WO2022004864A1 - ヒト長期造血幹細胞マーカー - Google Patents
ヒト長期造血幹細胞マーカー Download PDFInfo
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- WO2022004864A1 WO2022004864A1 PCT/JP2021/025050 JP2021025050W WO2022004864A1 WO 2022004864 A1 WO2022004864 A1 WO 2022004864A1 JP 2021025050 W JP2021025050 W JP 2021025050W WO 2022004864 A1 WO2022004864 A1 WO 2022004864A1
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- hematopoietic stem
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Definitions
- the present disclosure discloses a marker for concentrating or isolating hematopoietic stem cells and a method for utilizing the marker, a cell or cell population isolated or enriched by the marker, a method for utilizing the cell or cell population, the cell or cell.
- the present invention relates to a medicine containing a population, the cell or a classifier obtained by utilizing the cell population.
- Stem cells such as hematopoietic stem cells (HSCs) are known to have biological functions of self-renewal ability and pluripotency. It is known that among stem cells having self-renewal ability and pluripotency, there are cells that maintain the biological functions that characterize these stem cells for a long period of time and stem cells that lose their functions in a relatively short period of time. (Non-Patent Document 1).
- Hematopoietic stem cell is mainly present in the bone marrow, is defined as a blood cell having self-renewal ability and pluripotency, and is used for hematopoietic stem cell transplantation and the like. If high-quality hematopoietic stem cells such as long-term hematopoietic stem cells that maintain the properties of the stem cells for a long period of time can be appropriately isolated, it is conceivable that the hematopoietic stem cells can be proliferated in large quantities in vitro, but for a long period of time in humans. No specific marker has been known that enables proper isolation of hematopoietic stem cells (long-term HSC: LT-HSC).
- Non-Patent Document 2 the molecular markers specifically expressed on the surface of hematopoietic stem cells differ between mice and humans, and it has been desired to find markers that can concentrate and identify long-term hematopoietic stem cells in humans.
- Non-Patent Document 3 Attempts have been made to search for combinations of molecules specifically expressed on the surface of long-term hematopoietic stem cells in order to identify and isolate long-term hematopoietic stem cells. For example, in order to further concentrate hematopoietic stem cells from a CD34-positive hematopoietic stem cell population, it has been proposed to isolate the Thy1-positive fraction from the CD34-positive, CD38-negative, and CD45RA-negative fractions in the umbilical cord blood. It has been reported that fractions still contain cells that differ in their ability to maintain the properties of hematopoietic stem cells (Non-Patent Document 3).
- Non-Patent Document 4 Searching for such a combination of cell surface molecules generally requires trial and error involving a great deal of work for performing a cell function test. Therefore, it is desired to identify new cell surface molecule combinations for identifying long-term hematopoietic stem cells.
- the present disclosure discloses CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200, CD226.
- a long-term hematopoietic stem cell isolation marker consisting of 5 or more, or 1 or more selected from a combination of CD48 and CD34, a combination of CD48 and c-Kit, and a combination of CD48, CD34 and c-Kit, or a combination thereof.
- a concentration marker is provided.
- the present disclosure discloses CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200, CD226. , CD245, CD31, CD317, CD40, CD41, CD61, CD93, Notch1, CD352, Sigma-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48.
- the present disclosure is: The process of preparing a cell population containing hematopoietic stem cells, CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200, CD226, CD245, CD31, CD317, 1, 2, 3, 4 or 5 or more selected from CD40, CD41, CD61, CD93, Notch1, CD352, Sigma-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48, or CD48 and A step of isolating or concentrating cells using the expression mode of one or more selected from the combination of CD34, the combination of CD48 and c-Kit, and the combination of CD48, CD34 and c-Kit, or a combination thereof as an index.
- a method for producing an isolated or concentrated hematopoietic stem cells CD10
- the present disclosure is: The process of preparing a cell population containing hematopoietic stem cells, CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200, CD226, in each cell. 1, 2, 3, 4 or 5 selected from CD245, CD31, CD317, CD40, CD41, CD61, CD93, Notch1, CD352, Sigma-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48.
- the above, or a step of measuring the expression level of one or more selected from the combination of CD48 and CD34, the combination of CD48 and c-Kit, and the combination of CD48, CD34 and c-Kit, or a combination marker thereof. Provide a method for assessing the content and / or changes in the content of hematopoietic stem cells in the cell population.
- the present disclosure is: The process of preparing a cell population containing hematopoietic stem cells, CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, which are expressed in cells included in the cell population including hematopoietic stem cells.
- CD172a / b CD200, CD226, CD245, CD31, CD317, CD40, CD41, CD61, CD93, Notch1, CD352, Sigma-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48 1 , 2, 3, 4 or 5 or more, or one or more selected from a combination of CD48 and CD34, a combination of CD48 and c-Kit, and a combination of CD48, CD34 and c-Kit, or a combination of these markers.
- a method for testing a cell population containing hematopoietic stem cells which comprises a step of testing the quality of the cell population using the expression level as an index.
- the present disclosure is: The process of preparing a cell population containing hematopoietic stem cells, CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, which are expressed in cells included in the cell population including hematopoietic stem cells.
- CD172a / b CD200, CD226, CD245, CD31, CD317, CD40, CD41, CD61, CD93, Notch1, CD352, Sigma-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48 1 , 2, 3, 4 or 5 or more, or one or more selected from a combination of CD48 and CD34, a combination of CD48 and c-Kit, and a combination of CD48, CD34 and c-Kit, or a combination of these markers.
- a method for testing a cell population containing hematopoietic stem cells which comprises a step of testing the transplant suitability of the cell population using the expression level as an index.
- the present disclosure is: To predict the disease prognosis or health status of the subject, CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, which are expressed in cells contained in a cell population including hematopoietic stem cells derived from patients. Selected from CD135, CD172a / b, CD200, CD226, CD245, CD31, CD317, CD40, CD41, CD61, CD93, Notch1, CD352, Sigma-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48.
- the process of acquiring the expression level of the marker and Provided is a method for testing a cell population containing hematopoietic stem cells, which comprises a step of comparing the expression level of the marker with a control.
- the present disclosure is: The process of preparing a cell population containing hematopoietic stem cells, Steps to treat a cell population containing hematopoietic stem cells with the candidate compound and CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, expressed in the cells treated with the candidate compound.
- a method for screening a compound that modifies the characteristics of hematopoietic stem cells which comprises a step of selecting a candidate compound using the expression level of one or more or a combination of these markers as an index.
- the present disclosure comprises the step of preparing a cell population containing hematopoietic stem cells.
- a method for adjusting culture conditions that modifies the characteristics of hematopoietic stem cells which comprises a step of determining a method for adjusting culture conditions using an amount as an index.
- the present disclosure comprises the step of preparing a cell population containing hematopoietic stem cells.
- a method for producing an isolated or concentrated hematopoietic stem cell or a cell population containing a hematopoietic stem cell which comprises a step of adjusting the culture conditions so as to modify the characteristics of the hematopoietic stem cell.
- the present disclosure comprises the step of preparing a cell population containing hematopoietic stem cells.
- the process of determining the composition of culture aids and Provided is a method for producing a culture medium or a culture auxiliary additive that modifies the characteristics of hematopoietic stem cells which comprises a step of producing a culture medium or a culture auxiliary additive having the above-determined composition.
- the present disclosure is: The process of preparing the cell population and The process of treating the cell population with the test compound and CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, expressed in cells treated with the candidate compound. 1, 2, 3 selected from CD200, CD226, CD245, CD31, CD317, CD40, CD41, CD61, CD93, Notch1, CD352, Compound-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48.
- a method of screening a compound that induces hematopoietic stem cells including a step of selecting a candidate compound.
- the present disclosure discloses CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200, 1, 2, 3, 4 selected from CD226, CD245, CD31, CD317, CD40, CD41, CD61, CD93, Notch1, CD352, Siglec-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48.
- composition or kit for identifying, detecting, isolating or concentrating hematopoietic stem cells which comprises a reagent to be used.
- the present disclosure provides a method of producing modified hematopoietic stem cells by modifying the hematopoietic stem cells of the present disclosure.
- the present disclosure relates to a disease or disorder that is ameliorated or prevented by administration of hematopoietic stem cells, which comprises isolated or concentrated hematopoietic stem cells or a cell population containing hematopoietic stem cells or cells modified thereto.
- hematopoietic stem cells which comprises isolated or concentrated hematopoietic stem cells or a cell population containing hematopoietic stem cells or cells modified thereto.
- the disclosure comprises a method of assessing the physiological action of a test substance, comprising contacting the test substance with an isolated or concentrated hematopoietic stem cell or a cell population containing the hematopoietic stem cell or a cell modified thereto. offer.
- the present disclosure presents hematopoietic stem cells by a combination of physical, chemical, and biological characteristics obtained by analyzing isolated or enriched hematopoietic stem cells or cell populations containing hematopoietic stem cells.
- the present disclosure presents hematopoietic stem cells by a combination of physical, chemical, and biological characteristics obtained by analyzing isolated or concentrated hematopoietic stem cells or a cell population containing hematopoietic stem cells.
- the present disclosure discloses CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200.
- the present invention in one aspect, provides: [Item 1] CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200, CD226, CD245, CD31, CD317, One or more selected from CD40, CD41, CD61, CD93, Notch1, CD352, Sigma-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48, or a combination of CD48 and CD34, CD48 and c- A hematopoietic stem cell isolation marker or enrichment marker comprising a Kit combination and one or more selected from a combination of CD48, CD34 and c-Kit, or a combination thereof.
- a method for producing a cell population containing stem cells or hematopoietic stem cells [Item 6] 1 to be selected from the combination of CD48 (-) and CD34 (+), the combination of CD48 (-) and c-Kit (+), and the combination of CD48 (-), CD34 (+) and c-Kit (+) 1 Using the above expression pattern as an index, Item 5.
- Hematopoietic stem cells in the cell population comprising a combination, a combination of CD48 and c-Kit, and one or more selected from the combination of CD48, CD34 and c-Kit, or a step of measuring the expression level of a marker of these combinations. And / or a method for assessing changes in content.
- a method for testing a cell population containing hematopoietic stem cells which comprises the step of testing the quality of the cell population. [Item 10] 9.
- a method for testing a cell population containing hematopoietic stem cells comprising the step of comparing the expression level of the marker with a control for predicting the disease prognosis or health condition of a subject.
- the process of preparing a cell population containing hematopoietic stem cells The process of treating a cell population containing hematopoietic stem cells with a candidate compound, and CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, expressed in cells treated with the candidate compound.
- a method for screening compounds that modify the properties of hematopoietic stem cells including.
- the culture conditions are determined using the expression level of one or more selected from the combination of CD48 and CD34, the combination of CD48 and c-Kit, and the combination of CD48, CD34 and c-Kit, or the expression level of the marker of these combinations as an index.
- [Item 14] The process of preparing a cell population containing hematopoietic stem cells, A step of culturing a cell population containing hematopoietic stem cells in a medium having a composition to be evaluated or a medium containing a culture auxiliary additive to be evaluated, and CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200, CD226 expressed in cultured cells.
- the process of deciding and A method for producing a culture medium or a culture aid additive that modifies the characteristics of hematopoietic stem cells, comprising the step of producing a culture medium or a culture aid additive having the above-determined composition.
- [Item 15] The method according to any one of items 5 to 14, wherein the cell population containing the hematopoietic stem cells is a cell derived from a living body or a cell derived from an artificially acquired pluripotency.
- [Item 16] A single produced by the method according to any one of items 5 to 7, wherein the cell population containing the hematopoietic stem cells is a cell derived from a living body or a cell derived from an artificially acquired pluripotency cell.
- Methods for screening compounds that induce hematopoietic stem cells including.
- composition or kit [Item 19] The hematopoietic stem cell identified, isolated, or concentrated by the marker according to claim 1 or 2, or the isolated or concentrated hematopoietic stem cell or hematopoietic stem cell according to any one of items 3, 4 and 7. Physical characteristics, chemical characteristics, biology obtained by analyzing a cell population containing, or a cell population containing isolated or concentrated hematopoietic stem cells or hematopoietic stem cells produced by the method according to item 5 or 6. How to identify a combination of features.
- the hematopoietic stem cell identified, isolated, or concentrated by the marker according to claim 1 or 2, or the isolated or concentrated hematopoietic stem cell or hematopoietic stem cell according to any one of items 3, 4 and 7.
- Physical, chemical, and biology obtained by analyzing a cell population comprising, or a cell population containing isolated or concentrated hematopoietic stem cells or hematopoietic stem cells produced by the method according to item 5 or 6.
- Hematopoietic stem cells identified, isolated, or concentrated by the marker according to any one of claims 1 or 2, or isolated or concentrated hematopoietic cells according to any one of items 3, 4 and 7. Improvement or prevention by administration of hematopoietic stem cells, including a cell population comprising stem cells or hematopoietic stem cells, or a cell population comprising isolated or concentrated hematopoietic stem cells or hematopoietic stem cells produced by the method according to item 5 or 6. A drug for the prevention or treatment of a disease or disorder.
- hematopoietic stem cells Diseases or disorders that are ameliorated or prevented by administration of hematopoietic stem cells are metachromatic leukodystrophy (MLD), Gaucher's disease, Fabry's disease, mucopolysaccharidosis, HIV, ADA-SCID, X-SCID or salacemia.
- MLD metachromatic leukodystrophy
- Fabry's disease Fabry's disease
- mucopolysaccharidosis HIV
- ADA-SCID X-SCID
- salacemia The medicine according to item 21.
- 21 The pharmaceutical agent according to item 21, wherein the disease or disorder that is ameliorated or prevented by administration of hematopoietic stem cells is chronic granulomatous disease (CGD) or adrenoleukodystrophy (ALD).
- CCD chronic granulomatous disease
- ALD adrenoleukodystrophy
- the present disclosure has the effect of providing a marker for enriching or isolating hematopoietic stem cells and their use.
- FIG. 1 is an image that visualizes the cell arrangement spatial structure in which cells at close positions on the differentiation line occupy close positions, which are generated from data obtained by cell staining of a cord blood sample.
- FIG. 2 is an image that visualizes the expression level of CD34 in each cell in the cell arrangement space structure in which cells in close positions on the differentiation line occupy close positions, which is generated from data obtained by cell staining of a cord blood sample. be.
- FIG. 3 is a diagram showing the results of comparing the chimera rates in the transplantation test of CD34 (+) cells and CD34 ( ⁇ ) cells obtained from umbilical cord blood.
- FIG. 1 is an image that visualizes the cell arrangement spatial structure in which cells at close positions on the differentiation line occupy close positions, which are generated from data obtained by cell staining of a cord blood sample.
- FIG. 2 is an image that visualizes the expression level of CD34 in each cell in the cell arrangement space structure in which cells in close positions on the differentiation line occupy close positions, which is generated from data obtained by cell stain
- FIG. 4 is an image showing the result of arranging the cell arrangement of the CD34 (+) fraction in more detail in the cell arrangement space structure in which cells in close positions on the differentiation line occupy close positions.
- FIG. 5 is a diagram showing the proliferative / differentiating ability of hematopoietic stem cells of the cord blood fraction identified by CD34 (+) and CD38 ( ⁇ / +).
- FIG. 6 is a diagram showing an outline of the CFU assay at the 1-cell level.
