JP2016104711A - Pharmaceutical composition for use in assisting hematopoietic stem cell transplantation, and production method thereof - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition for use in assisting hematopoietic stem cell transplantation, and to provide a production method thereof.SOLUTION: The invention provides a cell formulation for use in assisting hematopoietic stem cell transplantation, the cell formulation containing cultures obtained by culturing hematopoietic stem cells under the presence of TPO and SCF; and a production method thereof.SELECTED DRAWING: None

Description

  The present invention relates to a pharmaceutical composition for use in assisting hematopoietic stem cell transplantation and a method for producing the same.

  Hematopoietic cell transplantation is performed as a radical therapy for many malignant diseases such as hematological malignancies such as leukemia and innate immune deficiency. As a cell source for hematopoietic stem cell transplantation, a cell source containing hematopoietic stem cells having both long-term bone marrow remodeling ability and multipotency is used. Demand for umbilical cord blood is particularly demanded because it is obtained non-invasively, hematopoietic stem cells are highly active, and acute graft-versus-host disease (acute GVHD) response is weak compared to transplants of other sources. Has increased.

Hematopoietic stem cell transplantation using umbilical cord blood requires umbilical cord blood containing 2 × 10 ^{7 to} 3 × 10 ⁷ nucleated cells per patient's body weight (that is, about 70 per kg for an adult patient weighing 70 kg). 1 × 10 ^{9 to} 2 × 10 ⁹ cells). However, the amount of umbilical cord blood obtained with one delivery is limited, and on average, the number of nucleated cells contained is about 7 × 10 ⁸ to 8 × 10 ⁸ cells per umbilical cord blood (ie, 70 kg). In an adult patient with body weight, it is only about 1 × 10 ⁷ cells per body weight).
For this reason, many umbilical cord blood is not used for transplantation and is dead stock. In addition, if there is no possibility that a sufficient amount of blood can be secured, the blood can be discarded without being preserved. Therefore, it is necessary to develop a method for utilizing cord blood that does not contain sufficient nucleated cells for hematopoietic stem cell transplantation.

  In order to overcome this problem, attempts have been made to mix and transplant a plurality of umbilical cord blood units. For any umbilical cord blood unit, 4 or more HLA antigens can be matched with the recipient. Necessary (Non-Patent Document 1). Therefore, the situation has not been improved yet.

In addition, even when the minimum necessary number of cells (2 × 10 ^{7 to} 3 × 10 ⁷ cells per patient body weight) is transplanted, hematopoietic recovery such as neutrophil count and platelet count in the recipient after umbilical cord blood transplantation It is known to be slow compared to transplantation of bone marrow or peripheral blood hematopoietic stem progenitor cells. Therefore, the development of methods that can promote hematopoietic recovery and avoid fatal complications has become an urgent issue in umbilical cord blood transplantation.

L.R. Fanning et al., Leukemia, 2008, 22: 1786-1790

  The present invention provides a cell preparation for use in assisting hematopoietic stem cell transplantation and a method for producing the same.

  The present inventors have found that the culture obtained by culturing hematopoietic stem cells in the presence of thrombopoietin (TPO) and stem cell factor (SCF) improves the success rate of bone marrow transplantation and umbilical cord blood transplantation, more specifically, Clarified that in recipients after hematopoietic stem cell transplantation, early hematopoiesis is supported during the period until hematopoiesis by transplanted bone marrow and umbilical cord blood rises, and can be eliminated after hematopoiesis has started. The present invention has been made based on such findings.

That is, according to the present invention, the following inventions are provided.
(1) A pharmaceutical composition for use in assisting hematopoietic stem cell transplantation,
A pharmaceutical composition comprising cells obtained by culturing hematopoietic stem cells in the presence of thrombopoietin (TPO) and stem cell factor (SCF).
(2) The pharmaceutical composition according to (1) above, comprising cells obtained by culturing hematopoietic stem cells in the presence of TPO and SCF for 5 to 10 days.
(3) The pharmaceutical composition according to (2) above, comprising cells obtained by culturing hematopoietic stem cells in the presence of TPO and SCF for 7 days.
(4) cord blood or bone marrow;
An implant material comprising the pharmaceutical composition according to any one of (1) to (3) above.
(5) a combined cell preparation for transplantation of hematopoietic stem cells,
Umbilical cord of a donor whose MHC fitness with the recipient is any of 0-2 antigen mismatch,
A combination of cells obtained by culturing umbilical cord blood of at least one individual, in which at least immune cells are substantially removed, in the presence of thrombopoietin (TPO) and stem cell factor (SCF); A combined cell preparation comprising.
(6) The cell obtained by culturing the hematopoietic stem cell fraction obtained from cord blood in the presence of thrombopoietin (TPO) and stem cell factor (SCF). The combined cell preparation described in 1.
(7) The combined cell preparation according to (5) or (6) above, wherein the cells obtained by the culture are any of 3-6 antigen mismatches with donor umbilical cord blood.
(8) The combined cell preparation according to any one of (5) to (7) above, wherein the MHC fitness between any two individuals is independently any of 3 to 6 antigen mismatches.
(9) The cell fraction comprising hematopoietic stem cells is obtained by collecting CD34-positive cells or CD34-positive and CD38-negative cells from cord blood, according to (5) to (8) above The combined cell preparation according to any one of the above.
(10) The combined cell preparation according to any one of (5) to (9), wherein the individual comprises 2 to 5 individuals.
(11) The combined cell preparation according to any one of (5) to (10) above, wherein the umbilical cord blood is human umbilical cord blood.
(12) A method for producing the pharmaceutical composition according to any one of (1) to (3) above,
Obtaining a hematopoietic stem cell fraction from bone marrow or cord blood;
Culturing a hematopoietic stem cell fraction in the presence of TPO and SCF to obtain cells.
(13) A method for producing a combined cell preparation for hematopoietic stem cell transplantation, comprising:
(A) selecting a plurality of umbilical cord blood from a stock of umbilical cord blood (except that the MHC fitness of all umbilical cord blood and the recipient is 0-2 antigen mismatch);
(B) substantially removing immune cells from each cord blood of all individuals selected in (a) and culturing in the presence of thrombopoietin (TPO) and stem cell factor (SCF);
(C) selecting one donor's umbilical cord blood from umbilical cord blood whose MHC fitness with the recipient is 0-2 antigen mismatch.
(14) Step (b) collects CD34-positive cells or CD34-positive and CD38-negative cells from each cord blood of all individuals selected in (a), and then the obtained cells are treated with thrombopoietin ( The method according to (12), which is performed by culturing in the presence of TPO) and stem cell factor (SCF).
(15) The method according to (12) or (13) above, wherein the umbilical cord blood is human umbilical cord blood.
(16) A cell preparation for hematopoietic stem cell transplantation in humans,
Umbilical cord of a donor whose MHC fitness with the recipient is any of 0-2 antigen mismatch,
A cell preparation comprising a combination of at least one umbilical cord blood with at least immune cells substantially removed from the umbilical cord blood.
(17) The cell preparation according to (16) above,
A cell preparation, which is at least one umbilical cord blood, wherein the umbilical cord blood from which at least immune cells are substantially removed is a hematopoietic stem cell fraction obtained from the umbilical cord blood.
(18) The cell preparation according to (16) or (17) above, wherein only donor umbilical cord blood whose MHC fitness with the recipient is any of 0 to 2 antigen mismatches contributes to long-term hematopoiesis of the recipient .
(19) A combination of at least two umbilical cord blood from which at least immune cells have been substantially removed, and promoting the engraftment of the donor umbilical cord blood to the recipient during hematopoietic stem cell transplantation For use in administering to recipients.
(20) The combination according to (19) above, wherein at least two umbilical cord blood, wherein at least immune cells are substantially removed, is a hematopoietic stem cell fraction.

