WO1993025216A1

WO1993025216A1 - Human primitive stem cells

Info

Publication number: WO1993025216A1
Application number: PCT/US1993/005229
Authority: WO
Inventors: Shian Huang; Leon Wmm Terstappen
Original assignee: Becton, Dickinson And Company
Priority date: 1992-06-08
Filing date: 1993-06-07
Publication date: 1993-12-23
Also published as: AU4402193A

Abstract

A population of cells comprising primitive human stem cells is disclosed. This population of cells is self-renewing and is capable of differentiating into both stromal stem cells and hematopoietic stem cells. This population of cells has the phenotype CD34?+/CD38-/HLA-DR-¿.

Description

HUMAN PRIMITIVE STEM CELLS

Field of the Invention

This invention relates to the identification and purification of human primitive stem cells. These primitive stem cells are capable of self-renewal and are capable of differentiating into both hematopoietic stem cells and into stromal stem cells that give rise to the hematopoietic microenvironment. The former stem cells also are capable of self-renewal and differentiation into precursor cells of all hematopoietic lineages. The latter cells develop into a complex reticulum of cells and extracellular matrix proteins which support hematopoiesis. These cells also are capable of self-renewal and differentiation into osteo progenitor cells, fibroblast precursors, endothelial cells, smooth muscle precursors and adipocytes. The invention more particularly relates to the use of three or more monoclonal antibodies to identify and purify such primitive stem cells.

Background of the Invention

Hematopoietic stem cells are defined as those cells that are capable of both self-renewal and differentiation into the two principle precursor components (i.e. , the myeloid and lymphoid lines) . Such cells are said to be "totipotent" . Further differentiation then occurs among these precursor cells to produce the monocyte, eosinophil, neutrophil, basophil, megakaryocytes and erythroid lineages from the myeloid line and T cells, B cells and N cells from the lymphoid line. In an article published in Scientific American in December 1991, Golde described "stem cells" and described research into isolating the "totipotent" stem cell, "pluripotent" stem cells and methods of using stem cells for therapeutic uses. Although a number of scientists have been exploring multiple means for isolating stem cells, the first breakthrough into stem cell isolation and identification did not come until the early 1980s. In U.S. Patent No. 4,714,680, Civin described a population of pluripotent lympho-hematopoietic cells which were substantially free of mature lymphoid and myeloid cells. Civin also described an antigen, MY-10 and a monoclonal antibody thereto, which was present on these cells. These cells made up about 1% of all cells in normal adult bone marrow, and generally comprise a mixture of totipotent, pluripotent stem cell's and lineage committed precursor cells with the latter cells predominating.

Since that time, MY-10 has been classified by the

International Workshop on Human Leukocyte Antigens as falling with - the cluster designated as "CD34". Anti-CD34 monoclonal antibodies are commercially available from a number of sources including Becton Dickinson Immunocytometry Systems ("BDIS").

Anti-CD34 monoclonal antibodies have been used for a number of purposes outlined in the ύolde article. Loken, Terstappen and their collaborators have published a series of papers describing the maturational stages for various components of the hematopoietic system (e.g. , T and B lymphocytes, erythroid cells and neutrophils) . The purpose of this work was to define, starting from the most mature ' cell and working backwards, the various maturational and developmental stages that a lineage committed cell goes through.

While the focus of this body of work has been on maturational stages, others have used anti-CD34 monoclonal antibodies to look for earlier non-lineage committed stem cells. In co-pending and commonly assigned U.S. patent application Serial Number 759,092, Terstappen described a subset of , human progenitor cells which were capable of self-renewal and differentiation into each of the various hematopoietic lineages (i.e. , a population of cells that include cells that are totipotent) . This population was characterized as being CD34⁺/CD38^~. See also Terstappen et a , Blood, 77:1218 (1991).

At roughly the same time, Tεuka oto et al. described a population of cells which also were capable of self-renewal and differentiation; however, this population of cells was

+ _ _ _ characterized as being CD34 /CD10 /CD19 /CD33 and Thy-1⁺. These so called CD34⁺/"Lineage^~" cells are described in U.S. Patent No. 5,061,620.