- FIG. 7 is a diagram showing the direction in which undifferentiated cells are located for a CD34-positive cell fraction on a cell arrangement space structure in which cells at close positions on the differentiation lineage occupy close positions.
- FIG. 8 is a diagram showing an example of the result of searching for a molecular marker whose expression is unevenly distributed along the differentiation direction on the cell arrangement space structure in which cells at close positions on the differentiation line occupy close positions.
- FIG. 9 is a diagram showing the proportion of cells exhibiting CFU-GEMM (correlation with the degree of enrichment of undifferentiated cells) in the CFU assay of cells identified by each marker.
- a cell population pre-concentrated with CD34 (+) from cord blood is gated with c-Kit (+) and CD34 (+) (fraction P1), and further CD48 (-) (P2 fraction).
- CD48 (+) (non-P2 fraction) is a diagram showing an example of a FACS plot when gating.
- FIG. 10 is a diagram showing an example of a FACS plot when gating.
- FIG. 11 shows a cell population pre-concentrated with CD34 (+) from bone marrow is gated with c-Kit (+) and CD34 (+) (fraction P1), and further CD48 (-) (P2 fraction).
- FIG. 12 shows a cell population pre-concentrated with CD34 (+) from mobilized peripheral blood gated with c-Kit (+) and CD34 (+) (fraction P1), and further CD48 (-) (P2 plot). It is a figure which shows an example of the FACS plot when gating with a minute) or a CD48 (+) (non-P2 fraction).
- FIG. 12 shows a cell population pre-concentrated with CD34 (+) from mobilized peripheral blood gated with c-Kit (+) and CD34 (+) (fraction P1), and further CD48 (-) (P2 plot).
- FIG. 12 shows a cell population pre-concentrated with CD34 (+) from mobilized peripheral blood gated with c-Kit (+) and CD34 (+) (fraction P1), and further CD48 (
- FIG. 13 is a diagram showing the results of comparing the number of CD34-positive cells contained in 1 ml of cord blood with the number of cells in the P2 fraction.
- FIG. 14 shows the cell fractions (P2) identified by c-Kit (+), CD34 (+), CD48 (-) and c-Kit (+), CD34 (+) isolated from cord blood.
- CD48 (+) is a diagram showing the results of comparison of the proliferative and differentiating ability of hematopoietic stem cells of the cell fraction (non-P2) identified by the CFU assay.
- FIG. 15 is a diagram showing the results of comparing the undifferentiated cells fraction P2 isolated from cord blood and the undifferentiated cell fraction non-P2 by a transplantation test.
- FIG. 14 shows the cell fractions (P2) identified by c-Kit (+), CD34 (+), CD48 (-) and c-Kit (+), CD34 (+) isolated from cord blood.
- CD48 (+) is a diagram showing the results of comparison of
- FIG. 16 shows cell fractions (P2) identified by c-Kit (+), CD34 (+), CD48 (-) and c-Kit (+), CD34 (+), isolated from bone marrow. It is a figure which shows the result of having compared the proliferation / differentiation ability of the hematopoietic stem cell of the cell fraction (non-P2) specified by CD48 (+) by the CFU assay. The results of the P1 fraction, which is the fraction specified by c-Kit (+) and CD34 (+), are also shown.
- FIG. 17 shows cell fractions (P2) identified by c-Kit (+), CD34 (+), CD48 (-) and c-Kit (+), CD34 (+) isolated from mobilized peripheral blood.
- FIG. 18 shows the proliferative and differentiating ability of hematopoietic stem cells by fractionating the CD34 (+) fraction into the c-Kit (+) fraction and the c-Kit (-) fraction for the cord blood sample.
- FIG. 19 is a diagram showing the enrichment ratio of additional markers. Data with a enrichment factor of more than 10 are shown as 10.
- FIG. 20 is a diagram showing the results of culturing the cells of the P2 fraction under the condition of adding or not adding UM171, and testing the total number of cells and the ratio of undifferentiated cells (P1) after 7 days.
- FIG. 21 is a diagram showing the results of culturing cells of the non-P2 fraction under the condition of adding or not adding UM171, and testing the total number of cells and the ratio of undifferentiated cells (P1) after 7 days.
- FIG. 20 is a diagram showing the results of culturing the cells of the P2 fraction under the condition of adding or not adding UM171, and testing the total number of cells and the ratio of undifferentiated cells (P1) after 7 days.
- differentiation has the meaning commonly used in the art, typically less specialized cells are transformed into more specialized cells such as, for example, nerve cells or muscle cells. Means the process of doing. "Differentiated” or “differentiated” is a relative term, and “differentiated cell” or “differentiated cell” goes further in the developmental pathway than the cell with which it is being compared, and more. It means that it is specialized.
- stem cell has the meaning commonly used in the art, typically having self-renewal ability at the single cell level and the ability to produce two or more different differentiated cells.
- stem cells can divide symmetrically into two stem cells, thus maintaining some stem cells in a cell population with stem cells, while other cells in the population are differentiated progeny. Cause only.
- hematopoietic stem cell has a meaning commonly used in the art, and typically has pluripotency capable of finally differentiating into all blood cells (erythrocytes, leukocytes, platelets, etc.). Means a cell. Hematopoietic stem cells may include intermediate stages of differentiation into progenitor cells or blast cells. "Progenitor cells” or “blasts” are used interchangeably in the present disclosure and have reduced potency, but are still capable of maturing into cells of a particular lineage (eg, bone marrow lineage or lymphatic lineage). It means a certain, maturing cell.
- a particular lineage eg, bone marrow lineage or lymphatic lineage
- long-term hematopoietic stem cell has a meaning usually used in the art, and typically means a hematopoietic stem cell that maintains self-renewal ability for a long period of time even after undergoing cell division. At least one of the two daughter cells produced by cell division from long-term hematopoietic stem cells retains the same hematopoietic stem cell traits as pre-division cells. In one embodiment, long-term hematopoietic stem cells do not lose any self-renewal ability due to cell division in a normal in vivo or in vitro culture environment. Also, in one embodiment, long-term hematopoietic stem cells maintain their self-renewal ability after 100, 50, 20 or 10 cell divisions.
- the self-replicating ability is maintained.
- the self-renewal ability is maintained by measuring the content of the undifferentiated cell population contained in the cell group after culturing for a certain period of time.
- the undifferentiated cell population can be identified, for example, by a combination of positive and negative known markers expressed on the cell surface.
- the fact that hematopoietic stem cells maintain their self-renewal ability for a long period of time even after undergoing cell division can be confirmed by maintaining their bone marrow remodeling ability for a long period of time.
- long-term hematopoietic stem cells can maintain bone marrow remodeling in a primary transplant recipient in known transplantation methods and, in a particularly preferred embodiment, also in a secondary transplant.
- Hematopoietic stem cells which maintain self-renewal ability only for a short period of time, can reconstruct bone marrow by in vitro or primary transplantation, but cannot maintain bone marrow reconstruction in the primary transplant recipient, and secondary. Bone marrow cannot be reconstructed and maintained by transplantation.
- the secondary transplantation means that the bone marrow-derived hematopoietic stem cells reconstructed by the primary transplantation are transplanted to yet another individual.
- the fact that hematopoietic stem cells maintain their self-renewal ability for a long period of time even after undergoing cell division means that when the cells are transplanted into another living body, they are 4 days, 5 days, 6 days, 7 days, 8 days. Continuously producing blood for days, 9 days, or 10 days or more, typically 500, 600, 700, 800, 900 or 1000 of neutrophils, a type of white blood cell, over the above period. It can be confirmed by producing more than 100 pieces / ⁇ L (mm 3).
- the hematopoietic stem cells or long-term hematopoietic stem cells of the present disclosure include hematopoietic stem cells or long-term hematopoietic stem cells derived from various animals.
- various mammals, animals belonging to primates, animals belonging to the order Monkey, or human-derived hematopoietic stem cells or long-term hematopoietic stem cells can be used in the present disclosure, or the present disclosure can be applied to these cells. ..
- the hematopoietic stem cells of the present disclosure can be isolated from a somatic cell population obtained from a living body, or from a cell group induced to differentiate from ES cells or induced pluripotent stem cells.
- isolated cell has a meaning commonly used in the art and typically means a cell taken from an organism in which it is originally found or a progeny of such a cell. Such cells may be cultured in vitro, eg, in the presence of other cells. In addition, such cells or cells that are descendants thereof may be later introduced into a second organism.
- isolated cell population has a meaning commonly used in the art, and typically means a population of cells taken out of and isolated from an inhomogeneous cell population.
- the isolated population may be a substantially pure cell population compared to the heterogeneous cell population from which the cells were isolated or concentrated, and is still heterogeneous. It may be a cell population.
- a cell population is "substantially pure" for a particular cell type has the meaning commonly used in the art, typically at least for the cells that make up the entire cell population in that cell population. About 50%, more preferably at least about 60%, 65%, 70%, 75%, 85%, 90%, 92%, 93%, most preferably at least about 95%, 96%, 97%, 98%, It means that 99% of the cells are composed of cells of the specific cell type.
- a "substantially pure" stem cell population is less than about 50%, more preferably about 40%, 30%, 20%, 15%, 10%, 8%, less than 7% of non-stem cells, most. It preferably means a population of cells containing about 5%, 4%, 3%, 2% or less than 1%.
- isolated hematopoietic stem cells in the present disclosure are at least about 50%, more preferably at least about 60%, 65%, 70%, 75% of the cells identified by the markers identified by the present disclosure. , 85%, 90%, 92%, 93%, most preferably at least about 95%, 96%, 97%, 98%, 99%.
- enrichment has the meaning commonly used in the art and typically in a population containing multiple organisms, cells, substances, data, a particular object of interest. It means increasing the content of organisms, cells, substances, data, etc. that have properties.
- a marker for concentrating long-term hematopoietic stem cells is that when a cell population containing long-term hematopoietic stem cells is divided into two fractions by the marker, long-term hematopoietic stem cells are unevenly present in either fraction. Means.
- identification refers to selecting a target such as an organism, cell, substance, or data having the above-mentioned properties from a population including a large number by a specific operation / evaluation method. Selected targets may or may not be physically separated from the population.
- a marker for isolating long-term hematopoietic stem cells is that when a cell population containing long-term hematopoietic stem cells is divided into two fractions by the marker, the long-term hematopoietic stem cells are biased to one of the fractions by a certain degree or more. It means that the number of long-term hematopoietic stem cells present in other fractions is below a certain level.
- long-term hematopoietic stem cells present in fraction A when a cell population containing long-term hematopoietic stem cells is fractionated into fractions A and B, long-term hematopoietic stem cells present in fraction A / (long-term hematopoietic stem cells present in fraction A + long-term hematopoietic stems present in fraction B) It means that the value of stem cell) x2 is 1.4, 1.5, 1.6, 1.7 or 1.8 or more.
- promoting proliferation of long-term hematopoietic stem cells has a meaning commonly used in the art, and typically, shortening the time required for cell division of long-term hematopoietic stem cells, that of long-term hematopoietic stem cells. It means increasing the number of cells, or suppressing the decrease in the content ratio of long-term hematopoietic stem cells due to proliferation, or maintaining or increasing the content ratio in a cell composition containing long-term hematopoietic stem cells and other cells. ..
- Hematopoietic stem cell markers of the present disclosure In general, various cells of a living body have various physics such as the intracellular content of proteins expressed from each gene, the types of proteins exposed on the cell surface, the state of post-translational modification of proteins, and cell morphology. It has a unique state in its physical properties, and the combination of these physical properties makes it possible to identify cells with unique biological functions. These physical properties can be quantified based on certain criteria by various known methods, and therefore, various cells having a certain biological function are a combination of numerical data regarding the quantitative physical properties. Can be specified with. Furthermore, cells can be identified in more detail by combining the combination of the quantitative physical property data and the numerical data obtained from the analysis of the biological property.
- cell differentiation has a hierarchy, and it is understood that cells more closely related in the cell differentiation hierarchy are more similar in their quantitative physical characteristic data.
- cell placement spatial structures based on the similarity of multiple numerical data selected from numerical data on quantitative physical properties and / or numerical data on biological properties, they are located in close proximity to the differentiation lineage. It is possible to obtain a cell arrangement space structure in which certain cells occupy close positions. Then, by using a known differentiation marker or the like, the differentiation direction on the cell arrangement space structure can be specified.
- the physics for identifying the cells at the specific differentiation stage by specifying the region where the cells at the specific differentiation stage exist by using a known differentiation marker or the like.
- a combination of numerical data of physical properties and / or range of biologically specific numerical data is obtained.
- the inventors of the present disclosure identify long-term hematopoietic stem cells with more undifferentiated cell characteristics by using analysis using the constructed cell arrangement spatial structure and further combining cell function analysis at the single cell level. We have succeeded in identifying the combination of marker molecules to be used very quickly and with high accuracy.
- Long-term hematopoietic stem cells with the more undifferentiated cellular properties of the present disclosure can be variously concentrated or isolated using the above markers, depending on the desired accuracy.
- long-term hematopoietic stem cells with the more undifferentiated cellular properties of the present disclosure can be concentrated or isolated by the expression level of a single marker molecule, and in another example, the expression level of multiple marker molecules. Depending on the combination, it can be concentrated or isolated.
- a fraction having an expression level of a certain level or less, a "negative" fraction for the expression of the marker molecule, and a fraction having an expression level of a certain level or more are used.
- the expression of the marker molecule may be referred to as a "positive” fraction.
- the threshold for distinguishing between “negative” and “positive” is usually determined by a person skilled in the art according to the equipment used, the purpose of work, and the like.
- cells can be fractionated based on more detailed differences in expression, for example, the "positive” fraction and the lesser expression of the marker molecule is "weak”.
- a "positive” fraction and a fraction with a lower expression level can be distinguished as a “strongly positive” fraction.
- Long-term hematopoietic stem cells are based on the expression level of a single marker molecule being classified as “negative”, “positive”, “weakly positive”, “strongly positive” or in a more detailed range, or with multiple markers. Concentrated or isolated based on a combination of molecular classifications.
- long-term hematopoietic stem cells with the more undifferentiated cellular properties of the present disclosure are CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200, CD226, CD245, CD31, CD317, CD40, CD41, CD61, CD93, Notch1, CD352, Sigma-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and One or more selected from 1, 2, 3, 4 or 5 or more selected from CD48, or one or more selected from a combination of CD48 and CD34, a combination of CD48 and c-Kit, and a combination of CD48, CD34 and c-Kit. These combinations are isolated or enriched by use as a long-term hematopoietic stem cell isolation marker or enrichment marker.
- long-term hematopoietic stem cells with the more undifferentiated cellular properties of the present disclosure are CD48 (-) and CD34 (+) combinations, CD48 (-) and c-Kit (+) combinations, and. Isolation is performed by using one or more selected from the combination of CD48 ( ⁇ ), CD34 (+) and c-Kit (+) as a long-term hematopoietic stem cell isolation marker.
- Isolation is performed by using the combination of CD48 ( ⁇ ), CD34 (+) and c-Kit (+) as a long-term hematopoietic stem cell isolation marker.
- CD48 ( ⁇ ), CD34 (+) and c-Kit (+) as a long-term hematopoietic stem cell isolation marker, it becomes possible to isolate long-term hematopoietic stem cells with extremely high purity.