FIG. 1 shows a scheme of a transplantation experiment for determining the number of hematopoietic stem cells necessary for hematopoietic recovery (survival). FIG. 2 shows the results of an experiment for determining the number of hematopoietic stem cells necessary for hematopoietic recovery (survival). FIG. 3 shows the results of an experiment for determining the number of total bone marrow cells required for recovery (survival) of hematopoiesis. FIG. 4 shows the results of recovery (survival) of the recipient's hematopoiesis when transplanted with hematopoietic stem cells having an MHC antigen different from that of the recipient. FIG. 5 shows the results of recovery (survival) of the recipient's hematopoiesis when a plurality of hematopoietic stem cells having MHC antigens different from the recipient are mixed and transplanted. FIG. 6 shows changes in the number of white blood cells and platelets after transplantation in a recipient when a plurality of hematopoietic stem cells each having a different MHC serotype from the recipient are transplanted. FIG. 7 shows the contribution of hematopoietic stem cells having various MHC serotypes to the hematopoietic system constructed in the recipient. FIG. 8 shows recovery of a recipient's hematopoiesis (survival) when all bone marrow cells having the same MHC serotype as the recipient and four hematopoietic stem cells having a different MHC serotype from the recipient are mixed and transplanted. The results are shown. FIG. 9 is a diagram showing the contribution of hematopoietic stem cells having various MHC serotypes to the hematopoietic system constructed in the recipient. FIG. 10 is a diagram showing a scheme in which a culture obtained by culturing hematopoietic stem cells in the presence of a hematopoietic factor is administered together with whole bone marrow. FIG. 11 is a diagram showing the analysis results of a culture obtained by culturing hematopoietic stem cells in the presence of hematopoietic factors. FIG. 12 is a graph showing changes in the chimera rate of blood cells derived from the obtained culture in recipients after whole bone marrow transplantation. FIG. 13 shows the contribution of hematopoietic stem cells cultured in the presence of hematopoietic factors to early hematopoiesis. FIG. 14 is a diagram showing the results of investigating determinants of chimerism in recipients after transplantation. FIG. 15 is a diagram showing the influence on bone marrow transplantation of a mixture of cultures obtained by culturing four types of allo-hematopoietic stem cells in the presence of hematopoietic factors. FIG. 16 is a graph showing the transition of the contribution ratio of cells derived from the culture in the erythrocytes of the recipient after transplantation. FIG. 17 is a graph showing the transition of the contribution ratio of cells derived from culture in the platelets of recipients after transplantation. FIG. 18 is a graph showing changes in the contribution ratio of cells derived from cultures in neutrophils of recipients after transplantation. FIG. 19 is a diagram showing the effect of a culture obtained by culturing in the presence of a hematopoietic factor on the transplantation result in a system in which both graft-versus-host reaction (GVH) and host-versus-graft reaction (HVG) occur. is there. FIG. 20 is a graph showing the transition of the contribution ratio of cells derived from the culture in the neutrophils of the transplanted recipient in the experimental system shown in FIG. 19A. FIG. 21 is a diagram showing a scheme of a transplantation experiment using human umbilical cord blood. FIG. 22 is a diagram (FIG. 22A) showing the percentage (%) of human cells in bone marrow after transplanting human umbilical cord blood to immunodeficient mice irradiated with sublethal dose radiation, and chimerism of each umbilical cord blood in human cells. It is a figure (FIG. 22B) which shows this. FIG. 23 is a diagram showing the time course of chimera rate in human cells of HLA-A2 positive cells used to assist hematopoietic stem cells.

Detailed description of the invention

  In the present specification, “umbilical cord blood” means blood obtained from a umbilical cord connecting a fetus and a mother and components thereof. “Saitai” refers to a cord that connects the fetus and placenta in mammals. Since umbilical cord blood contains a lot of hematopoietic stem cells, it is preferably used for hematopoietic stem cell transplantation. However, umbilical cord blood is rarely obtained in a sufficient amount for administration to adults, and in recent years when umbilical cord blood collection technology has improved, the number of transplant cases in adults has increased. The shortage is one of the biggest barriers to transplantation.

  As used herein, “umbilical cord blood stock” means umbilical cord blood stored in a umbilical cord blood bank. In many cases, umbilical cord blood stock is a cryopreserved umbilical cord blood collected at birth. In the present invention, a frost damage preservative may be added to the stock of umbilical cord blood. In the present invention, newly collected umbilical cord blood can be used in addition to the umbilical cord blood stock.

As used herein, “hematopoietic stem cell” is a cell having multipotency and self-renewal ability to differentiate into blood cells. Human hematopoietic stem cells can be obtained by, for example, fractionating from cord blood as CD34 positive cells. Human hematopoietic stem cells can also be obtained by, for example, fractionating from CD34 positive cells as CD38 negative cells (CD34 ⁺ CD38 ⁻ ). In addition, mouse hematopoietic stem cells are obtained, for example, by being fractionated from bone marrow as lineage marker negative, c-kit positive, and Sca-1 positive cells (hereinafter referred to as “KSL cells”). A lineage marker is a general term for marker molecules that are specifically expressed in mature blood cells of each lineage (such as lymphocytes, erythrocytes, neutrophils and macrophages).

