating ActivityThe percentage of engrafted mice were compared before and after ex-vivo culture of Lin- CD34-, lin- CD34-CD38- and lin-CD34-CD38+ fractions.
Purified fractions from 43 CB and 3 BM samples were cultured for 2 and 4 days in SF, SF supplemented with 5%FCS or 25% HWEC-CM and transplanted at various doses into 136 recipient NOD/SCID mice (Fig. 7). Mice transplanted with cultured lin-CD34-CD38+ cells were not engrafted consistent with the inability of this fraction to engraft at day 0 (limit of detection <
0.05%). Transplanted Lin-CD34- cells we capable of engrafting mice with as few as 8, 700 Lin-CD34- CB cells cultured for 4 days in SF conditions. A total of 13 out 29 mice transplanted with cultured Lin-CD34- cells (cell dose ranging from 8, 700 to 55, 000) were engrafted, indicating that ex-vivo culture significantly increases the frequency of SRC (1 in 30,000 versus 1 in 125,000 atday 0). The increase in SRC activity could be explained by the acquisition of the CD34 antigen due to a differentiation response of a subfraction of lin-CD34-CD38- cells (Fig.6). However, similar increases in SRC was observed after stimulation of Lin-CD34-CD38- cells for 4 days in the presence of SF+25% HWEC-CM which does not cause differentiation into CD34+CD38-cells. As many as 35 % of the mice transplanted with cultured lin-CD34-CD38-cells engraft compared to 9% at day 0 ( Fig. l). In contrast,5,000 and 10,000 lin-CD34+CD38- CB cells CO~ g 10-20 SRC cultured for 4 days in the presence of serum or the addition of 25% HWEC-CM were unable to engraft NOD/SCID confirming the absence of collt~ ting SRC from this fraction in Lin-CD34-CD38- repopulating cells (data not shown). The fact that repopulating cells within lin-CD34+CD38- and lin-CD34-CD38- fractions respond differently to the presence of 25%HWEC-CM, and that no phenotypic change of lin-CD34-CD38- cells occurred after 4 days of culture suggests that elements other than the acquisition of CD34 antigen must play a role in the increase of repopulating activity of Lin-CD34-CD38- cells.
Table OneHematopoietic Progenitor Activity of Lin-CD34-~ Lin-CD34-CD38-and Lin-CD34-CD38+
Phenotype of purified CFC/800 cellsFrequency of population LTC-IC
CD34-Lin- 8.9+3 <1/10,000 (n=2) (n=4) 5,501_1640(n=2) CD34-Lin-CD38- 2.7_2 ~1/2,000 (n=36) (n=4) CD34-Lin-CD38+ 77+31 <1/10,000 (n=10) (n=4) CD34+CD38- 250_46 >1/10 (n=4) (n=4) Table Two Effect of ex vivo culture on the number of clonogenic progenitors present in the lin- CD34-, lin-CD34-CD38- and lin-CD34-CD38+ cell fractions.
Phenotype of purified Da~l 0 After in vitro stimulation population CD34-Lin- 8.9 _3 Serum Free 10 _0.9 (n=4) (n=4) 5% Serum 76 _21 (n=2) CD34-Lin-CD38- 2.7_ 2 5% Serum 6.4 _4.5 (n=7) (n=36) Serum Free 1.2 _1 (n=9) 25% HWEC 0.5% _0.5 (n=7) CD34-Lin-CD38+ 77 _31 Serum Free 20 _10 (n=4) (n=10) 5% Serum 134_ 41 (n=4) *CFC/800 Input Cells References l.Till, J.E., McCulloch, E.A. 1980. Hemopoietic stem cell differentiation. Biochim.Biophys. Acta. 605(4):431-59.
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| CA002219869A CA2219869A1 (en) | 1997-10-31 | 1997-10-31 | Human cd-34 hematopoietic stem cells |
| CA 2307624 CA2307624A1 (en) | 1997-10-31 | 1998-10-30 | Hematopoietic stem cells |
| AU97320/98A AU9732098A (en) | 1997-10-31 | 1998-10-30 | Hematopoietic stem cells |
| PCT/CA1998/001012 WO1999023205A1 (en) | 1997-10-31 | 1998-10-30 | Hematopoietic stem cells |
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| CA002219869A CA2219869A1 (en) | 1997-10-31 | 1997-10-31 | Human cd-34 hematopoietic stem cells |
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| CA002219869A Abandoned CA2219869A1 (en) | 1997-10-31 | 1997-10-31 | Human cd-34 hematopoietic stem cells |
| CA 2307624 Abandoned CA2307624A1 (en) | 1997-10-31 | 1998-10-30 | Hematopoietic stem cells |
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| WO (1) | WO1999023205A1 (en) |
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| US7927587B2 (en) | 1999-08-05 | 2011-04-19 | Regents Of The University Of Minnesota | MAPC administration for the treatment of lysosomal storage disorders |
| US6632620B1 (en) | 2000-06-22 | 2003-10-14 | Andrew N. Makarovskiy | Compositions for identification and isolation of stem cells |
| US20040029188A1 (en) * | 2000-11-07 | 2004-02-12 | Eaves Connie J | Method for the detection of human hematopoietic short term repopulating cells |
| WO2003050251A2 (en) | 2001-12-07 | 2003-06-19 | Geron Corporation | Hematopoietic cells from human embryonic stem cells |
| US7799324B2 (en) | 2001-12-07 | 2010-09-21 | Geron Corporation | Using undifferentiated embryonic stem cells to control the immune system |
| AU2004296848A1 (en) | 2003-12-04 | 2005-06-23 | Regents Of The University Of Minnesota | Compositions and methods for the treatment of lysosomal storage disorders |
| AU2007321928A1 (en) | 2006-11-24 | 2008-05-29 | Regents Of The University Of Minnesota | Endodermal progenitor cells |
| EP2746769A1 (en) * | 2012-12-21 | 2014-06-25 | Stembios Technologies, Inc. | Method for evaluating effect of action on subject based on stem celldynamics |
| SG11201603166UA (en) | 2013-10-24 | 2016-05-30 | Ospedale San Raffaele Srl | Method |
| KR20210018437A (en) * | 2018-06-07 | 2021-02-17 | 더 브리검 앤드 우먼즈 하스피털, 인크. | Method for producing hematopoietic stem cells |
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| AU9732098A (en) | 1999-05-24 |
| CA2307624A1 (en) | 1999-05-14 |
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