- long-term hematopoietic stem cells having the more undifferentiated cellular properties of the present disclosure are CD84 (+), CD111 (+), CD150 (-), GPR56 (-), in addition to the combination of gene expression patterns described above. -), Notch-1 (-), CD52 (-), CD62L (+), CD71 (+), CD93 (-), CD122 (-), CD133 (-), CD275 (-), CD131 (-), Higher purity by combining one or more selected from CD326 (+), CD200 (-), CD200R (-), CD324 (+), CD40 (-), CD11c (-), CD318 (-). It is possible to concentrate long-term hematopoietic stem cells.
- the gene expression mode to be additionally combined is selected from CD111 (+), CD133 ( ⁇ ), CD131 ( ⁇ ), CD93 ( ⁇ ), CD122 ( ⁇ ), CD200 ( ⁇ ), CD324 (+).
- the gene expression mode to be additionally combined is selected from CD111 (+), CD133 ( ⁇ ), and CD131 ( ⁇ ).
- differentiated cells may be excluded from the cell population to be treated in advance.
- a marker generally called a Lineage marker that specifically identifies the differentiated cells can be used.
- a combination of CD11b, CD14, CD19, CD20, CD235ab, CD3, CD4, CD56, and CD8a can be used as the Lineage marker, but is not limited thereto.
- a marker molecule expressed on the cell surface is used as a detection target.
- mRNA transcribed from a gene or a precursor thereof, a peptide or protein translated from the mRNA, or an intermediate product, fragment, modification thereof, etc. can be used.
- the gene product used to identify the hematopoietic stem cells of the present disclosure is not limited to the gene product of the gene specifically described above. Expression or non-expression of one or more additional gene products can be used as an indicator to further classify the hematopoietic stem cells of the present disclosure. In addition, some of the above gene combinations can be replaced by other genes. Such genes can be selected from genes that are functionally or statistically similar to the expression patterns of the genes contained in the above gene combinations. As will be described later, by analyzing the gene expression state in the hematopoietic stem cells of the present disclosure, it is also possible to identify the upper addition or substitution gene.
- the present disclosure discloses CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, CD172a / b, CD200, CD226.
- the present disclosure is a combination of CD48 (-) and CD34 (+), a combination of CD48 (-) and c-Kit (+), and CD48 (-), CD34 (+) and c-Kit (+).
- CD84 (+), CD111 (+), CD150 ( ⁇ ), GPR56 ( ⁇ ), Notch-1 ( ⁇ ), CD52 ( ⁇ ), CD62L. (+), CD71 (+), CD93 (-), CD122 (-), CD133 (-), CD275 (-), CD131 (-), CD326 (+), CD200 (-), CD200R (-), CD324 Concentrate long-term hematopoietic stem cells with higher purity by combining one or more selected from (+), CD40 (-), CD11c (-), CD318 (-) as a long-term hematopoietic stem cell isolation marker or enrichment marker. Is possible.
- the gene expression mode to be additionally combined is selected from CD111 (+), CD133 ( ⁇ ), CD131 ( ⁇ ), CD93 ( ⁇ ), CD122 ( ⁇ ), CD200 ( ⁇ ), CD324 (+).
- the gene expression mode to be additionally combined is selected from CD111 (+), CD133 ( ⁇ ), and CD131 ( ⁇ ).
- the present disclosure also provides an isolated hematopoietic stem cell or a hematopoietic stem cell-containing cell population enriched with hematopoietic stem cells with more undifferentiated cellular properties.
- such hematopoietic stem cells or cell populations are CD10, CD111, CD112, CD131, CD143, CD244, CD275, CD328, CD62L, GPR56, CD133, CD36, CD370, CD84, CD326, CD114, CD116, CD135, 1 selected from CD172a / b, CD200, CD226, CD245, CD31, CD317, CD40, CD41, CD61, CD93, Notch1, CD352, Sigma-10, CD45RO, CD11c, CD52, CD318, CD49f, c-Kit and CD48 1 , 2, 3, 4 or 5 or more, or one or more selected from the combination of CD48 and CD34, the combination of CD48 and c-Kit, and the combination of CD48, CD34 and c-Kit, or the expression mode of these combinations.
- such hematopoietic stem cells or cell populations are CD48 ( ⁇ ) and CD34 (+) combinations, CD48 ( ⁇ ) and c—Kit (+) combinations, and CD48 ( ⁇ ), CD34 (+). And c-Kit (+) are identified by one or more expression modes selected from the combination.
- the cell or cell population expresses or does not express the molecule as a cell surface protein.
- the cell or cell population may be expressed as an mRNA or precursor thereof transcribed from a gene, a peptide or protein translated from the mRNA, or an intermediate product, fragment, modification thereof, or the like. It expresses the molecule.
- the present disclosure describes isolated or concentrated hematopoietic stem cells based on the mode of expression of marker molecules that isolate or concentrate long-term hematopoietic stems.
- a method for producing a cell population containing hematopoietic stem cells is provided.
- the method can be performed by operating a cell sorter linked to a flow cytometer based on the cell surface expression of a marker molecule that identifies long-term hematopoietic stem cells discovered in the present disclosure. Not limited to.
- This disclosure uses the marker molecule for isolating or concentrating long-term hematopoietic stem cells of the present disclosure as an index for cell compositions containing long-term hematopoietic stem cells. Provided is a method for evaluating the content and / or change in the content of long-term hematopoietic stem cells.
- a cell population containing hematopoietic stem cells is prepared, the expression level of a marker molecule for isolating or concentrating long-term hematopoietic stem cells is measured for the cells contained in the cell population, and the expression level is used as a marker in a target sample. Changes in the content and / or content of long-term hematopoietic stem cells by comparison with the expression level of the molecule or by comparison with the expression level of the marker molecule at another time point in the cell composition containing the long-term hematopoietic stem cells. evaluate.
- the cell population containing the hematopoietic stem cells may be cells derived from a living body or cells derived from artificially acquired pluripotency. Further, the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- the expression level of the marker molecule can be measured by a method known to those skilled in the art, for example, using a flow cytometer, an antibody panel, or the like, but is not limited thereto.
- the present disclosure provides a method for testing or evaluating the properties of a cell composition containing long-term hematopoietic stem cells, using the marker molecule for isolating or concentrating the long-term hematopoietic stem cells of the present disclosure as an index.
- a cell population containing hematopoietic stem cells is prepared, the expression level of a marker molecule for isolating or concentrating long-term hematopoietic stem cells is measured for the cells contained in the cell population, and the expression level is used as a marker in a control sample.
- the properties of the cell composition are tested or evaluated by comparison with the expression level of the molecule.
- the control sample may be a sample whose properties as a long-term hematopoietic stem cell-containing cell population have been confirmed by an in vivo or in vitro test, or may be a sample at another time point of the cell population to be tested or evaluated.
- the cell composition may be, but is not limited to, a sample used for hematopoietic stem cell transplantation, for example, a cord blood, bone marrow fluid, or peripheral blood-containing composition.
- the expression level of a marker molecule for isolating or concentrating long-term hematopoietic stem cells is used as an index for the cell composition before and after cell culture and before and after storage for a certain period of time. It is possible to test or evaluate to what extent the properties of long-term hematopoietic stem cells have changed or have not changed in the cell population before and after storage.
- the properties of the hematopoietic stem cell-containing composition may be of a quality that is evaluated according to certain criteria, and for example, quality such as hematopoietic ability maintenance ability, transplant compatibility, or storage stability can be evaluated.
- quality such as hematopoietic ability maintenance ability, transplant compatibility, or storage stability can be evaluated.
- a test transplant composition containing long-term hematopoietic stem cells and a standard composition that has been confirmed to be compatible with transplantation should be compared. Allows the transplant suitability of the test transplant composition to be tested or evaluated.
- the cell population containing the hematopoietic stem cells may be cells derived from a living body or cells derived from artificially acquired pluripotency cells, and can be obtained by culturing umbilical cord blood, bone marrow cells, peripheral blood and the like.
- the cell composition may be, but is not limited to.
- the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- the expression level of the marker molecule can be measured by a method known to those skilled in the art, for example, using a flow cytometer, an antibody panel, or the like, but is not limited thereto.
- the present disclosure provides a method for testing a hematopoietic stem cell-containing composition for predicting the disease prognosis or health status of a subject, using the marker molecule for isolating or concentrating the long-term hematopoietic stem cells of the present disclosure as an index.
- long-term hematopoietic stem cells are simply referred to for cells contained in a cell population containing hematopoietic stem cells obtained from a patient or a healthy person, or for cells contained in a cell population containing hematopoietic stem cells in a patient or a healthy person.
- the expression level of the marker molecule to be released or concentrated is measured, and the measured expression level is used as an index to predict the disease prognosis or future health condition of the subject.
- a cell population containing hematopoietic stem cells is obtained from a patient before and after treatment, and an increase or decrease in the proportion of long-term hematopoietic stem cells is tested to determine the proportion of long-term hematopoietic stem cells.
- a good prognosis for a patient can be predicted when it is increasing or when the decrease is suppressed.
- a cell population containing hematopoietic stem cells in the donor body when treatment is performed by allogeneic transplantation of a living body-derived hematopoietic stem cell-containing composition, a cell population containing hematopoietic stem cells in the donor body, hematopoietic stem cells obtained from the donor, for predicting the prognosis of the patient. It is also possible to test a cell population containing hematopoietic stem cells before or after transplantation, a cell population containing hematopoietic stem cells in the patient's body, or a cell population containing hematopoietic stem cells obtained from the patient.
- the health condition of the patient or a healthy person for example, grasping the health condition in the hematopoietic system or predicting the future health condition. Therefore, the expression level of marker molecules that isolate or concentrate long-term hematopoietic stem cells can be tested.
- any long-term hematopoietic stem cell-containing cell population such as myeloid tissue, cord blood, and peripheral blood can be used.
- the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- the expression level of the marker molecule can be measured by a method known to those skilled in the art, for example, using a flow cytometer, an antibody panel, or the like, but is not limited thereto.
- the present disclosure relates to isolated or concentrated hematopoietic stem cells or cell populations containing hematopoietic stem cells isolated or enriched by markers that isolate or concentrate long-term hematopoietic stem cells.
- the modification comprises inducing various differentiations of the isolated or concentrated hematopoietic stem cells or cell populations containing the hematopoietic stem cells.
- the culture conditions include the composition of the medium, the change of the medium composition, the specific additive contained in the medium, the method of adding the additive, the culture temperature or its modification mode, the culture humidity or its modification mode, the atmospheric pressure or its modification mode, It includes, but is not limited to, the structure or material of the culture vessel or a modification thereof, the structure or material of the culture medium or a modification thereof, and the like.
- the agent includes, but is not limited to, inorganic compounds, organic small molecule compounds, high molecular weight compounds, proteins and nucleic acids.
- the genome editing includes, but is not limited to, a method known to those skilled in the art, including, but is not limited to, a genome editing technique using a CRISPR-Cas system, a TALEN system, or the like. Modifications to long-term hematopoietic stem cell-containing cell populations include changes in the type or amount of molecules expressed on the cell surface, changes in the type or amount of genes transcribed intracellularly, changes in the genomic sequence of cells, or It can be confirmed by the changes in cell morphology and function associated with these, but it is not limited to these.
- the type or amount of any molecule expressed on the surface of the modified cell or the gene transcribed intracellularly is 0.1%, 0.5%, 1%, 2% compared to that before modification.
- the difference is 3%, 4%, 5%, 6%, 7%, 8%, 9% or 10% or more, the cell can be regarded as a modified cell.
- the gene transfer includes a method known to those skilled in the art, and includes, but is not limited to, a gene transfer technique using a viral vector, a gene transfer method using an electroporation method, and the like.
- fusion with the other cells includes those by a method known to those skilled in the art, and the fused cells are from somatic cell populations such as human or primate somatic cells, ES cells, induced pluripotent stem cells or Muse cells.
- Pluripotent stem cells and the like isolated by a specific method can be used, but are not limited thereto.
- the modified cell population can be used as a sample for studying the cell differentiation mechanism and the like, and can also be used for various purposes according to its characteristics.
- long-term hematopoietic stem cells are isolated or concentrated from the living tissue of a patient, and the cells are modified by gene transfer or deletion, expression level adjustment, etc. by genome editing or gene transfer using a vector.
- long-term hematopoietic stem cells existing in vivo are identified by the markers that isolate or concentrate the long-term hematopoietic stem cells of the present disclosure, and the gene is introduced into the cells by genome editing or gene transfer.
- the present invention provides a method for treating a disease, maintaining health, or promoting by producing long-term hematopoietic stem cells having added or modified functions by adding modifications such as deficiency and adjustment of expression level.
- the modification of these cells may be constant or temporary. For example, there may be one that modifies cells only for a specific period at a specific site in the body.
- the present disclosure analyzes long-term hematopoietic stem cell-containing cell populations isolated or concentrated by markers that isolate or concentrate long-term hematopoietic stem cells, and the physical, physical, of the cell populations.
- Provided is a method for obtaining information on chemical or biological characteristics.
- the physical, chemical, and biological characteristics of isolated or concentrated hematopoietic stem cells or hematopoietic stem cells can be evaluated and analyzed using methods known to those of skill in the art.
- the information obtained can be used, for example, in methods for identifying long-term hematopoietic stem cell-containing cell populations based on, for example, physical, chemical, biological, or combinations thereof.
- hematopoietic stem cells By comparing the physical characteristics, chemical characteristics, and biological characteristics of the hematopoietic stem cells obtained by using the markers of the present disclosure with the physical characteristics, chemical characteristics, and biological characteristics, the biological characteristics are concerned.
- a method for isolating or enriching characteristic-based hematopoietic stem cells can be evaluated or characterized. Through such evaluation and characterization, in one embodiment, hematopoietic stem cells are identified using the physical, chemical, and biological characteristics of the hematopoietic stem cells obtained by using the markers of the present disclosure as indicators, and the hematopoietic stem cells are identified.
- a method for producing a cell population containing is provided.
- the present disclosure describes isolated or enriched hematopoietic stem cells or cell populations containing hematopoietic stem cells, or various modifications thereof. It provides a method of evaluating the physiological effects of test substances such as compounds and drugs or environmental factors such as temperature and nutritional conditions on hematopoietic stem cells using cells.
- the isolated or concentrated hematopoietic stem cells or cell population containing hematopoietic stem cells of the present disclosure or various cells obtained by modifying the cells are contacted with a test substance such as a compound or a drug by a known method, or , Evaluate the physiological effects of environmental factors such as temperature and nutritional conditions on cells, such as changes in cell function, changes in proliferative state, and effects on survival.
- a test substance such as a compound or a drug by a known method
- a method for evaluating the effect of culture conditions on the proliferation of long-term hematopoietic stem cells This disclosure is a method for evaluating the effect of culture conditions on the proliferation of long-term hematopoietic stem cells using the marker molecule for isolating or concentrating the long-term hematopoietic stem cells of the present disclosure as an index. I will provide a.
- a cell population containing hematopoietic stem cells is prepared, a cell population containing hematopoietic stem cells is cultured according to the culture conditions to be evaluated, and the long-term hematopoietic stem cells of the present disclosure in the cultured cell population are obtained.
- the expression level of the marker molecule before and after culturing under the culture condition to be evaluated is compared, or the expression level of the marker molecule after culturing under the culture condition to be evaluated is compared. And the expression level of the marker molecule after culturing under the control culture conditions are compared.