Examples of the lineage marker include CD2, CD3, CD4, CD5, CD8, NK1.1, B220, TER-119, and Gr-1. A person skilled in the art can easily fractionate KSL cells based on the expression of a lineage marker. Mouse hematopoietic stem cells can also be obtained by fractionating as CD34 negative KSL cells or CD34 ^Low KSL cells using CD34 expression as an index (Science, 1996, 12; 273 (5272): 242-5). When hematopoietic stem cells are fractionated as described above, at least immune cells are substantially removed. Methods for fractionating cells using the above markers are well known in the field of hematology.

  As used herein, “hematopoietic stem cell fraction” means a hematopoietic stem cell fraction obtained from umbilical cord blood, bone marrow, etc. For example, in humans, a fraction obtained as CD34 positive cells, or a CD34 positive CD38 negative cell Means the fraction obtained. These fractions can be obtained by methods well known to those skilled in the art, for example, using cell sorter technology.

  As used herein, “transplanted hematopoietic stem cell” means a hematopoietic stem cell transplanted into a recipient.

  As used herein, “hematopoietic stem cell transplantation” means transplantation of donor (donor) hematopoietic stem cells to a patient (recipient). Hematopoietic stem cell transplantation includes bone marrow transplantation, peripheral blood stem cell transplantation, and umbilical cord blood transplantation. In hematopoietic stem cell transplantation, a treatment that causes bone marrow suppression is usually performed before transplantation to cause destruction of the patient's bone marrow and possibly malignant tumor, and then transplanted hematopoietic stem cells. Examples of treatments that cause bone marrow suppression include irradiation and administration of anticancer agents. Recipients in umbilical cord blood transplantation include leukemia patients, aplastic anemia patients, congenital immunodeficiency patients, and patients with inborn errors of metabolism such as mucopolysaccharidosis and adrenoleukodystrophy. Hematopoietic stem cell transplantation is performed in mammals, particularly humans. Generally, bone marrow, peripheral blood or cord blood to be transplanted is transplanted in a state containing immune cells.

  “Umbilical cord blood transplantation” is a type of hematopoietic stem cell transplantation and means transplanting umbilical cord blood as a cell source of hematopoietic stem cells.

  As used herein, “MHC” means major histocompatibility complex. MHC is a group of cell surface antigens that cause graft rejection in transplantation immunity, and is known as H-2 antigen in mice and HLA antigen in humans. In the case of human hematopoietic stem cell transplantation, among HLA loci, six types of HLA antigens encoded by paternal and maternal A, B and DR loci genes are assigned to donor and recipient. It is desirable to match as much as possible.

  As used herein, “MHC suitability” refers to how many serotypes in MHC do not match between a donor and a recipient. Especially in the case of humans, “MHC suitability” means HLA suitability and how many of the 6 HLA antigens are inconsistent between the donor and the recipient. A high fitness means that the number of mismatched antigens is small, and a low fitness means that the number of mismatched antigens is large. Antigen match means that the serotypes match.

  In umbilical cord blood transplants, donors and recipients can be performed in allogeneic or allogeneic relationships. According to current transplantation practices, umbilical cord blood transplants generally tolerate discrepancies of 2 antigens or less between donors and recipients. That is, between a donor and a recipient, for example, any of two antigen mismatches (ie, four antigen matches), one antigen mismatch (ie, five antigen matches), and zero antigen mismatch (ie, six antigen matches). Can be transplanted. In the case where graft-to-tumor reaction (GVL effect) is not taken into consideration, from the viewpoint of avoiding GVHD reaction or rejection, the higher the degree of MHC antigen compatibility between the donor and the recipient, the more preferable. More preferred is zero antigen mismatch. Conversely, for example, 3 antigen mismatch (ie 3 antigen match), 4 antigen mismatch (ie 2 antigen match), 5 antigen mismatch (ie 1 antigen match) and 6 antigen mismatch between donor and recipient. In the case of (ie, zero antigen match), cord blood transplantation becomes more difficult as the fitness decreases. The significance of the HLA match varies depending on the underlying disease. For aplastic anemia and first remission acute myeloid leukemia, the higher the HLA match, the better the transplantation results, but in high-risk cases such as non-remission acute leukemia There is also a report that the survival rate is high due to the suppression of recurrence due to the GVL effect even if HLA mismatch.

  As used herein, “immune cells are substantially removed” means that a process is performed in which immune cells are substantially removed, and as a result of being subjected to some other treatment, immune cells Is used in the sense that it is substantially removed. In the present specification, “the immune cells are substantially removed” means that the number of immune cells is, for example, 30% or less, 20% or less, 10% or less, 5% or less, 3%, compared to before removal. Or less than 1%. As used herein, “at least immune cells are substantially removed” means that cells other than immune cells are removed as long as hematopoietic stem cells remain and immune cells are substantially removed. Means good. Therefore, “at least immune cells are substantially removed” is used in the meaning including fractionation of hematopoietic stem cells (which results in substantial removal of immune cells). The method for fractionating hematopoietic stem cells is as described above. For example, when hematopoietic stem cells and hematopoietic progenitor cells are obtained from human cord blood, immune cells are substantially removed by collecting CD34-positive cells or CD34-positive CD38-negative cells. Those skilled in the art can collect or fractionate CD34-positive cells or CD34-positive CD38-negative cells according to conventional methods. Accordingly, “the cord blood from which at least immune cells have been substantially removed” includes, for example, a cell fraction comprising hematopoietic stem cells obtained from the cord blood (eg, human CD34 positive cells and human CD34 positive CD38 negative cells). ) May be preferably used.

  In this specification, “combination” means a combination of two or more things. In a combination, the objects of the combination may be provided in a mixed state (eg, a mixture or composition), or the objects of the combination are provided in a separate form (eg, a kit) without being mixed. Also good.