More recently, others have begun to try to subset CD34 cells from both peripheral blood and bone marrow. Bender et al. , Blood, 77:2591 (1991), have described a four color approach to stem cell identification. They have used fluorescently labeled anti-CD34, anti-CD33, anti-CD45, anti-CD19, anti-CD7, anti-CDIO, anti-CD3, anti-CD20, anti-CD14, anti-CDllb and anti-HLA-DR monoclonal antibodies. Using combinations of these antibodies, Bender et al. were able to identify a number of subsets. One subset was CD34⁺/HLA-DR^~. This subset had very small numbers of cells and no clear population with this phenotype was resolved. Bender et al. speculated on the ability of this population of cells to give rise to blast cell colonies or cells reconstituting long term cultures based upon prior work of others.

Sutherland et al . , Blood, 78:666 (1991), recently reported on the differential regulation of "primitive" hematopoietic cells in long term culture. They used anti-CD34 and anti-HLA-DR monoclonal antibodies to select cells that were CD34⁺ and HLA-DR^dιm or HLA-DR^~. These cells then were grown on a unique stromal cell line. The purpose was to establish a method of long term culture of such cells for the purposes of studying hematopoiesis and the effect of different growth factors on hematopoiesis.

Simmons et al. , Blood, 78:55 (1991), also recently reported on the "identification" of a stromal cell precursor in human bone marrow. Using an antibody they have designated as "Stro-1", Simmons et al. were able to use this antibody to remove stromal cells from bone marrow. The antigen recognized by this antibody was not present on colony forming progenitor cells but was present on a "subpopul tion of cells experiencing the [CD34] antigen." Thus, Simmons et al . described the ability of the antibody to separate out stromal cells from hematopoietic cells in bone marrow before culture.

Verfaille et al. , J. Exp. Med. , 172:509 (1990), has reported on an CD34⁺/HLA-DR⁺ and CD3 ⁺/HLA-DR^~ population of "primitive" progenitor cells. Taking adult marrow, Verfaille et al. depleted bone marrow of Lineage cells using multiple monoclonal antibodies. In a second step, fluorescently labelled CD34 and HLA-DR monoclonal antibodies were used to select HLA-DR⁺ and HLA-DR- populations that also are CD34 . Having isolated these two groups, Verfaille et al. reported that the HLA-DR⁺ cells were better in short term culture than the HLA-DR- cells. In long term culture, the reverse was true. There is no discussion or recognition of the ability of the CD34⁺/HLA-DR- cells to generate stromal stem cells.

In none of the references set forth above is there any indication as to the identification or nature of a single cell that is capable of self-renewal and gives rise to both hematopoietic stem cells and a stem cell thar gives rise to the hematopoietic microenvironment. Although this possibility has been speculated for a long period of time, see, e.g. , Hamilton et al. , Human Embryology, W. Hefer & Sons, Cambridge, pp. 77-86 (1945), there has been no suggestion as to the phenotype of these cells or means for their identification and isolation. Lacking such means also has precluded the ability of researchers to carry out therapies such as gene therapy and bone marrow transplantation. The present invention solves these problems.

Summary of the Invention

This invention comprises a population of stem cells that includes human primitive stem cells that are capable of self-renewal and are capable of differentiating into hematopoietic stem cells and into stromal stem cells that give rise to the hematopoietic microenvironment. This population of cells has the phenotype that is CD34⁺/CD38-/HLA-DR-. This population of cells lacks lineage committed antigens (i.e. , is CD33^~, CDIO-, CD5^~ and CD7l^") .

Cells having this phenotype can be identified in adult and fetal peripheral blood, bone marrow, thymus, liver or spleen. Preferably, these cells are identified in fetal tissues, and more preferably in fetal bone marrow.

Cells having this phenotype can be identified by using a combination of antibodies and selecting for the^" presence or absence of the antigens recognized by those antibodies on the cells. Preferably, the combination of antibodies comprises at least three monoclonal antibodies and more preferably comprises anti-CD34, anti-CD38 and anti-HLA-DR monoclonal antibodies.

Brief Description of the Figures

FIG. 1 comprises four dot plots of fetal bone marrow cells that have been labelled with anti-CD34 FITC (fluorescein isothiocyanate) , anti-HLA-DR APC (allophycocyanin) and anti-CD38 PE (phycoeryth in) monoclonal antibodies and then analyzed by mean of flow cytometry wherein A) is a plot of transformed orthogonal light scatter verus forward light scatter, B) is a plot of log PE versus log FITC fluorescence, C) is a plot of log APC versus log FITC fluorescence and D) is a plot of log PE versus log APC fluorescence and further wherein CD34⁺/CD38-/HLA-DR- cells were colored red, CD34⁺/CD38-/HLA-DR⁺ cells were colored yellow, CD34⁺/CD38⁺/HLA-DR- "^"cells were colored blue and CD34⁺/CD38⁺/HLA-DR⁺ were colored gray.