- the culture conditions to be evaluated were evaluated as the culture conditions for promoting the proliferation of long-term hematopoietic stem cells, and the proliferation of long-term hematopoietic stem cells occurred after the change of the culture conditions.
- the evaluation target culture condition is evaluated as a culture condition that suppresses the proliferation of long-term hematopoietic stem cells.
- the method for evaluating whether or not long-term hematopoietic stem cells are specifically modified is not limited, and those skilled in the art can use known methods. For example, shortening or prolonging the time required for cell division of a long-term hematopoietic stem cell identified by a marker molecule that isolates or concentrates the long-term hematopoietic stem cell of the present disclosure, for example, of a long-term hematopoietic stem cell identified by the marker molecule of the present disclosure.
- the content ratio of the long-term hematopoietic stem cells specified by the marker molecule of the present disclosure is decreased, maintained or increased in the cell composition containing the long-term hematopoietic stem cells, and the like.
- long-term hematopoietic stem cells are specifically modified.
- the cell population containing the hematopoietic stem cells may be cells derived from a living body or cells derived from artificially acquired pluripotency. Further, the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- factors constituting the cell culture conditions factors usually considered in the art can be evaluated, and for example, the medium composition, temperature, humidity, atmospheric composition, culture substrate, etc. may be changed for the test. However, it is not limited to these.
- the present disclosure provides a method for screening compounds that modify the properties of long-term hematopoietic stem cells using a marker molecule that isolates or concentrates long-term hematopoietic stem cells as an index.
- a cell population containing hematopoietic stem cells is typically prepared, and the cells or cell population treated with the candidate compound are compared with the untreated cells or cell population, and the long-term hematopoietic stem cells of the present disclosure are compared.
- a compound that shortens or prolongs the time required for long-term hematopoietic stem cells to divide a compound that increases the number of long-term hematopoietic stem cells, and a cell composition containing long-term hematopoietic stem cells, the content ratio of long-term hematopoietic stem cells is reduced.
- Compounds that maintain or increase, compounds that prolong or shorten the survival time of long-term hematopoietic stem cells, compounds that suppress or promote cell death of long-term hematopoietic stem cells, modifications of long-term hematopoietic stem cells (gene transfer, genome editing, drug processing efficiency, etc.) Compounds that promote or suppress, compounds that differentiate long-term hematopoietic stem cells into specific cell types, compounds that suppress differentiation into specific cell types, compounds that suppress or promote the differentiation of long-term hematopoietic stem cells, differentiation into specific cell types It is possible to obtain a compound or the like that suppresses.
- the cell population containing the hematopoietic stem cells may be cells derived from a living body or cells derived from artificially acquired pluripotency. Further, the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- the present disclosure provides a method for screening compounds that induce long-term hematopoietic stem cells using a marker molecule that isolates or concentrates long-term hematopoietic stem cells as an index.
- a marker molecule that isolates or concentrates long-term hematopoietic stem cells as an index.
- cells other than long-term hematopoietic stem cells are typically prepared, the cells or cell populations are treated with a candidate compound, and long-term hematopoietic stem cells in the treated cells or cell populations are isolated or Using the marker to concentrate as an index, obtain a compound that induces long-term hematopoietic stem cells.
- the cells other than long-term hematopoietic stem cells are not particularly limited, and for example, cells derived from a living body, cells artificially acquired pluripotency, cells modified such as gene transfer, and the like can be used.
- cells having artificial pluripotency such as ES cells and iPS cells can be used, but the cells are not limited thereto.
- the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- Culture medium that modifies the characteristics of long-term hematopoietic stem cells The present disclosure describes a method for producing a culture medium or a culture auxiliary additive that specifically modifies the characteristics of long-term hematopoietic stem cells using a marker molecule that isolates or concentrates long-term hematopoietic stem cells as an index.
- a culture medium or a culture auxiliary additive produced by the method.
- a cell population containing hematopoietic stem cells is prepared, and the cell population containing hematopoietic stem cells is cultured in a medium having a composition to be evaluated or a medium containing a culture auxiliary additive to be evaluated, and the long-term period of the present disclosure is disclosed.
- the growth state of the cells before and after the change of the culture conditions is evaluated, and the composition of the culture medium or the culture auxiliary additive that specifically modifies the characteristics of the long-term hematopoietic stem cells is determined.
- Culture media and culture aid additives are configured, for example, as solid or liquid compositions.
- the culture medium can also include a particular physical structure, such as shape, material, volume, etc., in its composition.
- the method for evaluating whether or not the characteristics of long-term hematopoietic stem cells are specifically modified is not limited, and those skilled in the art can use known methods. For example, shortening or prolonging the time required for cell division of a long-term hematopoietic stem cell identified by a marker molecule that isolates or concentrates the long-term hematopoietic stem cell of the present disclosure, for example, of a long-term hematopoietic stem cell identified by the marker molecule of the present disclosure.
- the content ratio of the long-term hematopoietic stem cells specified by the marker molecule of the present disclosure is decreased, maintained or increased in the cell composition containing the long-term hematopoietic stem cells, and the like.
- the cell population containing the hematopoietic stem cells may be cells derived from a living body or cells derived from artificially acquired pluripotency. Further, the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- the present disclosure provides a method for adjusting the culture conditions for promoting the proliferation of long-term hematopoietic stem cell using the marker molecule for isolating or concentrating the long-term hematopoietic stem cell of the present disclosure as an index.
- a cell population containing hematopoietic stem cells is prepared, a cell population containing hematopoietic stem cells is cultured under the adjusted culture conditions, and the marker molecule is used as an index before and after the change of the culture conditions.
- the proliferation status of the cells is evaluated, and the culture conditions are adjusted so that the proliferation of long-term hematopoietic stem cells is promoted.
- factors constituting cell culture conditions factors usually considered in the art can be evaluated. For example, medium components, culture aids, temperature, humidity, atmospheric composition, culture substrate shape, material, surface treatment, culture vessel volume, shape, material, surface treatment, culture vessel, medium, or physics added to the cells themselves.
- Target stimuli, changes over time, etc. can be evaluated as factors, but the factors are not limited to these.
- the method for evaluating whether or not the proliferation of long-term hematopoietic stem cells is promoted is not limited, and a person skilled in the art can use a known method. For example, the time required for cell division of long-term hematopoietic stem cells identified by the marker molecule that isolates or concentrates the long-term hematopoietic stem cells of the present disclosure is shortened, and the number of cells of the long-term hematopoietic stem cells identified by the marker molecule of the present disclosure.
- long-term hematopoietic hematopoiesis is obtained when the content ratio of long-term hematopoietic stem cells specified by the marker molecule of the present disclosure is maintained or increased. It is evaluated that the proliferation of stem cells was promoted.
- the cell population containing the hematopoietic stem cells may be cells derived from a living body or cells derived from artificially acquired pluripotency. Further, the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- the present disclosure adjusts the culture conditions using the long-term hematopoietic stem cell isolation or enrichment marker molecule of the present disclosure as an index.
- a method for producing isolated or concentrated hematopoietic stem cells or a cell population containing hematopoietic stem cells is provided.
- a cell population containing hematopoietic stem cells is prepared, and the proliferation of long-term hematopoietic stem cells is promoted by a culture condition adjusted in advance to promote the proliferation of long-term hematopoietic stem cells, for example, the above-mentioned method for adjusting the culture conditions.
- a culture condition adjusted in advance to promote the proliferation of long-term hematopoietic stem cells
- the above-mentioned method for adjusting the culture conditions By culturing a cell population containing hematopoietic stem cells under the culture conditions adjusted so as to be produced, an isolated or concentrated hematopoietic stem cell or a cell population containing hematopoietic stem cells is produced.
- the growth status of the cell is monitored by a marker molecule for isolating or concentrating the long-term hematopoietic stem cell of the present disclosure, and the long-term growth state is monitored.
- a method for producing isolated or concentrated hematopoietic stem cells or a cell population containing hematopoietic stem cells is produced.
- the method for evaluating whether or not the proliferation of long-term hematopoietic stem cells is promoted is not limited, and a person skilled in the art can use a known method. For example, the time required for cell division of long-term hematopoietic stem cells identified by the marker molecule that isolates or concentrates the long-term hematopoietic stem cells of the present disclosure is shortened, and the number of cells of the long-term hematopoietic stem cells identified by the marker molecule of the present disclosure.
- long-term hematopoietic hematopoiesis is obtained when the content ratio of long-term hematopoietic stem cells specified by the marker molecule of the present disclosure is maintained or increased. It is evaluated that the proliferation of stem cells was promoted.
- the cell population containing the hematopoietic stem cells may be cells derived from a living body or cells derived from artificially acquired pluripotency. Further, the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- the factors constituting the cell culture conditions factors usually considered in the art can be evaluated, and for example, the medium composition, temperature, humidity, atmospheric composition, culture substrate, etc. may be changed for the test. However, it is not limited to these.
- the culture conditions may be adjusted so as to promote the proliferation of long-term hematopoietic stem cells from the start of culture of the cell population collected from the living body, and the culture of long-term hematopoietic stem cells is optimized for a certain period of time. After culturing under no conditions, the culture conditions may be adjusted so as to promote the proliferation of long-term hematopoietic stem cells after the number of cells has increased to some extent.
- cells are selected using a known method such as a cell sorter using a molecular marker for isolating or concentrating the long-term hematopoietic stem cells of the present disclosure as an index, and further adjusted if necessary.
- the culture can be continued under the same culture conditions.
- a plurality of different methods can be combined, and the same method or different methods can be repeatedly applied.
- the present disclosure provides a gene modification method for imparting the characteristics of long-term hematopoietic stem cells to cells using a marker for isolating or concentrating long-term hematopoietic stem cells as an index.
- a genetically modified cell modified with a candidate gene is prepared, and the marker for isolating or concentrating the long-term hematopoietic stem cells of the present disclosure in the genetically modified cell is not subjected to genetic modification. Compare with markers that isolate or concentrate long-term hematopoietic stem cells of the present disclosure in cells.
- the markers that isolate or concentrate the long-term hematopoietic stem cells of the present disclosure before and after gene modification in the same cells are compared.
- a genetic modification that gives a more positive result for the marker that isolates or concentrates the long-term hematopoietic stem cells of the present disclosure is identified as a genetic modification that imparts the characteristics of long-term hematopoietic stem cells to the cells.
- the gene modification can be performed by various methods known to those skilled in the art, for example, permanent or transient gene transfer using nucleic acids or other vectors, gene or gene expression on the genome by genome editing. Deletions, additions, mutations, etc. of controlling factors can be utilized.
- the mutation includes, but is not limited to, deletion, addition, substitution, etc. of a base in a base sequence constituting a gene or a factor controlling gene expression.
- the gene-modified cell may be a cell derived from a living body or a cell derived from an artificially acquired pluripotency cell.
- the cells tested for the marker may be all or part of the cells contained in the cell population.
- compositions or Kits for Identifying, Isolating or Concentrating Long-Term Hematopoietic Stem Cells comprising reagents that quantify or detect marker molecules that identify, isolate or concentrate long-term hematopoietic stem cells.
- a composition or kit for release or concentration is provided.
- the reagent for quantifying or detecting the marker molecule a reagent usually used in the art can be used, and for example, an antibody labeled with a fluorescent dye, a nucleic acid, a low molecular weight compound, a high molecular weight compound and the like can be used. Not limited to these.
- compositions or kits for identifying, isolating or concentrating long-term hematopoietic stem cells of the present disclosure can be used for identifying, isolating or concentrating long-term hematopoietic stem cells in a cell population collected from a living body, as well as raw.
- the composition or kit for identifying long-term hematopoietic stem cells can further contain a drug for modifying the function of long-term hematopoietic stem cells, and is a therapeutic agent for a disease treated by modifying the function of long-term hematopoietic stem cells. , Or provide a treatment method.
- long-term hematopoietic stem cells or cells obtained by modifying them
- the present disclosure describes long-term hematopoietic stem cells isolated or concentrated by a marker for isolating or concentrating long-term hematopoietic stem cells, or various cells obtained by modifying the long-term hematopoietic stem cells.
- a drug for treating a disease by administration to a patient a drug used for preemptive medical treatment to a disease reserve army, a drug used for preventive medical treatment or health maintenance (improvement) for a healthy person, or a treatment using the drug.
- long-term hematopoietic stem cells of the present disclosure or various cells obtained by modifying them are used as a pharmaceutical
- long-term hematopoietic stem cells obtained from a cell population derived from a patient or various cells obtained by modifying the cells are administered to the patient.
- long-term hematopoietic stem cells obtained from a cell population derived from another donor or various cells obtained by modifying the stem cells may be administered to the patient.
- other drugs or treatment methods can be used in combination.
- a hematopoietic stem cell-containing cell population derived from activated cells long-term hematopoietic stem cells isolated or concentrated by the markers that isolate or concentrate the long-term hematopoietic stem cells of the present disclosure are used to treat the disorder in patients with hematopoietic disorders. It can be administered as a medicine to do so.
- long-term hematopoietic stem cells with a marker that isolates or concentrates the long-term hematopoietic stem cells of the present disclosure from a population of hematopoietic stem cell-containing cells obtained from a patient's biological tissue with or without an artificial pluripotency step.
- the cell population containing the hematopoietic stem cells may be cells derived from a living body or cells derived from artificially acquired pluripotency. Further, the cell for measuring the expression level of the marker molecule may be all or a part of the cells contained in the cell population.
- CCD chronic granulomatosis
- ALD adrenoleukodystrophy
- MLD metachromatic leukodystrophy
- Gauche's disease and Fabry disease.
- Diseases mucopolysaccharidosis, HIV, ADA-SCID, X-SCID, salacemia, but not limited to these.
- the gene-modified cells can be produced by various methods known to those skilled in the art, for example, permanent or transient gene transfer using nucleic acids or other vectors, genes on the genome by genome editing, or Deletions, additions, mutations, etc. of factors that control gene expression can be utilized.
- the mutation includes, but is not limited to, deletion, addition, substitution, etc. of a base in a base sequence constituting a gene or a factor controlling gene expression.
- the gene-modified cell may be a cell derived from a living body or a cell derived from an artificially acquired pluripotency cell.
- the cells tested for the marker may be all or part of the cells contained in the cell population.
- the collected data was analyzed using FlowJo (manufactured by BD) or an algorithm developed by the applicant (also disclosed in PCT / JP2019 / 051270, PCT / JP2020 / 025915, etc.).
- the algorithm generates a cell arrangement space structure based on the similarity of a plurality of numerical data selected from numerical data related to quantitative physical properties and / or numerical data related to biological properties obtained by cell staining or the like. By doing so, a cell arrangement space structure in which cells in close positions on the differentiation lineage occupy close positions is generated, and a differentiation direction on the cell arrangement space structure is specified by using a known differentiation marker or the like. It is an algorithm.
- Antibodies to the following were used for cell staining: human CD56, human CD3, human CD16, human CD19, human CD14, human CD45, mouse Gr-1, mouse CD45, and Propidium Iodide Solution (manufactured by BioLegend) for cell staining. Using.
- IMDM Insulin-Transferrin-Selenium_Ethanolamine
- ITS-X Insulin-Transferrin-Selenium_Ethanolamine
- Pencillin-Streptomycin-Glutamin manufactured by Gibco.