  The combined cell preparation of the present invention can be obtained by removing umbilical cord blood from which at least immune cells derived from a plurality of individuals have been removed, or a hematopoietic stem cell fraction derived from a plurality of individuals in the presence of thrombopoietin (TPO) and stem cell factor (SCF). The culture obtained by culture | cultivating in is comprised. Therefore, in the present specification, the “combination cell preparation” is obtained by culturing a hematopoietic stem cell fraction derived from umbilical cord blood derived from a plurality of individuals in the presence of thrombopoietin (TPO) and stem cell factor (SCF). Cell preparation comprising a combination of different cultures. The combined cell preparation of the present invention may comprise umbilical cord blood from which at least immune cells derived from a plurality of individuals have been removed, or a hematopoietic stem cell fraction derived from a plurality of individuals. That is, in the combined cell preparation of the present invention, cord blood from which at least immune cells derived from a plurality of individuals have been removed, or hematopoietic stem cell fraction derived from a plurality of individuals is present in the presence of thrombopoietin (TPO) and stem cell factor (SCF). You may culture | cultivate under and use for a cell formulation, and you may use for a cell formulation, without culturing.

  As used herein, “engraftment promoter” means an agent that promotes the engraftment of transplanted hematopoietic stem cells into the recipient body after hematopoietic stem cell transplantation. As used herein, “engraftment” means that the transplanted hematopoietic stem cells begin to make blood cells in the recipient's body. In current medical practice, engraftment is generally judged by neutrophil recovery. Specifically, in humans, the number of neutrophils exceeded 500 / μL for 3 consecutive days. In this case, it can be determined that the first day exceeding 500 / μL has been engrafted. As used herein, “engraftment promotion” means increasing the proportion of recipients that exhibit engraftment.

Currently, hematopoietic stem cell transplantation by umbilical cord blood transplantation is performed. The number of nucleated cells contained in human umbilical cord blood is dispersed with a peak of about 7 × 10 ⁸ to 8 × 10 ⁸ cells per unit. For adult transplantation, 1 × 10 ⁹ to 2 × 10 ⁹ cells (about 2 × 10 ^{7 to} 3 × 10 ⁷ cells per kg body weight) are recommended, but cord blood containing cells exceeding this number not many. Many umbilical cord blood whose nucleated cell count is less than this recommended cell count is either stored as dead stock without being used, or is discarded.

  According to the present invention, hematopoietic stem cells of the transplanted donor's umbilical cord blood from other individual's umbilical cord blood (where immune cells are substantially removed), By mixing a culture obtained by culturing the hematopoietic stem cells in the presence of thrombopoietin (TPO) and stem cell factor (SCF), the donor cord blood does not contain the number of cells necessary for transplantation alone. Even in this case, it was found that donor-derived transplanted hematopoietic stem cells can be engrafted. Therefore, according to the present invention, umbilical cord blood that could not be used due to a lack of cells is used by mixing with umbilical cord blood-derived cultures of other individuals. Under such circumstances, as described below, for example, in the present invention, even cord blood in the case of premature birth (the gestational age is 22 to 36 weeks) or a low birth weight infant can be used. Further, according to the present invention, it is possible to select umbilical cord blood without worrying about the MHC suitability in relation to the recipient, and the range of use of umbilical cord blood is further expanded.

  Accordingly, in one aspect of the present invention, a combined cell preparation for transplantation of hematopoietic stem cells, wherein the donor's cord blood has an MHC fitness with the recipient of any of 0-2 antigen mismatch, and at least one individual. A combination of cells (cell culture) obtained by culturing in the presence of thrombopoietin (TPO) and stem cell factor (SCF) after at least immune cells are substantially removed, A combined cell preparation is provided. Here, it is preferable that T cell is contained in the cord blood of the donor whose MHC compatibility with a recipient is either 0-2 antigen mismatch. By including T cells in the cord blood, as the donor's hematopoiesis rises, hematopoiesis originating from other individuals can be gradually eliminated immunologically, and the final recipient's hematopoietic system is added to the recipe. This is because it is possible to start with a donor-derived hematopoietic system whose MHC compatibility with the ent is any one of 0 to 2 antigen mismatches. In one embodiment, cord blood-derived cells from which at least immune cells have been substantially removed or cells obtained by culturing the cells have an MHC compatibility with the recipient of any of 3-6 antigen mismatches. In the combined cell preparation for transplantation of hematopoietic stem cells of the present invention, hematopoietic stem cells derived from all individuals can temporarily contribute to the hematopoiesis of the recipient and temporarily support the hematopoiesis of the recipient. For example, donor umbilical cord blood whose MHC fitness with the recipient is any of 0-2 antigen mismatches contributes to the recipient's hematopoietic system, and cells from other individuals with low MHC fitness can be removed. .

  In one embodiment, the combined preparation for transplantation of hematopoietic stem cells of the present invention has at least immune cells substantially from donor cord blood whose MHC fitness is any of 4-6 antigen mismatch, or 5 antigen mismatch or 6 antigen mismatch. Cells, which are then removed and then cultured in the presence of thrombopoietin (TPO) and stem cell factor (SCF) as a combination. In one embodiment, the combined preparation for transplantation of hematopoietic stem cells of the present invention has an MHC fitness between any of the remaining individuals and the recipient, except for one donor, each having 3 to 6 antigen mismatches.

  When the combined cell preparation for transplantation of hematopoietic stem cells of the present invention contains two or more umbilical cord blood, the degree of MHC compatibility between individuals may be 3-6 antigen mismatch. Therefore, in this embodiment, donor umbilical cord blood can be selected without being constrained by the condition that the MHC fitness between individuals must be any of 0 to 2 antigen mismatches.

  In one aspect, the combined cell preparation for transplantation of hematopoietic stem cells of the present invention includes, but is not limited to, cells derived from 2 to 10 individuals, cells derived from 2 to 5 individuals or 3 to 5 individuals. Including cells from the combination.

  In one embodiment, immune cells are substantially removed from the cord blood of any donor whose MHC fitness with the recipient is 0-2 antigen mismatch, but the donor-derived T cells are contaminated. Moreover, in a certain aspect, the umbilical cord blood of the donor whose MHC compatibility with a recipient is 0-2 antigen mismatch is transplanted as it is. In these embodiments, umbilical cord blood from individuals with MHC compatibility with recipients of any of 3-6 antigen mismatches is removed from the recipient body after contributing to early hematopoiesis until donor hematopoiesis is established. Is done.