FIG. 2 comprises two photomicrographs of cells taken from a well after 17 days of culture of a single CD34⁺/CD38^~/HLA-DR^~ cell with A) being taken at magnification 200x and B) being taken at 400x magnification. The photograph shows a bone/cartilage-like structure with longitudinally arranged cell arrays surrounded by stromal cells. The presence of hematopoietic-like colonies at structure B is indicated with arrows.

FIG. 3 comprises four photomicrographs of cells A) from a typical blast colony taken from a well after 14 days of culture of cells depicted and dispersed from a colony shown in FIG. 2 (magnification 200x) , B) blast colony cells stained with a Wright Giemsa stain (magnification l,000x), C) second generation colonies taken from a cell after 14 days of culture of a dispersed and replated blast colony (magnification 200x) and D) a composition of photomicrographs of Wright Giemsa stained cells generated from colonies originated from replated blast colonies (magnification l,000x) showing Neutrophil (n) , Eosinophil (eo), Basophil (b) , Erythrocytes (e) , Macrophage (ma), Monocyte (m) , Megakaryocyte (me), platelets (p) , and Lymphocytes (1). The lower right corner shows, a lymphoid colony immunofluorescently stained with CD10 FITC. FIG. 4 is a schematic representation of the early development of the hematopoietic system.

Detailed Description of the Invention

Fetal bone marrows were obtained from aborted fetuses 16-22 weeks of gestational age and used following the guideline of the institutional review board of Stanford University Medical Center on the use of Human Subjects in Medical Research. Bone marrow cells were isolated by flushing intramedullary cavities of the femurs with RPMI 1640 with 10% FCS (fetal calf serum) followed by density gradient separation. The cells were immunofluorescently labeled with anti-CD34 (anti-HPCA-2 FITC, BDIS), anti-CD38 (an i-Leu 17 PE, BDIS) and anti-HLA-DR biotin followed by an incubation with Streptavidin Allophycocyanin (BDIS) .

Cell sorting was performed on a FACStar Plus (BDIS) using the Automated Cell Deposition Unit. The cells colored red, yellow, blue and gray in FIG. 1 were sorted singly into 96 well flat bottom plates. In the liquid culture system each well contained a 200 μl mixture of alpha medium, 12.5% HS (horse serum), 12.5% FCS, 10^~4 M 2-mercaptoethanol, 2 mM L-glutamine, 0.2 mM i-inositol, 20μM folic acid, antibiotics,

2.5 ng/ml b-FGF, 10 ng/ml IGF-1 (Collaborative BioMedical P Prroodduuccttss)) .. AAllll ccuullttuurreess wweerree iinnccuulbated in 5% CO 2 in air at 37°C in a fully humidified incubator

In the liquid culture system for the blast colony assay, each well contained the ^r ?-dium described above and 10 ng/ml rhIL-3, 500 U/ml rhIL-6, 10 ng/ml rhGM-CSF, (Collaborative BioMedical Products), 2.5 U/ml rhEPO (Amgen) and 50 ng/ml human stem cell growth factor (Genzyme) . The plates were observed between days 7 and 14 for the appearance of . colonies. Replating of colonies was performed by dispersion of the individual colonies into 96 well flat bottom plates containing the culture medium. Repetitive replating of colonies was performed under identical conditions.

Flow cytometric analysis was performed on a FACStar Plus equipped with an Argon ion laser tuned at 488nm and a Helium Neon laser (633nm). Data acquisition was performed with the Lysis 2.0 software (BDIS) with gates on light scattering and CD34 cells. Forward light scattering, orthogonal light scattering and 3 fluorescence signals were determined for each cell and the 10,000 cells were stored in listmode data files. The analysis of the five dimensional data was performed with the PAINT-A-Gate^Plus software (BDIS).

In previous experiments, it was shown that expression of α-

CD38 on CD34 human progenitor cells is correlated with lineage commitment of the progenitors and only the non-lineage committed CD34 /CD38- cells were capable of extensive self-renewal. See U.S. Serial No. 759,092. HLA-DR was employed to further subdivide these non-lineage committed cells.