- CD34 + CD117CD48-fraction is a fraction mainly composed of long-term hematopoietic stem cells.
- Example 1 Generation of cell arrangement space structure in which cells in close positions on the differentiation lineage occupy close positions Red blood cells are removed and concentrated from fresh umbilical cord blood obtained from a living body, and a cell population containing hematopoietic stem cells Got A CD34-positive cell fraction was roughly purified from the cell population containing the hematopoietic stem cells using EASYSep Human Cord Blood CD34 Positive Selection Kit II (manufactured by STEMCELL Technologies).
- the antibodies contained in CD11b, CD14, CD19, CD20, CD235ab, CD3, CD4, CD56, CD8a, CD34, CD38, CD90, CD45RA, CD49f and LEGENDSclean Human PE Kit manufactured by BioLegend
- Dead cell staining was performed using the cells, and the staining intensity of each antibody was quantified for each cell.
- a cell arrangement space structure was generated in which cells in close positions on the differentiation lineage occupy close positions. A visualization of a part of the structure is illustrated in FIGS. 1 and 2.
- FIG. 1 and 2 A visualization of a part of the structure is illustrated in FIGS. 1 and 2.
- FIG. 1 shows a two-dimensional image in which the positions of living cells after staining of dead cells are assigned to a three-dimensional space consisting of a synthetic axis 1, a synthetic axis 2, and a synthetic axis 3 generated from a combination of quantified data. It is a thing.
- FIG. 2 is a visualization of the expression level of CD34, which is known to exhibit a characteristic expression mode in hematopoietic stem cells, for each cell assigned a position to the spatial structure. CD34-positive cells are unevenly distributed in a part of the spatial structure.
- Example 2 Relationship between long-term hematopoietic stem cells and CD34 expression pattern CD3 (-) CD34 (-) (CD3 negative, CD34 negative) isolated from human umbilical cord blood to investigate the relationship between long-term hematopoietic stem cell and CD34 expression pattern. ) Cells or CD3 (-) CD34 (+) (CD3 negative, CD34 positive) cells were transplanted into immunodeficient mice and the chimeric rate after transplantation was tested. When the chimera rate was tested 4 weeks, 8 weeks and 12 weeks after transplantation, CD3 (-) CD34 (+) cells had significantly higher survival rates and chimeras than CD3 (-) CD34 (-) cells. Shown the rate. The results are shown in FIG. The negative and positive categories of each marker were based on the cell sorting results of BD FACSAria III used. This result indicates that long-term hematopoietic stem cells are associated with the expression mode of CD34 (+).
- fraction P6 CD34 (+), CD38 (+)
- fraction P7 CD34 (+), CD38 (CD34 (+), CD38 (+)
- fraction P8 CD34 (+), CD38 (-), CD45RA (-), CD90 (-)
- fraction P9 CD34 (+), CD38.
- CFU assays were performed on (-), CD45RA (+) and CD90 (-)) to test the proliferation and differentiation potential of hematopoietic stem cells in each fraction.
- Example 3 Cell function analysis at the 1-cell level A CFU assay was performed at the 1-cell level to confirm the undifferentiated nature of the cells identified by the markers for identifying human long-term hematopoietic stem cells. An example is shown in FIG. By combining the cell function analysis results at the one-cell level with the index sort analysis, it becomes possible to link the undifferentiated state of each cell with the expression mode of each marker. By arranging the relationship between the marker obtained by the analysis and the undifferentiated property on the cell arrangement space structure in which cells in close positions on the differentiation lineage occupy close positions, cell differentiation on the cell arrangement space structure is performed. The direction was fixed. FIG. 7 shows an example of the results of the analysis of CD34-positive cells.
- Example 4 Marker for enriching or isolating long-term hematopoietic stem cells
- CD34-positive cells are expressed along a differentiation direction determined by the cell arrangement spatial structure in which cells in close positions on the differentiation line occupy close positions.
- the proliferative and differentiating ability of the hematopoietic stem cells of the cells identified by these markers was confirmed by the CFU assay, the cells identified by these markers had higher hematopoietic stem cell proliferation and differentiation than the control (CD34 (+) cells). It showed the ability (Fig. 9).
- the above marker for concentrating long-term hematopoietic stem cells in the cell population pre-concentrated with CD34 (+) is CD34. Even when applied to a cell population that has not been pre-concentrated with (+), each can be used alone as a marker for concentrating long-term hematopoietic stem cells.
- Example 5 Marker for specifying long-term hematopoietic stem cells in more detail Among the above-specified markers, cells specified by a marker consisting of a combination of c-Kit (+), CD34 (+), and CD48 (-). The characteristics of the were analyzed in more detail. Cell populations pre-concentrated with CD34 (+) from cord blood are gated with c-Kit (+) and CD34 (+) (fraction P1), and further gated with CD48 (-) (fraction P2). An example of the FACS plot is shown in FIG. Of the fractions P1, most of the cells were fractionated into CD48 (+) (fraction non-P2) and only some were fractionated into P2.
- FIG. 12 shows the results of comparing the cell numbers of the CD34-positive fraction and the P2 fraction contained in 1 ml of umbilical cord blood. It is confirmed that the cells fractionated into the P2 fraction are a small part of the cell population pre-concentrated with CD34 (+). It should be noted that since most of the cord blood is CD34 (-), the proportion of cells in the P2 fraction in the entire cord blood sample is extremely low.
- P2 Cell fractions (P2) specified by c-Kit (+), CD34 (+), CD48 (-) and cell images specified by c-Kit (+), CD34 (+), CD48 (+).
- the proliferation and differentiation potential of hematopoietic stem cells in minutes (non-P2) was compared by CFU assay. As a result, it was confirmed that the P2 fraction has an extremely high ability to proliferate and differentiate hematopoietic stem cells (Fig. 14).
- the undifferentiated state of the cell fraction P2 and the cell fraction non-P2 were compared by a transplantation test.
- Cell transplantation was performed 12 hours or 24 hours after irradiation, and the chimera rate in human blood cells (human CD45 positive cells) in the bone marrow and the chimera rate in human granulocyte cells (hGra) at 20 weeks after transplantation. It was confirmed that the cell fraction P2 showed an extremely high chimera rate in all the results (FIG. 15).
- CD34 (+), CD48 (+) identified cell fractions (non-P2) were obtained and the proliferative and differentiating potential of hematopoietic stem cells was compared by CFU assay.
- the P2 fraction has an extremely high ability to proliferate and differentiate hematopoietic stem cells.
- the results of the bone marrow cell sample are shown in FIG. 16 and the mobilized peripheral blood sample is shown in FIG.
- the results of the P1 fraction, which is the fraction specified by c-Kit (+) and CD34 (+), are also shown.
- the CD34 (+) fraction was fractionated into c-Kit (+) and c-Kit (-) fractions, and the proliferative and differentiating ability of hematopoietic stem cells for each was measured by the CFU assay.
- the c-Kit (+) fraction showed almost the same proliferation and differentiation ability of hematopoietic stem cells as the CD34 (+) fraction, whereas the c-Kit (-) fraction showed that of hematopoietic stem cells. It showed almost no growth / differentiation ability (Fig. 18).
- a marker consisting of a combination of CD48 (-) and CD34 (+) and a combination of CD48 (-) and c-Kit (+) is also used as a marker capable of efficiently concentrating or isolating long-term hematopoietic stem cells. It is understood to get.
- Example 6 Additional Marker An additional marker for further fractionating the P2 fraction by analysis using the cell arrangement spatial structure in which cells in close positions on the above differentiation line occupy close positions. was identified. Specifically, in the above analysis, a marker that is unevenly distributed in the differentiation direction or the undifferentiation direction is selected as a candidate, and cells of the P2 fraction are divided into a test fraction and a control fraction based on the expression level of the marker, and each of them is divided into two. The proliferation and differentiation potential of hematopoietic stem cells in the fraction was compared by CFU assay. From the obtained results, the efficiency of long-term hematopoietic stem cell concentration in the test fraction was quantified by the following formula.
- Concentration ratio CFU-GEMM occupies the colonies generated from the test colony / CFU-GEMM occupies the colonies generated from the control fraction CFU-GEMM occupies the colonies generated from the test fraction and the control fraction If they are the same, the concentration ratio is 1.
- each marker indicates the one having a relatively high expression level as (+) and the one having a relatively low expression level as (-) when the above P2 is divided into two, and are presented by the cell sorter. Note that it has a different meaning than negative, positive, etc.