In one aspect, in the combined cell preparation for transplantation of hematopoietic stem cells of the present invention, the donor umbilical cord blood whose MHC fitness with the recipient is any of 0 to 2 antigen mismatches is the number of cells measured at the time of umbilical cord blood collection. It contains only nucleated cells of 2 × 10 ⁸ to 1 × 10 ⁹ cells, and in some cases 2 × 10 ^{8 to} 5 × 10 ⁸ cells. Therefore, according to the present invention, it is possible to use umbilical cord blood obtained at the time of delivery of preterm infants in addition to full-term birth.

  Accordingly, in one aspect, in the combined cell preparation of the present invention, donor umbilical cord blood whose MHC fitness with the recipient is between 0 and 2 antigen mismatches is obtained at the time of delivery of a full-term or preterm infant Can be umbilical cord blood. “Full-term delivery” refers to births from 37 weeks 0 days of pregnancy to 41 weeks 6 weeks of pregnancy. “Premature birth” refers to childbirth before full term birth, that is, childbirth from pregnancy 22 weeks 0 days to pregnancy 36 weeks 6 weeks.

  Similarly, in a certain embodiment, in the combined cell preparation for transplantation of hematopoietic stem cells of the present invention, donor umbilical cord blood whose MHC fitness with the recipient is any of 0 to 2 antigens does not match with a birth weight of 3000 g or less. Umbilical cord blood obtained at the birth of a baby or low birth weight infant. “Low birth weight infant” refers to a baby whose birth weight is less than 2500 g.

The number of individuals from which umbilical cord blood is used in the combined cell preparation for transplantation of hematopoietic stem cells of the present invention can be determined using the following indicators. That is, the number of cells contained in the cord blood combined in the combined cell preparation for transplantation of hematopoietic stem cells of the present invention is the total number of nucleated cells at the time of cord blood collection, preferably 1 × 10 ⁹ cells or more, more preferably 2 ×. 10 ⁹ cells or more, more preferably 3 × 10 ⁹ cells or more, and even more preferably 4 × 10 ⁹ cells or more. As the number of nucleated cells increases, the survival rate of transplanted cells improves.

Thus, in some embodiments, in combination cellular preparation for hematopoietic stem cell transplantation of the present invention, cord blood of the donor is the total number of nucleated cells was measured during the cord blood collection, 2 × 10 ⁸ cells or more, 5 × 10 ⁸ cells The above includes nucleated cells of 1 × 10 ⁹ cells or more, 2 × 10 ⁹ cells or more, 3 × 10 ⁹ cells or more, or 4 × 10 ⁹ cells or more.

  As described above, the present invention pioneers the use of cord blood with a small number of nucleated cells as a hematopoietic stem cell transplant preparation. A problem in transplanting hematopoietic stem cells of umbilical cord blood is that the nucleated cells often do not contain a sufficient amount of nucleated cells for transplantation. However, in the present invention, regardless of the MHC suitability, the umbilical cord blood of the donor and the umbilical cord blood or hematopoietic stem cell fraction from which at least immune cells have been removed are cultured in the presence of TPO and SCF. By transplanting in combination with the cell culture obtained in this manner, hematopoietic stem cell transplantation can be engrafted even using cord blood with a small number of nucleated cells.

Therefore, in the combined cell preparation for transplantation of hematopoietic stem cells of the present invention, a donor whose MHC fitness with the recipient is 0-2 antigen mismatch is a donor whose MHC fitness with the recipient is 1 antigen mismatch. More preferably, it can be a donor with zero antigen mismatch. In some embodiments, donor-derived umbilical cord blood that has zero or one antigen mismatch in MHC compatibility with the recipient is 2 × 10 ⁸ to 1 × 10 ⁹ cells, optionally 2 × 10 ^{8 to} 5 × 10. Contains only ⁸ nucleated cells.

  In one embodiment, the combined cell preparation for hematopoietic stem cell transplantation of the present invention is intended for administration to humans. In the combined cell preparation for hematopoietic stem cell transplantation of the present invention, preferably, both the donor and the recipient are human.

  In one aspect, the present invention provides a pharmaceutical composition for use in assisting hematopoietic stem cell transplantation. The pharmaceutical composition for use in assisting hematopoietic stem cell transplantation according to the present invention comprises culturing cord blood from which hematopoietic stem cells or immune cells have been substantially removed in the presence of thrombopoietin (TPO) and stem cell factor (SCF). Cell obtained. As used herein, “assisting hematopoietic stem cell transplantation” means improving the survival rate of transplanted cells in a recipient after transplantation in hematopoietic stem cell transplantation. As used herein, “assisting hematopoietic stem cell transplantation” includes not requiring that the cells contained in the pharmaceutical composition of the present invention be engrafted in the recipient. In one embodiment of the present invention, cells contained in the pharmaceutical composition of the present invention are removed from the recipient's body by transplanted bone marrow or umbilical cord blood.

  In the present specification, the “pharmaceutical composition for use in assisting hematopoietic stem cell transplantation” is used in the meaning of “engraftment promoter”, or transplanted hematopoietic stem cells after hematopoietic stem cell transplantation are given to the recipient. It is used to mean a pharmaceutical composition used to maintain the recipient's hematopoiesis during the period until engraftment. As used herein, “a pharmaceutical composition for use in assisting hematopoietic stem cell transplantation” increases the survival rate of the recipient after hematopoietic stem cell transplantation, but the cells themselves contained in the composition are It also means a pharmaceutical composition that does not engraft in long-term hematopoiesis (eg, hematopoiesis after umbilical cord blood or bone marrow contributes to hematopoiesis). Long-term hematopoiesis is, for example, hematopoiesis after 4 weeks after transplantation, after 5 weeks, after 6 weeks, after February, after half a year, or after one year.

  In one embodiment of the present invention, the pharmaceutical composition of the present invention comprises cells obtained by culturing cord blood from which hematopoietic stem cells or immune cells have been substantially removed in the presence of TPO and SCF for 6 to 8 days. In a more specific aspect of the present invention, the pharmaceutical composition of the present invention comprises cells obtained by culturing hematopoietic stem cells in the presence of TPO and SCF for 5 to 10 days (eg, 7 days).

In an aspect of the present invention, the TPO concentration is 20 to 200 ng / mL, more preferably 30 to 150 ng / mL, and further preferably 40 to 100 ng / mL,
For example, it can be 50 ng / mL.

  In one embodiment of the present invention, the SCF concentration is 20 to 200 ng / mL, more preferably 30 to 150 ng / mL, and further preferably 40 to 100 ng / mL, for example, about 50 ng / mL. be able to.