Referring to FIG. 1, in three color immunofluorescence experiments, the expression of CD38 and HLA-DR was assessed on CD34 cells in eight samples of human fetal bone marrow. Four CD34 cell populations were identified: 1) CD38-/HLA-DR~ (4%, SD 3); 2) CD38^~/HLA-DR⁺, (4%, SD 2); 3) CD38⁺/HLA-DR- (3%, SD 2); and 4) CD38⁺/HLA-DR⁺ (89%, SD 5). FIG. 1 illustrates a typical flow cytometric analysis of CD34 fetal bone marrow cells; the CD34⁺/CD38-/HLA-DR-,cells were colored red, the CD34⁺/CD38-/HLA-DR⁺ . cells were colored yellow; the CD34⁺/CD38⁺/HLA-DR- cells were colored blue; and the CD34⁺/CD38⁺/HLA-DR⁺ cells were colored gray. Phenotyping and cell sorting studies revealed that the CD38 cell populations were heterogeneous in morphology and expressed lineage associated antigens containing myeloblaεtε, erythroblasts, lymphoblasts as well as megakaryoblasts. The CD38-/HLA-DR cells were homogeneous, primitive blast cells by morphology. In contrast, primitive blasts were admixed with primitive mesenchymal elements in the CD38 /HLA-DR cell population.

The four CD34⁺ cell fractions based on HLA-DR and CD38 expression (colored red, yellow, blue and gray in FIG. 1), were sorted singly in 96 well plates and assayed for their ability to form colonies in alpha medium containing IL-3, IL-6, SCF, GM-CSF, EPO, IGF-1, b-FGF, 12.5% HS and 12.5% FCS. In eight experiments, an average of 6% of the HLA-DR-, CD38- cells; 50% of the CD38-/HLA-DR⁺ cells; 35% of the CD38⁺/HLA-DR^~ cells and 16% of the CD38⁺/HLA-DR⁺ cells gave rise to hematopoietic colonies 14 days after cell sorting. In control experiments in which cells were sorted into the medium with 12.5% HS and 12.5% FCS, no colony formation was found. See Table I.

TABLE I

Clonal Plating Efficiency and Clonal Expanding Efficiency of Single Cell Culture in CD34+ Subpopulations

- 10-

Replating of the -formed colonies confirmed that only the colonies originating from the CD34 /CD38 subsets could reform colonies after repetitive replating. See Table II.

TABLE II

Clonal Replating Efficiency and Clonal Expanding Efficiency of CD34+ Subpopulations

The presence of primitive mesenchymal elements in the CD38-/HLA-DR^~ cell population indicates that this subset includes the primitive stem cells which gives rise to both the hematopoietic microenvironment and hematopoietic stem cells. To confirm this, the presence of stem cells which were not dependent of hematopoietic growth factors was examined.

The four CD34 cell fractions based on HLA-DR and CD38 expression (colored red, yellow, blue and gray in FIG. 1), again were sorted singly in 96 well plated in alpha medium containing only 1GF-1, b-FGF, 12.5% HS and 12.5% FCS. 1-5% of the single CD34⁺/CD38-/HLA-DR- cells produced randomly distributed ellipsoid shaped cells after 7-10 days of cell culture. The single CD34⁺/CD38^~/HLA-DR⁺, and the CD38⁺ subsets did not. Between 10-13 days of culture spindle cells appeared in these wells which gradually formed organized cell structures with longitudinally arranged cellular arrays. At day 15 to 17 the structures emerged as complex composites of bone/cartilage structures surrounded by stromal cells. Colonies of hematopoietic cells appeared after formation of the microenvironment. FIG.s 2A and 2B illustrate such a structure obtained after 17 days of cell culture of a single CD34⁺/CD38-/HLA-DR^" cell. The position of the cell colonies is indicated with arrows in FIG. 2A and shown with a higher magnification in FIG. 2B.

To verify the hematopoietic origin of the colonies, these were selected, dispersed, and replated in alpha medium, containing HS, FCS, IL-3, IL-6, GM-CSF, b-FGF, IGF-1, Epo and SCF. Two weeks after replating typical hematopoietic blast colonies could be found, see FIG.s 3A and 3B. Parts of the colonies were sacrificed for morphologic examination or to probe for cell surface antigen expression, whereas the remaining cells were dispersed and replated. The replated cells were cultured in identical media and were evaluated for colony formation after two weeks of culture and gave rise to a variety of second generation hematopoietic colonies. A typical example is shown in FIG. 3C.