- additional markers include: CD84 (+), CD111 (+), CD150 (-), GPR56 (-), Notch-1 (-), CD52 (-), CD62L (+), CD71 (+), CD93 (-), CD122 (-) ), CD133 (-), CD275 (-), CD131 (-), CD326 (+), CD200 (-), CD200R (-), CD324 (+), CD40 (-), CD11c (-), CD318 (-). ).
- Example 7 Identification of long-term hematopoietic stem cells after in vitro culture
- the 300 cells contained in the P2 fraction or the non-P2 fraction were cultured under the conditions of addition or no addition of UM171, and the total number of cells and the ratio of undifferentiated cells (P1) after 7 days were tested.
- the results of the P2 fraction are shown in FIG. 20, and the results of the non-P2 fraction are shown in FIG. From this study, it is understood that the markers that isolate or concentrate the long-term hematopoietic stem cells of the present disclosure can identify long-term hematopoietic stem cells after in vitro culture. It can be further confirmed by functional tests such as CFU assay that the markers for isolating or concentrating long-term hematopoietic stem cells of the present disclosure can identify long-term hematopoietic stem cells after in vitro culture.
- Example 8 Gene transfer into isolated or concentrated long-term hematopoietic stem cells
- Long-term hematopoietic stem cells isolated or concentrated by the markers that isolate or concentrate the long-term hematopoietic stem cells of the present disclosure are characteristic of long-term hematopoietic stem cells even after gene transfer.
- a functional test by CFU assay is performed to confirm whether or not the disease is maintained. Specifically, the GFP reporter gene was introduced into cells contained in the P2 fraction, non-P2 fraction, or DC34-positive fraction using a lentiviral vector, and a CFU assay was performed on the cells into which these genes were introduced. This is performed to confirm that the gene-introduced cells derived from the P2 fraction maintain their proliferative and differentiating ability.
- the present disclosure describes long-term hematopoietic stem cells, a method for isolating or concentrating long-term hematopoietic stem cells, a method for producing isolated or concentrated long-term hematopoietic stem cells or a cell population containing long-term hematopoietic stem cells, isolated or concentrated long-term hematopoietic stem cells or long-term.
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Abstract
Description
造血幹細胞を含む細胞集団を準備する工程と、
CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせの発現様式を指標として、細胞を単離または濃縮する工程と
を含む、単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団の製造方法を提供する。
造血幹細胞を含む細胞集団を準備する工程と、
各細胞における各細胞のCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせマーカーの発現量を測定する工程と
を含む、前記細胞集団における造血幹細胞の含有量および/または含有量の変化を評価する方法を提供する。
造血幹細胞を含む細胞集団を準備する工程と、
前記造血幹細胞を含む細胞集団に含まれる細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、前記細胞集団の品質を試験する工程と
を含む、造血幹細胞を含む細胞集団の試験方法を提供する。
造血幹細胞を含む細胞集団を準備する工程と、
前記造血幹細胞を含む細胞集団に含まれる細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、前記細胞集団の移植適合性を試験する工程と
を含む、造血幹細胞を含む細胞集団の試験方法を提供する。
被験者の疾患予後予測または健康状況予想のために、
患者由来の造血幹細胞を含む細胞集団に含まれる細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を取得する工程と、
前記マーカーの発現量を対照と比較する工程と
を含む、造血幹細胞を含む細胞集団を試験する方法を提供する。
造血幹細胞を含む細胞集団を準備する工程と、
候補化合物により造血幹細胞を含む細胞集団を処理する工程と
候補化合物により処理された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、候補化合物を選択する工程と
を含む、造血幹細胞の特性を修飾する化合物のスクリーニング方法を提供する。
造血幹細胞を含む細胞集団を準備する工程と、
調整された培養条件により造血幹細胞を含む細胞集団を培養する工程と、
調整された培養条件により培養された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、培養条件の調整方法を決定する工程と
を含む、造血幹細胞の特性を修飾する培養条件の調整方法を提供する。
造血幹細胞を含む細胞集団を準備する工程と、
前記細胞集団に含まれる細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせの発現量を指標として、造血幹細胞の特性を修飾するように培養条件を調整する工程と
を含む、単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団の製造方法を提供する。
造血幹細胞を含む細胞集団を準備する工程と、
評価対象の構成を有する培地、または評価対象の培養補助添加物を含む培地により、造血幹細胞を含む細胞集団を培養する工程と、
培養された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせマーカーの発現量を指標として培養培地または培養補助添加物の構成を決定する工程と、
上記決定された構成を有する培養培地または培養補助添加物を製造する工程と
を含む、造血幹細胞の特性を修飾する培養培地または培養補助添加物を製造する方法を提供する。
細胞集団を準備する工程と、
被験化合物により細胞集団を処理する工程と、
候補化合物により処理された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせマーカーの発現量を指標として、候補化合物を選択する工程と
を含む、造血幹細胞を誘導する化合物のスクリーニング方法を提供する。
[項目1]
CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせからなる、造血幹細胞単離マーカーまたは濃縮マーカー。
[項目2]
CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上からなる、項目1に記載の造血幹細胞単離マーカーまたは濃縮マーカー。
[項目3]
CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせの発現様式によって特定される、単離された造血幹細胞または造血幹細胞が濃縮された造血幹細胞含有細胞集団。
[項目4]
CD48(-)およびCD34(+)の組み合わせ、CD48(-)およびc-Kit(+)の組み合わせ、ならびにCD48(-)、CD34(+)およびc-Kit(+)の組み合わせから選択される1以上の発現様式によって特定される、項目3に記載の単離された造血幹細胞または造血幹細胞が濃縮された造血幹細胞含有細胞集団。
[項目5]
造血幹細胞を含む細胞集団を準備する工程と、
CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせの発現様式を指標として細胞を単離または濃縮する工程と
を含む、単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団の製造方法。
[項目6]
CD48(-)およびCD34(+)の組み合わせ、CD48(-)およびc-Kit(+)の組み合わせ、ならびにCD48(-)、CD34(+)およびc-Kit(+)の組み合わせから選択される1以上の発現様式を指標とする、
項目5に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団の製造方法。
[項目7]
さらに修飾を含む、請求項1または2に記載のマーカーにより同定、単離、または濃縮された造血幹細胞、または、項目3または4に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団、または項目5または6のいずれか一項に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団。
[項目8]
造血幹細胞を含む細胞集団を準備する工程と、
各細胞における各細胞のCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を測定する工程と
を含む、前記細胞集団における造血幹細胞の含有量および/または含有量の変化を評価する方法。
[項目9]
造血幹細胞を含む細胞集団を準備する工程と、
前記造血幹細胞を含む細胞集団に含まれる細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD48、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、前記細胞集団の品質を試験する工程と
を含む、造血幹細胞を含む細胞集団の試験方法。
[項目10]
前記品質が造血能維持能力、移植適合性、または保存安定性である、項目9に記載の試験方法。
[項目11]
被験者由来の造血幹細胞を含む細胞集団に含まれる細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を取得する工程と、
被験者の疾患予後予測または健康状況予想のために、前記マーカーの発現量を対照と比較する工程と
を含む、造血幹細胞を含む細胞集団の試験方法。
[項目12]
造血幹細胞を含む細胞集団を準備する工程と、
候補化合物により造血幹細胞を含む細胞集団を処理する工程と、
候補化合物により処理された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、候補化合物を選択する工程と
を含む、造血幹細胞の特性を修飾する化合物のスクリーニング方法。
[項目13]
造血幹細胞を含む細胞集団を準備する工程と、
調整された培養条件により造血幹細胞を含む細胞集団を培養する工程と、
調整された培養条件により培養された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として培養条件を決定する工程と
を含む、造血幹細胞の特性を修飾する培養条件の調整方法。
[項目14]
造血幹細胞を含む細胞集団を準備する工程と、
評価対象の構成を有する培地、または評価対象の培養補助添加物を含む培地により、造血幹細胞を含む細胞集団を培養する工程と、
培養された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として培養培地または培養補助添加物の構成を決定する工程と、
上記決定された構成を有する培養培地または培養補助添加物を製造する工程と
を含む、造血幹細胞の特性を修飾する培養培地または培養補助添加物を製造する方法。
[項目15]
前記造血幹細胞を含む細胞集団が、生体由来の細胞または人工的に多能性を獲得した細胞から誘導された細胞である、項目5~14のいずれか一項に記載の方法。
[項目16]
前記造血幹細胞を含む細胞集団が、生体由来の細胞または人工的に多能性を獲得した細胞から誘導された細胞である、項目5~7のいずれか一項に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団。
[項目17]
細胞集団を準備する工程と、
被験化合物により細胞集団を処理する工程と、
候補化合物により処理された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、候補化合物を選択する工程と
を含む、造血幹細胞を誘導する化合物のスクリーニング方法。
[項目18]
CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせの発現量を定量または検出する試薬を含む、造血幹細胞を同定、検出、単離または濃縮するための組成物またはキット。
[項目19]
請求項1または2に記載のマーカーにより同定、単離、または濃縮された造血幹細胞、または、項目3、4および7のいずれか一項に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団、または項目5または6に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団を分析することで得られた物理的特徴、化学的特徴、生物学的特徴の組み合わせを特定する方法。
[項目20]
請求項1または2に記載のマーカーにより同定、単離、または濃縮された造血幹細胞、または、項目3、4および7のいずれか一項に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団、または項目5または6に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団を分析することで得られた物理的特徴、化学的特徴、生物学的特徴の組み合わせを指標として造血幹細胞を同定する分類器を製造する方法。
[項目21]
請求項1または2のいずれか一項に記載のマーカーにより同定、単離、または濃縮された造血幹細胞、または、項目3、4および7のいずれか一項に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団、または項目5または6に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団を含む、造血幹細胞を投与することで改善または予防される疾患または障害を予防または治療するための医薬。
[項目22]
造血幹細胞を投与することで改善または予防される疾患または障害が、異染性白質ジストロフィー(MLD)、ゴーシェ病、ファブリー病、ムコ多糖症、HIV、ADA-SCID、X-SCIDまたはサラセミアである、項目21に記載の医薬。
[項目23]
造血幹細胞を投与することで改善または予防される疾患または障害が、慢性肉芽腫症(CGD)または副腎白質ジストロフィー(ALD)である、項目21に記載の医薬。
本開示において、複数の数値の範囲が示された場合、それら複数の範囲の任意の下限値および上限値の組み合わせからなる範囲も同様に意味する。
一般に生体の各種細胞は、各遺伝子から発現されるタンパク質の細胞内含量、細胞表面に露出されるタンパク質の種類、タンパク質の翻訳後修飾の状態、細胞形態等の様々な物理的特性において固有の状態を有しており、これらの物理的特性の組み合わせによって、固有の生物学的機能を有する細胞を特定することが可能である。これらの物理的特性は公知の各種方法により一定の基準に基づいて定量化することが可能であり、したがって、一定の生物学的機能を有する各種細胞は、その定量物理的特性に関する数値データの組み合わせで特定することができる。さらに、当該定量物理的特性データの組み合わせと、生物学的特性に関する分析から得られた数値データを組み合わせることにより、より詳細に細胞を特定することができる。
好ましくは、追加して組み合わされる遺伝子発現様式は、CD111(+)、CD133(-)、CD131(-)、CD93(-)、CD122(-)、CD200(-)、CD324(+)から選択される。より好ましくは、追加して組み合わされる遺伝子発現様式は、CD111(+)、CD133(-)、CD131(-)から選択される。
好ましくは、追加して組み合わされる遺伝子発現様式は、CD111(+)、CD133(-)、CD131(-)、CD93(-)、CD122(-)、CD200(-)、CD324(+)から選択される。より好ましくは、追加して組み合わされる遺伝子発現様式は、CD111(+)、CD133(-)、CD131(-)から選択される。
本開示はまた、より未分化な細胞特性を有する、単離された造血幹細胞または造血幹細胞が濃縮された造血幹細胞含有細胞集団を提供する。一態様において、このような造血幹細胞または細胞集団は、CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1、2、3、4または5以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせの発現様式によって特定される。一態様において、このような造血幹細胞または細胞集団は、CD48(-)およびCD34(+)の組み合わせ、CD48(-)およびc-Kit(+)の組み合わせ、ならびにCD48(-)、CD34(+)およびc-Kit(+)の組み合わせから選択される1以上の発現様式によって特定される。典型的には、当該細胞または細胞集団は、細胞表面タンパク質として前記分子を発現しているまたは発現していない。他の非限定的な例として、当該細胞または細胞集団は、遺伝子から転写されるmRNAもしくはその前駆体、当該mRNAから翻訳されるペプチド、タンパク質、またはこれらの中間産物、断片、修飾物などとして、前記分子を発現している。
本開示は、長期造血幹細胞を単離または濃縮するマーカー分子の発現様式に基づいて、単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団の製造方法を提供する。
一例において、前記方法は、本開示において発見された長期造血幹細胞を特定するマーカー分子の細胞表面発現に基づいて、フローサイトメーターに連結されたセルソーターを動作させることによって実施することができるが、これに限定されない。
本開示は、長期造血幹細胞を含有する細胞組成物について、本開示の長期造血幹細胞を単離または濃縮するマーカー分子を指標として、長期造血幹細胞の含有量および/または含有量の変化を評価する方法を提供する。典型的には、造血幹細胞を含む細胞集団を準備し、当該細胞集団に含まれる細胞について、長期造血幹細胞を単離または濃縮するマーカー分子の発現量を測定し、当該発現量を対象サンプルにおけるマーカー分子の発現量と比較し、あるいは、前記長期造血幹細胞を含有する細胞組成物の別の時点におけるマーカー分子の発現量と比較することにより、長期造血幹細胞の含有量および/または含有量の変化を評価する。