  In one embodiment of the present invention, the TPO concentration and the SCF concentration are 20 to 200 ng / mL, more preferably 30 to 150 ng / mL, still more preferably 40 to 100 ng / mL, such as about 50 ng / mL. mL.

  The pharmaceutical composition for use in assisting hematopoietic stem cell transplantation of the present invention is administered to a recipient in combination with bone marrow or umbilical blood during bone marrow transplantation or umbilical cord blood transplantation. In the pharmaceutical composition for use in assisting hematopoietic stem cell transplantation according to the present invention, it is obtained by culturing umbilical cord blood or hematopoietic stem cells from which immune cells are substantially removed or any of these in the presence of TPO and SCF. The cells can support the recipient's hematopoiesis for a period of time until hematopoiesis is established by transplanted bone marrow or cord blood (for example, about 2 to 4 weeks after transplantation).

  In the pharmaceutical composition for use in assisting the hematopoietic stem cell transplantation of the present invention, the cultured hematopoietic stem cells may have an allo relationship with the transplanted bone marrow or umbilical cord blood. In the case of Allo, cell components derived from cultured hematopoietic stem cells are removed from the recipient body in the process of hematopoiesis caused by transplanted bone marrow or umbilical cord blood. Without being bound by the following theory, this removal of cells from the body suggests that transplanted bone marrow or umbilical cord blood-derived T cells may be involved. In one embodiment of the present invention, the cultured hematopoietic stem cells may be 3-6 antigen mismatches with the transplanted bone marrow or umbilical cord blood and MHC compatibility, and are derived from the donor by immune cells (particularly T cells) included in the donor. Hematopoiesis is established and removed from the recipient body.

  Therefore, the pharmaceutical composition for use in assisting the hematopoietic stem cell transplantation of the present invention is a T cell, specifically, a T cell capable of immunologically eliminating the hematopoietic stem cell contained in the pharmaceutical composition. Can be administered to a recipient in combination with a composition comprising The T cells capable of immunologically excluding the cell preparation can be T cells that are allo-cultured from the hematopoietic stem cells to be cultured. Of course, the T cells that can be combined are those that are immunologically compatible with the donor and may not inhibit hematopoiesis from donor cord blood-derived hematopoietic stem cells that contribute to long-term hematopoiesis.

  As described above, in the pharmaceutical composition for use in assisting the hematopoietic stem cell transplantation of the present invention, the cultured hematopoietic stem cells may have an allo relationship with the transplanted bone marrow or umbilical cord blood. Therefore, the pharmaceutical composition for use in assisting hematopoietic stem cell transplantation of the present invention can be produced in large quantities in a single lot and administered to various recipients regardless of the MHC suitability. is there.

  In one aspect, the pharmaceutical composition for use in assisting hematopoietic stem cell transplantation of the present invention comprises bone marrow or umbilical blood from which immune cells have been removed during bone marrow transplantation or umbilical cord blood transplantation, The hematopoietic cells contained in the pharmaceutical composition can be administered to the recipient in combination with T cells that can be immunologically excluded.

  As used herein, “administered in combination” includes both simultaneous administration and sequential administration. When administered at the same time, the recipient may be administered as a mixture or as separate compositions.

  The cell preparation may contain an excipient such as a buffer in addition to the cells.

  In addition, the pharmaceutical composition for use in assisting hematopoietic stem cell transplantation of the present invention can be administered to a recipient in combination with bone marrow or umbilical cord. Accordingly, in one aspect of the present invention there is provided a transplant material comprising cord blood or bone marrow and a pharmaceutical composition for use in assisting the hematopoietic stem cell transplant of the present invention. The transplant material of the present invention is a transplant material for hematopoietic stem cell transplantation.

  In one embodiment, in the combined cell preparation for transplantation of hematopoietic stem cells of the present invention, at least immune cells are removed from the blood, or hematopoietic stem cell fraction obtained from the blood, or any of these, Cells obtained by culturing in the presence of poietin (TPO) and stem cell factor (SCF) may also be used. The culture conditions of hematopoietic stem cells, for example, the number of culture days and the concentration of each factor are as described above.

  According to the examples described below, the cell preparation obtained by combining hematopoietic stem cells derived from a plurality of individual cord blood activated the recipient's hematopoiesis regardless of the degree of MHC compatibility with the recipient. Therefore, according to the present invention, a cell preparation for hematopoietic stem cell activation is provided.

  More specifically, a cell preparation for activating hematopoietic stem cells, which comprises umbilical cord blood and at least immune cells are substantially removed from the umbilical cord blood, is provided. In some embodiments, the MHC fitness between the individual from whom the cord blood is derived and the recipient is any of 3-6 antigen mismatches.

  As used herein, activation of hematopoietic stem cells means activation of proliferation and / or differentiation of hematopoietic stem cells.

  The cell preparation for activating hematopoietic stem cells of the present invention is advantageous in that it can be administered regardless of the MHC suitability. That is, the cell preparation for activating hematopoietic stem cells of the present invention can be mass-produced and can be administered to various patients regardless of the MHC suitability.

  In one embodiment, the cell preparation for activating hematopoietic stem cells of the present invention is obtained from cord blood from which at least immune cells derived from one or more further individuals have been removed or from one or more individual cord blood. Or a cell culture obtained by culturing the cord blood or hematopoietic stem cell fraction in the presence of TPO and SCF. In one embodiment, the cell preparation for activating hematopoietic stem cells of the present invention is a cord blood from which at least immune cells derived from a plurality of individuals whose MHC compatibility with the recipient is any of 3 to 6 antigen mismatches are removed. Or a combined cell preparation comprising a cell culture obtained by culturing the cord blood in the presence of TPO and SCF. In certain embodiments, the cell preparation for hematopoietic stem cell activation of the present invention comprises a cell culture derived from 2 to 10, 2 to 5, or 3 to 5 umbilical cord blood.

  When the cell preparation for activating hematopoietic stem cells of the present invention contains two or more umbilical cord blood or a culture thereof, the MHC fitness between any two individuals may be 3-6 antigen mismatches. Good.