The second generation colonies were processed similar to the first generation colonies and gave rise to third generation colonies and these could also give rise to fourth generation colonies. Morphology of the sacrificed colonies showed that the percentage of blasts within the colonies decreased gradually in the second, third and fourth generations. The oύtcome of the lineage commitment of the colonies was not predictable and followed a random pattern. Neutrophils, eosinophils, basophils, erythocytes, monocyteε, macrophages, megakaryocyteε, plateletε and B-lymphocyteε could be found whether in the firεt, εecond, third, or fourth generations, εee FIG. 3D. Identity of cells which appeared as lymphoblastε by morphology waε confirmed by immunofluorescence cell surface staining with either CD10 FITC or CD19 PE. No cells were found which were differentiεrted into the T lymphocyte lineage (using CD7 and CD3). These data indicate that the cells depicted from the surface illustrated in FIG. 2 do have properties expected from the hematopoietic stem cells (i.e. , extensive self-renewal capacity and capable to differentiate into each of the hematopoietic cell lineages).

Self-renewal ability of the formed structures was studied by disrupting and removing the structures and the supernatent, and replenishing of the media with serum, IGF-1 and b-FGF. After 2 to 5 days of culture only degenerated cells and debris could be found; however, the identical differentiation process developed as for the originally sorted single cells. After 15 to 17 days of culture similar structures appeared including the presence of hematopoietic cell colonies. The disruption of the micro-structure could be repeated and both the structure and the colonies reappeared in an identical timely fashion. These results provide direct evidence for the existence of a primitive stem cell which is capable of self-renewal and which can differentiate into cells capable of forming both the hematopoietic microenvironment and hematopoietic stem cells of human bone marrow. The observation that the structures repeatedly reconstructed themselves after disruption and supplementation with the media, IGF-1 and b-FGF, indicates that the primitive stem cell has a high self-renewal ability. The data also indicate that in early development of the hematopoietic system, primitive stem cells first generate stromal cells of the cell lineages constituting the hematopoietic microenvironment. This microenvironment then induces the primitive stem cells to differentiate into hematopoietic stem cells.

A schema of the observed development of the hematopoietic system is illustrated in FIG; 4.

Isolation of a population of stem cells that contain at least 1% and more preferably 5% of primitive stem cells can be accomplished in a number of ways. Each method involves at least the use of CD34, CD38 and HLA-DR monoclonal antibodies. Selection of cells that are CD34⁺/CD38^~/HLA-DR- can be performed by means of flow cytometry as described above wherein each of the antibodies is labelled with a different fluorescent dye. Alternatively, selection can be accomplished by sequential positive selection for CD34 cells followed by depletion of CD38⁺ and HLA-DR⁺ cells. In this embodiment, CD34 antibodies can be labelled with a binding agent, such as a magnetic or polymeric bead or biotin, and in the posi ive selection step the cells can be attached to the binding agent in a column or plate. The cells can be released from the binding agent by enzymatic means, see, e.g. , U.S. Patent No. 5,081,030. The selected cells then can be passed over or through another plate or plates having CD38 and HLA-DR antibodies labelled with a binding agent. Those CD34 cells that do not bind comprise the CD34⁺/CD38-/HLA-DR- subset. In any embodiment, cells bearing lineage committed antigens can be depleted first as a preliminary step.

All publications and patent applications mentioned in this specification are indicative of the level of ordinary skill in the art to which this invention pertains. All publications and patent applications are herein incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

It will be apparent to one of ordinary skill in the art that many changes and modifications can be made in the invention without departing from the spirit or scope of the appended claims.

Claims

Claims :

1. A population of cellε comprising human primitive stem cells.

The population of cells of claim 1 wherein such cells are CD34⁺/CD38 /HLA-DR^"

3. A population of human primitive stem cells that are capable of self-renewal and are capable of differentiating into stromal stem cellε that give rise to the hematopoietic microenvironment and into hematopoietic stem cells.

4. A substantially pure population of cells wherein such cells are CD34⁺/CD38-/HLA-DR^~.

5. A method of isolating the population of cells comprising human primitive stem cells of claim 4 said method comprising the steps of positively selecting for CD34 expression and negatively selecting for expression of CD38 and HLA-DR on the CD34⁺ cells.

6. The method of claim 5 wherein the selection method uses anti-CD34, anti-CD38 and anti-HLA-DR monoclonal antibodies.

7. The method of claim 5 wherein depletion of cells bearing lineage committed antigens is carried out as a preliminary step.

8. The method of claim 5 wherein the selection method is carried out by means of flow cytometry.