本開示は、長期造血幹細胞を含有する細胞組成物について、本開示の長期造血幹細胞を単離または濃縮するマーカー分子を指標として、当該細胞組成物の性質を試験または評価する方法を提供する。典型的には、造血幹細胞を含む細胞集団を準備し、当該細胞集団に含まれる細胞について、長期造血幹細胞を単離または濃縮するマーカー分子の発現量を測定し、当該発現量を対照サンプルにおけるマーカー分子の発現量と比較することにより、細胞組成物の性質を試験または評価する。当該対照サンプルは、インビボまたはインビトロ試験によって長期造血幹細胞含有細胞集団としての性質が確認されたサンプルであってもよく、試験または評価対象の細胞集団の別の時間点におけるサンプルであってもよい。
例えば当該細胞組成物は造血幹細胞移植に用いる検体、例えば臍帯血、骨髄液、末梢血含有組成物であってもよいが、これらに限定されない。例えば、長期造血幹細胞を含有する細胞組成物において、細胞の培養前後、一定期間の保存前後の細胞組成物について、長期造血幹細胞を単離または濃縮するマーカー分子の発現量を指標として、培養前後または保存前後の細胞集団において、長期造血幹細胞としての性質がどの程度変化または変化していないかを試験または評価することができる。
本開示は、本開示の長期造血幹細胞を単離または濃縮するマーカー分子を指標として、被験者の疾患予後予想または健康状況予想のために、造血幹細胞含有組成物を試験する方を提供する。典型的には、患者または健常人から取得された造血幹細胞を含む細胞集団に含まれる細胞について、あるいは、患者または健常人体内の造血幹細胞を含む細胞集団に含まれる細胞について、長期造血幹細胞を単離または濃縮するマーカー分子の発現量を測定し、当該測定された発現量を指標として、被験者の疾患予後または将来の健康状況を予想する。例えば、患者の予後予測または治療効果の確認のために、治療前および治療後の患者から造血幹細胞を含む細胞集団を取得し、長期造血幹細胞の割合の増減を試験し、長期造血幹細胞の割合が増加しているとき、あるいは減少が抑制されているときに、患者の良好な予後を予測することができる。別の態様において、生体由来造血幹細胞含有組成物を他家移植することで治療を行う場合に、患者の予後予想のために、ドナー体内の造血幹細胞を含む細胞集団、ドナーから得られた造血幹細胞を含む細胞集団、移植前あるいは移植後の患者体内の造血幹細胞を含む細胞集団、または、患者から得られた造血幹細胞を含む細胞集団を試験することもできる。別の態様において、患者又は健常人から取得された造血幹細胞を含む細胞集団に含まれる細胞について、患者または健常人の健康状態、例えば造血系における健康状態の把握、または将来の健康状況の予測のために、長期造血幹細胞を単離または濃縮するマーカー分子の発現量試験することができる。
本開示は、長期造血幹細胞を単離または濃縮するマーカーにより単離または濃縮された、単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団を修飾する方法を提供する。当該修飾は、前記単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団を各種分化誘導することを含む。例えば、特定の培養条件による培養、特定の薬剤または薬剤の組み合わせによる処理、ゲノム編集、遺伝子導入、mRNA、アンチセンスオリゴヌクレオチド、アプタマーなどの細胞内導入、他の細胞との融合等の処理により、長期造血幹細胞含有細胞集団を修飾することができるがこれらに限定されない。当該培養条件は、培地の組成、培地組成の変更、培地に含まれる特定の添加物、添加物の添加方法、培養温度またはその変更様式、培養湿度またはその変更様式、大気圧またはその変更様式、培養容器の構造もしくは素材またはそれらの変更様式、培養足場の構造もしくは素材またはそれらの変更様式等を含むが、これらに限定されない。当該薬剤は、無機化合物、有機低分子化合物、高分子化合物、タンパク質、核酸を含むが、これらに限定されない。前記ゲノム編集は当業者に公知の方法によるものを含み、例えば、CRISPR-Cas系、TALEN系等を用いたゲノム編集技術を含むが、これらに限定されない。長期造血幹細胞含有細胞集団が修飾されたことは、細胞表面に発現する分子の種類または量の変化、細胞内で転写される遺伝子の種類または量の変化、細胞のゲノム塩基配列の変化、または、これらに伴う細胞形態、機能の変化などにより確認することができるが、これらに限定されない。例えば、修飾後の細胞表面に発現する任意の分子または細胞内で転写される遺伝子の種類または量が、修飾前のものと比して0.1%、0.5%、1%、2%、3%、4%、5%、6%、7%、8%、9%または10%以上異なる場合に、当該細胞を修飾された細胞とすることができる。また、当該遺伝子導入は当業者に公知の方法によるものを含み、ウイルスベクターを用いた遺伝子導入技術、電気穿孔法を用いた遺伝子導入法等含むが、これらに限定されない。また、当該他の細胞との融合は当業者に公知の方法によるものを含み、融合する細胞は、ヒト又は霊長類の体細胞、ES細胞、人工多能性幹細胞またはMuse細胞など体細胞集団から特定の方法で単離された多能性幹細胞などを使用できるが、これらに限定されない。当該修飾された細胞集団は、細胞の分化メカニズム等を研究するための試料として用いることができるほか、その特性に合わせて、様々な用途に使用することができる。一例において、患者の生体組織から長期造血幹細胞を単離または濃縮し、当該細胞に対してゲノム編集またはベクターによる遺伝子導入等により、遺伝子の導入もしくは欠損、発現量の調整等の修飾を行った後に、機能の修飾された長期造血幹細胞として患者体内に戻すことにより、疾患治療などに利用することができる。また、別の例において、生体内に存在する長期造血幹細胞を本開示の長期造血幹細胞を単離または濃縮するマーカーにより同定し、ゲノム編集や遺伝子導入によって当該細胞に対して生体内で遺伝子の導入もしくは欠損、発現量の調整等の修飾を加えることにより、機能が付加または改変された長期造血幹細胞を生じさせることによる、疾患治療もしくは健康維持、増進方法などを提供する。一態様において、これら細胞の修飾は、恒常的なものであってもよく、一時的なものであってもよい。例えば、体内の特定に部位において、特定の期間のみ細胞を修飾するものあってもよい。
本開示は、長期造血幹細胞を単離または濃縮するマーカーにより単離または濃縮された長期造血幹細胞含有細胞集団を分析し、当該細胞集団の物理的、化学的または生物学的特徴に関する情報を得る方法を提供する。単離または濃縮された造血幹細胞または造血幹細の物理的特徴、化学的特徴、生物学的特徴は、当業者に公知の方法を用いることにより評価、分析することができる。得られた情報は、例えば物理的特徴、化学的特徴、生物学的特徴またはこれらの組み合わせに基づいて長期造血幹細胞含有細胞集団を特定するための方法に使用することができる。
一例において、上記の物理的特徴、化学的特徴、生物学的特徴は、造血幹細胞を単離または濃縮する方法を開発する際の陽性対照として利用することができる。例えば、細胞の生物学的特徴、例えば特定の化学物質に対する増殖応答などの特徴のみに基づいて造血幹細胞を単離または濃縮する場合に、当該方法によって単離または濃縮された細胞または細胞集団の物理的特徴、化学的特徴、生物学的特徴と、本開示のマーカーを利用して得られた造血幹細胞の物理的特徴、化学的特徴、生物学的特徴とを比較することにより、当該生物学的特徴に基づく造血幹細胞を単離または濃縮する方法を評価または特徴付けることができる。このような評価、特徴付けを通じて、一態様において、本開示のマーカーを利用して得られた造血幹細胞の物理的特徴、化学的特徴、生物学的特徴を指標として造血幹細胞を同定し、造血幹細胞を含有する細胞集団を製造する方法を提供する。
上記の物理的特徴、化学的特徴、生物学的特徴は、一例において、造血幹細胞または造血幹細胞を単離または濃縮するための分類器を作成するために使用される。例えば、分類器を機械学習により構成する場合、本開示の単離または濃縮された造血幹細胞または造血幹細胞から得た物理的特徴、化学的特徴、生物学的特徴またはこれらの組み合わせまたはその一部を、機械学習のための教師データとして使用することができる。
本開示は、単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団、またはこれを修飾して得られる各種細胞を用いて、化合物および薬剤などの被験物質、または温度および栄養条件などの環境要因が造血幹細胞に及ぼす生理作用を評価する方法を提供する。
当該方法では、本開示の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団またはこれを修飾して得られる各種細胞と、化合物、薬剤などの被験物質を公知の方法により接触させ、または、温度および栄養条件などの環境要因が細胞にもたらす生理作用、例えば、細胞機能の変化、増殖状態の変化、生存に与える影響などを評価する。
本開示は、長期造血幹細胞の増殖に対する培養条件の影響を、本開示の長期造血幹細胞を単離または濃縮するマーカー分子を指標として評価する方法を提供する。本開示の評価方法では、典型的には、造血幹細胞を含む細胞集団を準備し、評価対象の培養条件により造血幹細胞を含む細胞集団を培養し、培養後の細胞集団における本開示の長期造血幹細胞を特定するマーカー分子の発現量を試験する工程を含み、評価対象の培養条件における培養前後の前記マーカー分子の発現量を比較、または、評価対象の培養条件における培養後の前記マーカー分子の発現量と、対照となる培養条件における培養後の前記マーカー分子の発現量を比較する。比較の結果、長期造血幹細胞の増殖が促進されていた場合に、当該評価対象培養条件を、長期造血幹細胞の増殖を促進する培養条件として評価し、培養条件の変更後において長期造血幹細胞の増殖が抑制されていた場合に、当該評価対象培養条件を、長期造血幹細胞の増殖を抑制する培養条件として評価する。
本開示は、長期造血幹細胞を単離または濃縮するマーカー分子を指標として、長期造血幹細胞の特性を修飾する化合物をスクリーニングする方法を提供する。本開示のスクリーニング方法では、典型的には、造血幹細胞を含む細胞集団を準備し、候補化合物により処理した細胞または細胞集団と、未処理の細胞または細胞集団を比較し、本開示の長期造血幹細胞を単離または濃縮するマーカー分子によって特定される長期造血幹細胞の特性を修飾する化合物を取得する。例えば、長期造血幹細胞が細胞分裂に要する時間を短縮または延長する化合物、長期造血幹細胞の細胞数を増加させる化合物、長期造血幹細胞を含有する細胞組成物において、長期造血幹細胞の含有割合を、減少、維持または増加させる化合物、長期造血幹細胞の生存期間を延長または短縮する化合物、長期造血幹細胞の細胞死を抑制または促進する化合物、長期造血幹細胞の修飾(遺伝子導入、ゲノム編集、薬剤処理効率等)を促進または抑制する化合物、長期造血幹細胞を特定の細胞種に分化させる化合物、特定の細胞種への分化を抑制する化合物、長期造血幹細胞の分化を抑制または促進する化合物、特定の細胞種への分化を抑制する化合物等を取得することができる。
本開示は、長期造血幹細胞を単離または濃縮するマーカー分子を指標として、長期造血幹細胞を誘導する化合物をスクリーニングする方法を提供する。本開示のスクリーニング方法では、典型的には、長期造血幹細胞以外の細胞を準備し、前記細胞または細胞集団を候補化合物により処理し、処理後の細胞または細胞集団における、長期造血幹細胞を単離または濃縮するマーカーを指標として、長期造血幹細胞を誘導する化合物を取得する。長期造血幹細胞以外の細胞としては特に限定されず、例えば、生体由来の細胞、人工的に多能性を獲得した細胞、遺伝子導入などの修飾を受けた細胞などを用いることができる。例えば、ES細胞、iPS細胞などの人工的に多能性を獲得した細胞などを用いることができるがこれらに限定されない。
本開示は、長期造血幹細胞を単離または濃縮するマーカー分子を指標として長期造血幹細胞の特性を特異的に修飾する培養培地または培養補助添加物の製造方法および当該方法により製造された培養培地または培養補助添加物を提供する。典型的には、造血幹細胞を含む細胞集団を準備し、評価対象の構成を有する培地、または評価対象の培養補助添加物を含む培地により、造血幹細胞を含む細胞集団を培養し、本開示の長期造血幹細胞を単離または濃縮するマーカー分子を指標として、培養条件変更前後の細胞の増殖状態を評価し、長期造血幹細胞の特性を特異的に修飾する培養培地または培養補助添加物の構成を決定し、当該構成を有する培養培地または培養補助添加物を製造する。培養培地および培養補助添加物は例えば固体または液体組成物として構成される。培養培地はまた、特定の物理的構造、例えば、形状、素材、容量などをその構成に含むことができる。
本開示は、長期造血幹細胞の増殖を促進させる培養条件を、本開示の長期造血幹細胞を単離または濃縮するマーカー分子を指標として調整する方法を提供する。本開示の調整方法では、典型的には、造血幹細胞を含む細胞集団を準備し、調整された培養条件により造血幹細胞を含む細胞集団を培養し、前記マーカー分子を指標として、培養条件変更前後の細胞の増殖状態を評価し、長期造血幹細胞の増殖が促進されるように培養条件を調整する。
細胞の培養条件を構成する因子としては、当分野において通常考慮される因子を評価対象とすることがでる。例えば、培地成分、培養補助添加物、温度、湿度、大気構成、培養基質の形状、素材、表面処理、培養容器の容量、形状、素材、表面処理、培養容器、培地、または細胞自体に加える物理的刺激、これらの経時的な変更などを因子として評価することができるが、これらに限定されない。
本開示は、本開示の長期造血幹細胞単離または濃縮マーカー分子を指標として培養条件を調整する工程を含む、単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団の製造方法を提供する。典型的には、造血幹細胞を含む細胞集団を準備し、あらかじめ長期造血幹細胞の増殖を促進させるように調整された培養条件、例えば、上記の培養条件の調整方法により、長期造血幹細胞の増殖を促進させるように調整されている培養条件により、造血幹細胞を含む細胞集団を培養することで、単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団を製造する。
本開示は、長期造血幹細胞を単離または濃縮するマーカーを指標として、細胞に長期造血幹細胞の特性を付与する遺伝子改変方法を提供する。典型的には、試験する細胞の一部について、候補遺伝子を改変した遺伝子改変細胞を準備し、前記遺伝子改変細胞における本開示の長期造血幹細胞を単離または濃縮するマーカーと、遺伝子改変を行わなかった細胞における本開示の長期造血幹細胞を単離または濃縮するマーカーとを比較する。また、別の態様において、同一細胞における遺伝子改変前後における本開示の長期造血幹細胞を単離または濃縮するマーカーを比較する。比較の結果、本開示の長期造血幹細胞を単離または濃縮するマーカーについてより陽性の結果を与える遺伝子改変を、細胞に長期造血幹細胞の特性を付与する遺伝子改変として同定する。
本開示は、長期造血幹細胞を同定、単離または濃縮するマーカー分子を定量又は検出する試薬を含む、長期造血幹細胞を同定、単離または濃縮するための組成物またはキットを提供する。マーカー分子を定量又は検出する試薬としては、当分野において通常用いられる試薬を用いることができ、例えば蛍光色素で標識された抗体、核酸、低分子化合物、高分子化合物などを用いることができるが、これらに限定されない。本開示の長期造血幹細胞を同定、単離または濃縮するための組成物またはキットは、生体から採取された細胞集団において、長期造血幹細胞を同定、単離または濃縮するのに利用できるものの他、生体内において、長期造血幹細胞を同定するための組成物またはキットを含む。長期造血幹細胞を同定するための組成物またはキットは、さらに長期造血幹細胞の機能を修飾するための薬剤等を含むことができ、長期造血幹細胞の機能を修飾することで治療される疾患の治療薬、あるいは、治療方法を提供する。
本開示は、長期造血幹細胞を単離または濃縮するマーカーにより単離または濃縮された長期造血幹細胞、またはこれを修飾して得られる各種細胞を含有する、患者に投与することにより疾患を治療するための医薬、疾患予備軍への先制医療に用いる医薬、もしくは健常人に対する予防医療もしくは健康維持(改善)に用いる医薬または当該医薬を用いる治療法または療法を提供する。本開示の長期造血幹細胞またはこれを修飾して得られる各種細胞を医薬として使用する場合、患者由来の細胞集団から得た長期造血幹細胞またはこれを修飾して得られる各種細胞を当該患者に投与してもよく、他の提供者由来の細胞集団から得た長期造血幹細胞またはこれを修飾して得られる各種細胞を当該患者に投与してもよい。また、他の薬剤、または治療方法を併用することができる。例えば、患者または提供者から、本開示の長期造血幹細胞を単離または濃縮するマーカーにより単離または濃縮された長期造血幹細胞、あるいは、患者または提供者から得られた体細胞から生成された人工多能化細胞から誘導された造血幹細胞含有細胞集団から、本開示の長期造血幹細胞を単離または濃縮するマーカーにより単離または濃縮された長期造血幹細胞を、造血障害を有する患者に、当該障害を治療するための医薬として投与することができる。別の例として、患者の生体組織から、人工多能化工程を経て、または経ずに得られた造血幹細胞含有細胞集団から、本開示の長期造血幹細胞を単離または濃縮するマーカーにより長期造血幹細胞を単離または濃縮し、当該長期造血幹細胞に治療に有益な遺伝子の導入、ゲノム編集等の修飾を行った後に、疾患を治療するための医薬として患者に投与することができる。
(1)細胞ソース
臍帯血として、新鮮臍帯血(神戸市立医療センター中央市民病院、および足立病院(神戸市)より提供)を使用した。骨髄液として、凍結細胞、ヒト骨髄CD34+前駆細胞、2M-101A(Lonza Group AG, Basel, Switzerland)を使用した。末梢血として、凍結細胞、Mobilizedヒト末梢血CD34+造血幹細胞、ポジティブ選択、4Y-101C(Lonza Group AG,Basel,Switzerland)を使用した。
上記より得られた細胞ソースを用い、EasySep RBC Depletion Reagent(STEMCELL Technologies社製)を用い赤血球除去、濃縮処理を行い、造血幹細胞を含む細胞集団を得た。
上記で得られた造血幹細胞を含む細胞集団を、EASYSep Human Cord Blood CD34 Positive Selection Kit II(STEMCELL Technologies社製)を用い、CD34陽性細胞分画を粗精製した。
上記で得られた造血幹細胞および前駆細胞を濃縮した細胞集団(CD34陽性濃縮細胞分画)に対し、以下の抗体を加え細胞染色を行った。抗体による細胞染色は氷上にて30分間インキュベートにて行った。CD11b,CD14,CD19,CD20,CD235ab,CD3,CD4,CD56,CD8a,CD34,CD38,CD90,CD45RA,CD49fおよびLEGENDScreen Human PE Kit(BioLegend社製)に含まれる抗体。
細胞染色後のサンプルを、セルストレーナー付きFACSチューブ(FALCON社製)を用いて濾過した後、死細胞染色を行った。死細胞染色は、Propidium Iodide Solution (BioLegend社製)、SYTOX-Red(ThermoFisher SCIENTIFIC社製)等を用いた。細胞の解析およびソートは、FACSAria IIもしくはIII(BD社製)を使用した。採取されたデータは、FlowJo(BD社製)、もしくは出願人が開発したアルゴリズム(PCT/JP2019/051270、PCT/JP2020/025915等にも開示)を用いて解析した。なお、当該アルゴリズムは、細胞染色などによって得られる定量物理的特性に関する数値データおよび/または生物学的特性に関する数値データから選択される複数の数値データの類似性に基づいて、細胞配置空間構造を生成することにより、分化系統上近接した位置にある細胞が近接した位置を占める細胞配置空間構造を生成し、既知の分化マーカー等を利用することにより、当該細胞配置空間構造上の分化方向を特定するアルゴリズムである。
細胞染色の解析結果から、長期造血幹細胞を含むと思われる候補分画を特定し、MethoCult H4435 Enriched (STEMCELL Technologies社製) 25ulを各ウェルに播種した96ウェルプレートに1細胞ずつソートを行った。細胞ソートを行ったウェルに対して、Iscove’s MDM with 2% FBS (STEMCELL Technologies社製)を12.5ul添加した。プレートの最外のウェルには乾燥防止に滅菌水を200ul添加した。その後、細胞はCO2インキュベーターで、37℃、5%CO2の条件で2週間培養し、形成されたコロニーは光学顕微鏡(KEYENCE社製)にて撮像し、コロニー数を目視にてカウントし、コロニー形態を目視にて判別した。コロニーの形態から、骨髄系前駆細胞由来のColonyFormationUnit-Granulocyte、Erythroid、Macrophage、Megakaryocyte(CFU-GEMM)及び、赤芽球バーストコロニー形成細胞BurstFormingUnit-Erythroid(BFU-E)、顆粒球・マクロファージコロニー形成細胞ColonyFormationgUnit-Granulocyte、Macrophage(CFU-GM)を判別しカウントした。