  The cell preparation for activating hematopoietic stem cells of the present invention can activate transplanted hematopoietic stem cells. Accordingly, in one embodiment, the cell preparation for activating hematopoietic stem cells of the present invention is used in combination with a cell preparation for transplanting hematopoietic stem cells. That is, the cell preparation for activating hematopoietic stem cells of the present invention in this embodiment is an engraftment promoter for transplanted proliferating stem cells. Here, the cell preparation for transplantation of hematopoietic stem cells used in combination is preferably cord blood from which at least immune cells of an individual whose MHC compatibility with the recipient is any of 0 to 2 antigen mismatches are removed or A hematopoietic stem cell fraction, or a cell culture obtained by culturing any of these in the presence of TPO and SCF. The transplant-proliferating stem cell engraftment agent of the present invention may be mixed with donor umbilical cord blood and administered to the recipient, or may be administered to the recipient before, at the same time or after transplantation of the donor umbilical cord blood. Also good. In one embodiment of the present invention, the cells contained in the engraftment promoter of the present invention are temporarily used for early hematopoiesis of the recipient even if the MHC compatibility with the recipient is any of 3 to 6 antigen mismatches. And assists in the construction of the hematopoietic system by the donor's cord blood, but can eventually disappear from the recipient's body by the immune system constructed by the donor's cord blood.

  The pharmaceutical composition, combined cell preparation and cell preparation of the present invention may be intended to be used in combination with an immunosuppressive agent or may not be intended to be used in combination with an immunosuppressive agent.

  The pharmaceutical compositions, combination cell preparations and cell preparations of the present invention may contain one or more pharmaceutically acceptable carriers.

  The pharmaceutical compositions, combined cell preparations and cell preparations of the present invention are subject to hematopoietic stem cell transplantation (ie, recipients), including but not limited to acute lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, juvenile Aplastic monoaneous leukemia, myelodysplastic syndrome, malignant lymphoma, multiple myeloma, adrenocortical dystrophy, megakaryocyte hematopoietic thrombocytopenia, congenital erythroblastic fistula, Fanconi anemia , Hereditary neuropathy, mucopolysaccharidosis, congenital neutropenia, thalassemia, X chromosome lymphoproliferative syndrome recipient.

In the present invention, a method for producing a pharmaceutical composition for use in assisting hematopoietic stem cell transplantation, comprising:
Obtaining a hematopoietic stem cell fraction from bone marrow or cord blood;
A method comprising culturing a hematopoietic stem cell fraction in the presence of TPO and SCF to obtain cells is provided.

In the present invention, a method for producing a pharmaceutical composition for use in assisting hematopoietic stem cell transplantation, comprising:
Removing at least immune cells from bone marrow or cord blood;
A method comprising culturing the resulting bone marrow or umbilical cord blood in the presence of TPO and SCF to obtain cells is provided.

  The hematopoietic stem cell fraction can be obtained by methods well known to those skilled in the art, for example, as human CD34 positive cells and human CD34 positive CD38 negative cells. Cultivation in the presence of TPO and SCF is as described in the description of the cell preparation for use in assisting hematopoietic stem cell transplantation.

  In the combined cell preparation for transplantation of hematopoietic stem cells of the present invention, the umbilical cord of one individual is the umbilical cord of a donor whose MHC compatibility with the recipient is 0-2 antigen mismatch, and the umbilical cord of other individuals Blood can be selected regardless of the MHC suitability with the recipient. In this case, the produced combined cell preparation can engraft in the recipient transplanted hematopoietic stem cells derived from a donor whose MHC compatibility with the recipient is any of 0 to 2 antigen mismatches. This is because the donor-derived transplanted hematopoietic stem cells contain T cells, and in the long term, MHC compatibility with the recipient is any one of 0 to 2 antigen mismatches from cord blood other than donor-derived transplanted hematopoietic stem cells. This is probably because hematopoiesis is suppressed.

  Accordingly, in a further aspect of the present invention, a method for producing a combined cell preparation for hematopoietic stem cell transplantation is provided. That is, in the method for producing a combined cell preparation for transplantation of hematopoietic stem cells of the present invention, the cord blood selected in the following (c) is further added to the cord blood selected in the following (a) and the immune cells are substantially removed. Can be combined.

Specifically, the method for producing the combined cell preparation for hematopoietic stem cell transplantation of the present invention comprises:
(A) selecting a plurality of umbilical cord blood from a stock of umbilical cord blood (except that the MHC fitness of all umbilical cord blood and the recipient is 0-2 antigen mismatch);
(B) substantially removing immune cells from each cord blood of all individuals selected in (a) and culturing in the presence of thrombopoietin (TPO) and stem cell factor (SCF);
(C) A method comprising selecting a donor's umbilical cord blood from a donor's umbilical cord blood whose MHC fitness with a recipient is 0-2 antigen mismatch. The combined cell preparation obtained by this method can engraft the hematopoietic stem cells derived from cord blood selected in (c) and can be used for hematopoietic stem cell transplantation.

  In the above step (a), it is not restricted by the condition that the MHC fitness between the selected umbilical cord blood and the recipient must be 0-2 antigen mismatch, and the umbilical cord blood of a plurality of individuals is collected. You can choose from.

  In one embodiment, in the step (a), the MHC fitness between all umbilical cord blood is 0 in addition to the case where the MHC suitability between all umbilical cord blood and the recipient is 0-2 antigen mismatch. The case of antigen mismatch, any of 0-1 antigen mismatch, or 0-2 antigen mismatch is excluded.

  In one embodiment, the above step (b) collects CD34-positive cells or CD34-positive CD38-negative cells from each cord blood of all the donors selected in (a), and then the obtained cells are treated with thrombopoietin ( It is performed by culturing in the presence of TPO) and stem cell factor (SCF).

  According to the present invention, cord blood can be selected regardless of the degree of MHC compatibility with the recipient in order to produce a combined cell preparation for hematopoietic stem cell activation. Therefore, it is not necessary for all umbilical cord blood to meet the requirement that the degree of MHC compatibility with the recipient must be either 0-2 antigen mismatch. In particular, any cord blood may have 3-6 antigen mismatches with the recipient for MHC compatibility.

Accordingly, in a further aspect of the present invention, there is provided a method for producing a combined cell preparation for hematopoietic stem cell activation comprising:
(A) selecting a plurality of umbilical cord blood from a stock of umbilical cord blood (except that the MHC fitness of all umbilical cord blood and the recipient is 0-2 antigen mismatch);
(B) substantially removing immune cells from each cord blood of all individuals selected in (a) and culturing in the presence of thrombopoietin (TPO) and stem cell factor (SCF) A method is provided.