新鮮もしくは培養後の細胞を下記方法にて、免疫不全マウスへ移植を行い、造血能を評価した。BD FACSAriaIIIにてソートした細胞分画を2.5Gy放射線照射したNOGマウス(NOD/Shi-scid, IL-2RyKO Jic)に尾静脈もしくは骨髄内投与した。移植後、4週間毎に末梢血もしくは骨髄細胞を採取し、必要に応じてLysing Buffer(BD社製)を用いて赤血球を破砕した後、FACS Cantoにてドナー由来キメリズムを測定した。細胞染色に下記に対する抗体を用いた:ヒトCD56,ヒトCD3,ヒトCD16,ヒトCD19,ヒトCD14,ヒトCD45,マウスGr-1,マウスCD45,死細胞染色にはPropidium Iodide Solution (BioLegend社製)を用いた。
200ulのIMDM(GIBCO社製)、1% Insulin-Transferrin-Selenium_Ethanolamine(ITS-X)(Gibco社製)、1% Penicillin-Streeptomycin-Glutamin)(Gibco社製)、50ng/ml human Stem Cell Factor (PeproTech社製)、human Thrombopoietin (PeproTech社製)を添加したU底96ウェルプレート(CORNING社製)に、FACSAriaIIIを用いて候補細胞分画300細胞/ウェルとなるようにソートした。ソートした細胞は、CO2インキュベーターで、37℃、5%CO2の条件で1週間培養した。培養後、CD11b,CD14,CD19,CD20,CD235ab,CD3,CD4,CD56,CD8a,CD34,CD38,CD117,CD48をマーカーとして、生細胞数、CD34+CD117+分画数、CD34+CD117CD48-分画数、CD34+CD117+CD48+数、生細胞率、CD34+CD117+分画率、CD34+CD117CD48-分画率、CD34+CD117+CD48+率等を、FlowJo(BD)を用いて解析した。CD34+CD117CD48-分画は、長期造血幹細胞を主とする分画である。
レポーター遺伝子を含むレンチウイルスLenti-LabelerTM virus(フナコシ社製)を用いて感染実験を行った。レトロネクチン(Clontech社製)にてコーティングした96ウェルプレート(CORNING社製)に1x10e8IFU/mlのタイターを有するウイルス液を添加し、ウイルスがコーティングされたプレートを作製する。各ウェルに候補細胞分画より1000細胞以上ソートし播種する。IMDM/5ng/ml SCF/5ng/ml TPO/1% Pen/St/Glu 200ul中にて24時間感染させる。感染させた細胞は、その後細胞培養、CFUアッセイ、移植実験等を行う。
生体から得られた新鮮臍帯血に対して赤血球除去、濃縮処理を行い、造血幹細胞を含む細胞集団を得た。当該造血幹細胞を含む細胞集団に対して、EASYSep Human Cord Blood CD34 Positive Selection Kit II(STEMCELL Technologies社製)を用い、CD34陽性細胞分画を粗精製した。当該CD34陽性細胞分画について、CD11b,CD14,CD19,CD20,CD235ab,CD3,CD4,CD56,CD8a,CD34,CD38,CD90,CD45RA,CD49fおよびLEGENDScreen Human PE Kit(BioLegend社製)に含まれる抗体を用いて死細胞染色を行い、各細胞について、各抗体の染色強度を定量化した。当該定量化データを用いて、分化系統上近接した位置にある細胞が近接した位置を占める細胞配置空間構造を生成した。当該構造の一部を可視化したものを図1、図2に例示する。図1は、死細胞染色後、生細胞の位置を、定量化データの組み合わせから生成された合成軸1、合成軸2、合成軸3からなる3次元空間に割り当てたものを2次元画像として示したものである。図2は、当該空間構造に位置を割り当てられた各細胞について、造血幹細胞において特徴的な発現様式を示すことの知られたCD34の発現量を可視化したものである。CD34陽性の細胞は、当該空間構造の一部に偏在している。
長期造血幹細胞とCD34の発現様式の関連を調べるために、ヒト臍帯血から分離されたCD3(-)CD34(-)(CD3陰性、CD34陰性)細胞又はCD3(-)CD34(+)(CD3陰性、CD34陽性)細胞を免疫不全マウスへ移植し、移植後のキメラ率を試験した。移植後4週間、8週間および12週間後にキメラ率を試験したところ、CD3(-)CD34(+)細胞はCD3(-)CD34(-)細胞に比して、著明に高い生存率およびキメラ率を示した。結果を図3に示す。なお、各マーカーの陰性、陽性の区分は、使用したBD FACSAriaIIIの細胞ソート結果に従った。本結果は、長期造血幹細胞とCD34(+)の発現様式が関連していることを示している。
そこで改めてCD34(+)画分におけるCD38(+)およびCD38(-)画分の未分化特性を確認した。具体的には、CD38(+)である画分P6(CD34(+)、CD38(+))、従来提案されたCD38(-)画分に関係する画分P7(CD34(+)、CD38(-)、CD45RA(-)、CD90(+))、画分P8(CD34(+)、CD38(-)、CD45RA(-)、CD90(-))、および画分P9(CD34(+)、CD38(-)、CD45RA(+)およびCD90(-))について、CFUアッセイを行い、それぞれの画分における造血幹細胞の増殖・分化能を試験した。その結果、従来提案されたCD38(-)マーカーによって画分されるP7およびP9は、極めて低い造血幹細胞の増殖・分化能を示した(図5)。一方、CD38(+)である画分P6の細胞も高い造血幹細胞の増殖・分化能を示したことから、従来報告されたCD34(+)、CD38(-)画分のみならず、CD34(+)画分全体に長期造血幹細胞が分布することが理解された。したがって、CD34(+)画分に存在する長期造血幹細胞を単離、濃縮し得る新たなマーカーの探索をする必要が認められた。
ヒト長期造血幹細胞を特定するためのマーカーで特定される細胞の未分化性を確認するために、1細胞レベルでCFUアッセイを行った。一例を図6に示す。当該1細胞レベルでの細胞機能解析結果を、インデックスソート分析と組み合わせることにより、各1細胞の未分化性と各マーカーの発現様式を紐づけることが可能となる。当該分析によって得られたマーカーと未分化特性の関連を、分化系統上近接した位置にある細胞が近接した位置を占める細胞配置空間構造上に配置することにより、当該細胞配置空間構造上の細胞分化方向を確定した。CD34陽性細胞を対象に、当該分析を行った結果の一例を図7に示す。
CD34陽性細胞について、分化系統上近接した位置にある細胞が近接した位置を占める細胞配置空間構造上確定された分化方向に沿って発現が偏在する分子マーカーを特定した。一例を図8に示す。より未分化方向に偏在して発現するマーカー分子、より分化方向に偏在して発現するマーカー分子を特定した。
これらのマーカーによって特定される細胞の造血幹細胞の増殖・分化能をCFUアッセイによって確認したところ、これらマーカーによって特定される細胞は、対照(CD34(+)細胞)よりも高い造血幹細胞の増殖・分化能を示した(図9)。
CD10、CD111、CD112、CD131、CD143、CD18、CD244、CD275、CD328、CD48、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD105、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD92、CD318、CD49f、c―Kit、CD34
上記特定されたマーカーのうち、c-Kit(+)、CD34(+)、CD48(-)の組み合わせからなるマーカーで特定される細胞の特性をより詳細に分析した。
臍帯血からCD34(+)で予備濃縮された細胞集団を、c-Kit(+)およびCD34(+)でゲーティングし(画分P1)、さらにCD48(-)でゲーティング(画分P2)した際のFACSプロットの一例を図10に示す。画分P1のうち、大半の細胞はCD48(+)に画分(画分non-P2)され、一部のみがP2に画分された。
実際に、臍帯血サンプルについてCD34(+)画分をc-Kit(+)画分とc-Kit(-)画分に分画し、それぞれについて造血幹細胞の増殖・分化能を、CFUアッセイによって比較したところ、c-Kit(+)画分はCD34(+)画分とほぼ同様の造血幹細胞の増殖・分化能を示したのに対して、c-Kit(-)画分は造血幹細胞の増殖・分化能をほとんど示さなかった(図18)。したがって、CD48(-)とCD34(+)の組み合わせ、CD48(-)とc-Kit(+)の組み合わせからなるマーカーも、長期造血幹細胞を効率的に濃縮し、あるいは単離し得るマーカーとして使用し得ることが理解される。
上記分化系統上近接した位置にある細胞が近接した位置を占める細胞配置空間構造を利用した分析により、P2画分を更に詳細に画分するための追加的なマーカーを特定した。具体的には、上記分析において分化方向または未分化方向に偏在するマーカーを候補とし、同マーカーの発現量に基づいてP2画分の細胞を被験画分と対照画分に2分し、それぞれの画分の造血幹細胞の増殖・分化能を、CFUアッセイによって比較した。
得られた結果から、被験画分に長期造血幹細胞が濃縮される効率を以下の式により定量化した。
濃縮倍率=被験画分から生じたコロニーのうちCFU-GEMMが占める割合/対照画分から生じたコロニーのうちCFU-GEMMが占める割合
被験画分と対照画分から生じたコロニーのうちCFU-GEMMが占める割合が同一の場合、濃縮倍率は1となる。
これら追加的なマーカーは以下を含む。
CD84(+)、CD111(+)、CD150(-)、GPR56(-)、Notch-1(-)、CD52(-)、CD62L(+)、CD71(+)、CD93(-)、CD122(-)、CD133(-)、CD275(-)、CD131(-)、CD326(+)、CD200(-)、CD200R(-)、CD324(+)、CD40(-)、CD11c(-)、CD318(-)。
本開示の長期造血幹細胞を単離または濃縮するマーカーが、体外培養後の長期造血幹細胞を同定し得るか否かを確認するための試験を行った。造血幹細胞の増殖を促進することの知られた化合物UM171を用いて培養した細胞集団について、c-Kit(+)、CD34(+)およびCD48(-)のマーカーセットで同定される細胞の割合を試験した。P2画分またはnon-P2画分に含まれる300細胞について、UM171添加または非添加の条件で培養し、7日後の総細胞数および未分化細胞(P1)の比率を試験した。P2画分の結果を図20に、non-P2画分の結果を図21に示す。本試験により、本開示の長期造血幹細胞を単離または濃縮するマーカーは、体外培養後の長期造血幹細胞を同定し得ることが理解される。本開示の長期造血幹細胞を単離または濃縮するマーカーが、体外培養後の長期造血幹細胞を同定し得ることは、さらにCFUアッセイ等の機能試験によっても確認することができる。 臍帯血サンプルからc-Kit(+)、CD34(+)およびCD48(-)のマーカーセットで分画したP2画分の細胞を7日間培養した後、培養後の細胞集団をc-Kit(+)、CD34(+)およびCD48(-)のマーカーセットを用いてP2画分(培養後)およびnon-P2画分(培養後)に分画し、それぞれにつき、CFUアッセイを行った。その結果、培養後細胞集団中はすべてP2画分(培養後)に分画され、non-P2画分(培養後)に分画される細胞は確認されなかった。P2画分(培養後)についてCFUアッセイを行ったところ、P2画分(培養後)からは培養前のP2画分と同様に、未分化細胞(CFU-GEMM細胞)の高い含有割合が確認された(図22)。
これらの結果は、本開示の長期造血幹細胞を単離または濃縮するマーカーが、体外培養後の長期造血幹細胞を同定し得ることを示している。
本開示の長期造血幹細胞を単離または濃縮するマーカーによって単離または濃縮された長期造血幹細胞が、遺伝子導入後も長期造血幹細胞の特性を維持しているか否かを確認するために、CFUアッセイによる機能試験を行う。具体的には、P2画分、non-P2画分、またはDC34陽性画分に含まれる細胞に対して、レンチウイルスベクターによりGFPレポーター遺伝子を遺伝子導入し、これら遺伝子導入された細胞についてCFUアッセイを行い、P2画分由来の遺伝子導入細胞が増殖・分化能を維持していることを確認する。
Claims (23)
- CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせからなる、造血幹細胞単離マーカーまたは濃縮マーカー。
- CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上からなる、請求項1に記載の造血幹細胞単離マーカーまたは濃縮マーカー。
- CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせの発現様式によって特定される、単離された造血幹細胞または造血幹細胞が濃縮された造血幹細胞含有細胞集団。
- CD48(-)およびCD34(+)の組み合わせ、CD48(-)およびc-Kit(+)の組み合わせ、ならびにCD48(-)、CD34(+)およびc-Kit(+)の組み合わせから選択される1以上の発現様式によって特定される、請求項3に記載の単離された造血幹細胞または造血幹細胞が濃縮された造血幹細胞含有細胞集団。
- 造血幹細胞を含む細胞集団を準備する工程と、
CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせの発現様式を指標として細胞を単離または濃縮する工程と
を含む、単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団の製造方法。 - CD48(-)およびCD34(+)の組み合わせ、CD48(-)およびc-Kit(+)の組み合わせ、ならびにCD48(-)、CD34(+)およびc-Kit(+)の組み合わせから選択される1以上の発現様式を指標とする、
請求項5に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団の製造方法。 - さらに修飾を含む、請求項1または2に記載のマーカーにより同定、単離、または濃縮された造血幹細胞、または、請求項3または4に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団、または請求項5または6のいずれか一項に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団。
- 造血幹細胞を含む細胞集団を準備する工程と、
各細胞における各細胞のCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を測定する工程と
を含む、前記細胞集団における造血幹細胞の含有量および/または含有量の変化を評価する方法。 - 造血幹細胞を含む細胞集団を準備する工程と、
前記造血幹細胞を含む細胞集団に含まれる細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD48、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、前記細胞集団の品質を試験する工程と
を含む、造血幹細胞を含む細胞集団の試験方法。 - 前記品質が造血能維持能力、移植適合性、または保存安定性である、請求項9に記載の試験方法。
- 被験者由来の造血幹細胞を含む細胞集団に含まれる細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を取得する工程と、
被験者の疾患予後予測または健康状況予想のために、前記マーカーの発現量を対照と比較する工程と
を含む、造血幹細胞を含む細胞集団の試験方法。 - 造血幹細胞を含む細胞集団を準備する工程と、
候補化合物により造血幹細胞を含む細胞集団を処理する工程と、
候補化合物により処理された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、候補化合物を選択する工程と
を含む、造血幹細胞の特性を修飾する化合物のスクリーニング方法。 - 造血幹細胞を含む細胞集団を準備する工程と、
調整された培養条件により造血幹細胞を含む細胞集団を培養する工程と、
調整された培養条件により培養された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として培養条件を決定する工程と
を含む、造血幹細胞の特性を修飾する培養条件の調整方法。 - 造血幹細胞を含む細胞集団を準備する工程と、
評価対象の構成を有する培地、または評価対象の培養補助添加物を含む培地により、造血幹細胞を含む細胞集団を培養する工程と、
培養された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として培養培地または培養補助添加物の構成を決定する工程と、
上記決定された構成を有する培養培地または培養補助添加物を製造する工程と
を含む、造血幹細胞の特性を修飾する培養培地または培養補助添加物を製造する方法。 - 前記造血幹細胞を含む細胞集団が、生体由来の細胞または人工的に多能性を獲得した細胞から誘導された細胞である、請求項5~14のいずれか一項に記載の方法。
- 前記造血幹細胞を含む細胞集団が、生体由来の細胞または人工的に多能性を獲得した細胞から誘導された細胞である、請求項5~7のいずれか一項に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団。
- 細胞集団を準備する工程と、
被験化合物により細胞集団を処理する工程と、
候補化合物により処理された細胞に発現するCD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせのマーカーの発現量を指標として、候補化合物を選択する工程と
を含む、造血幹細胞を誘導する化合物のスクリーニング方法。 - CD10、CD111、CD112、CD131、CD143、CD244、CD275、CD328、CD62L、GPR56、CD133、CD36、CD370、CD84、CD326、CD114、CD116、CD135、CD172a/b、CD200、CD226、CD245、CD31、CD317、CD40、CD41、CD61、CD93、Notch1、CD352、Siglec-10、CD45RO、CD11c、CD52、CD318、CD49f、c―KitおよびCD48から選択される1以上、もしくは、CD48およびCD34の組み合わせ、CD48およびc-Kitの組み合わせ、ならびにCD48、CD34およびc-Kitの組み合わせから選択される1以上、またはこれらの組み合わせの発現量を定量または検出する試薬を含む、造血幹細胞を同定、検出、単離または濃縮するための組成物またはキット。
- 請求項1または2に記載のマーカーにより同定、単離、または濃縮された造血幹細胞、または、請求項3、4および7のいずれか一項に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団、または請求項5または6に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団を分析することで得られた物理的特徴、化学的特徴、生物学的特徴の組み合わせを特定する方法。
- 請求項1または2に記載のマーカーにより同定、単離、または濃縮された造血幹細胞、または、請求項3、4および7のいずれか一項に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団、または請求項5または6に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団を分析することで得られた物理的特徴、化学的特徴、生物学的特徴の組み合わせを指標として造血幹細胞を同定する分類器を製造する方法。
- 請求項1または2のいずれか一項に記載のマーカーにより同定、単離、または濃縮された造血幹細胞、または、請求項3、4および7のいずれか一項に記載の単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団、または請求項5または6に記載の方法で製造された単離または濃縮された造血幹細胞または造血幹細胞を含む細胞集団を含む、造血幹細胞を投与することで改善または予防される疾患または障害を予防または治療するための医薬。
- 造血幹細胞を投与することで改善または予防される疾患または障害が、異染性白質ジストロフィー(MLD)、ゴーシェ病、ファブリー病、ムコ多糖症、HIV、ADA-SCID、X-SCIDまたはサラセミアである、請求項21に記載の医薬。
- 造血幹細胞を投与することで改善または予防される疾患または障害が、慢性肉芽腫症(CGD)または副腎白質ジストロフィー(ALD)である、請求項21に記載の医薬。
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WO2023222565A1 (en) * | 2022-05-16 | 2023-11-23 | Institut National de la Santé et de la Recherche Médicale | Methods for assessing the exhaustion of hematopoietic stems cells induced by chronic inflammation |
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