  In the above step (a), it is not restricted by the condition that the MHC fitness between the selected umbilical cord blood and the recipient must be 0-2 antigen mismatch, and the umbilical cord blood of a plurality of individuals is collected. You can choose from. In this case, the produced combined cell preparation is advantageous in that it can be administered regardless of the MHC suitability.

  In one aspect, in the step (a), the MHC suitability between all umbilical cord blood and the recipient is excluded from cases where the MHC suitability is any of 0 to 2 antigens, and the MHC suitability between all umbilical cord blood is excluded. Are either 0 antigen mismatches, 0-1 antigen mismatches, or 0-2 antigen mismatches.

In one embodiment, in the step (b), immune cells are removed by collecting CD34 positive cells or CD34 positive and CD38 negative cells. In one embodiment, in step (b) above, after immune cells are removed by collecting CD34 positive cells or CD34 positive and CD38 negative cells,
The obtained cells may be cultured in the presence of thrombopoietin (TPO) and stem cell factor (SCF).

  In one embodiment, the above production method of the present invention can be used to produce the pharmaceutical composition and the combined cell preparation of the present invention.

  In one aspect, the production method of the present invention is a method for producing a combined cell preparation for activating transplanted hematopoietic stem cells. In one aspect, the production method of the present invention is a method for producing a combined cell preparation for autologous hematopoietic stem cell activation in an administration subject. In some embodiments, the subject of administration is a patient with aplastic anemia.

  As used herein, “unrestricted by the requirement that the MHC fitness with the recipient must be 0-2 antigen mismatch” means “unrestricted by this condition”. It means you may. For example, the pharmaceutical composition, cell preparation or combined cell preparation for activating hematopoietic stem cells of the present invention may be selected without examining or knowing the MHC serotype of umbilical cord blood in (a). Alternatively, the combined cell preparation for hematopoietic stem cell transplantation or hematopoietic stem cell activation of the present invention may be selected based on any other conditions in (a). For example, the donor may select a donor with MHC suitability of 0-2 antigens that does not match the recipient, and the others without being particularly aware of the MHC suitability.

  In a further aspect of the present invention, there is provided a method of administering hematopoietic stem cells to a subject in need thereof, comprising donor umbilical cord blood whose MHC fitness with the subject is 0-2 antigen mismatch, and at least one person Administration of a cord blood or hematopoietic stem cell fraction from which at least immune cells of the individual have been substantially removed, or a combination with a cell culture obtained by culturing either of these in the presence of TPO and SCF. Is provided. In some embodiments, the cell culture has any 3-6 antigen mismatch with MHC compatibility with the recipient. The subject may be a subject undergoing hematopoietic stem cell transplantation (ie, a recipient), such as, but not limited to, acute lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, juvenile myelomonocytic leukemia, myelodysplastic syndrome, Malignant lymphoma, multiple myeloma, adrenocortical dystrophy, megakaryocyte hematopoietic thrombocytopenia, congenital erythroblast fistula, Fanconi anemia, and other aplastic anemia A patient with neutropenia, thalassemia, X-chromosome lymphoproliferative syndrome. If the subject has a cancer, such as lymphoma or leukemia, remove the cancer cells from the body by performing surgical, chemotherapy and / or radiation therapy on the subject before transplanting the hematopoietic stem cells be able to. The subject can be a human.

  In one aspect, a method of activating transplanted hematopoietic stem cells, wherein the subject is in need of at least one individual's cord blood, wherein at least immune cells are substantially removed, or cord blood Or a cell culture obtained by culturing any of these in the presence of TPO and SCF. In some embodiments, the cell culture has any 3-6 antigen mismatch with MHC compatibility with the recipient. The subject may be a subject undergoing hematopoietic stem cell transplantation (ie, a recipient), such as, but not limited to, acute lymphocytic leukemia, acute myeloid leukemia, chronic myelogenous leukemia, juvenile myelomonocytic leukemia, myelodysplastic syndrome, Malignant lymphoma, multiple myeloma, adrenocortical dystrophy, megakaryocyte hematopoietic thrombocytopenia, congenital erythroblast fistula, Fanconi anemia, and other aplastic anemia A patient with neutropenia, thalassemia, X-chromosome lymphoproliferative syndrome. If the subject has a cancer, such as lymphoma or leukemia, remove the cancer cells from the body by performing surgical, chemotherapy and / or radiation therapy on the subject before transplanting the hematopoietic stem cells be able to. The subject can be a human.

  In certain embodiments of the invention, the cord blood to be administered is a cell preparation or a combined cell preparation of the invention. In certain embodiments of the invention, cord blood is required with the pharmaceutical composition of the invention.

  In certain embodiments of the invention, the combination of cord blood to be administered can be administered separately, eg, co-administered, sequentially or sequentially.

Example 1 Calculation of Necessary Cell Numbers of KSL Cells and Total Bone Marrow Cells Necessary for Survival of Lethal Dose Irradiated Mice Mice irradiated with 4.9 Gy radiation twice cause bone marrow suppression and die. In this example, KSL cells or whole bone marrow cells, which are hematopoietic stem cells, were transplanted as a model of umbilical cord blood transplantation into mice irradiated with 9.8 Gy radiation, and the number of cells necessary for the survival of the mice was examined.

Each mouse strain was obtained as follows. C57BL / 6 (B6, H2 ^b , CD45.2), CBF1 (Balb / c
× B6 F1, H2 ^{b / d} , CD45.2), DBA / 2 (H2 ^d , CD45.2), B6C3F1 (B6 × C3HHe F1, H2 ^{b / k} , CD45.2) strains were purchased from Japan SLC . B6-CD45.1 (H2 ^b ) and B6-CD45.1 / 45.2 (H2 ^b ) lines are purchased from Sankyo Lab Service, and DBA / 1 (H2 ^q , CD45.2) lines are purchased from Charles River Japan. did. B6D1F1 (H2 ^{b / q} , CD45.2) strain is prepared by crossing male DBA / 1 and female B6-CD45.2, and BDF1 (H2 ^{b / d} , CD45.1 / CD45.2) strain is Each was produced by crossing male B6-CD45.1 and female DBA2. All animal experiments were prepared according to the guidelines of the Animal Experiment Committee of the University of Tokyo Medical Research Institute and used for experiments.

The recipient mice, C57BL / 6 (B6, H2 b, CD45.2) were used. Recipient mice were ingested acidic water (pH 2.5) from 7 days before the day of transplantation, irradiated with 4.9 Gy radiation twice (total 9.8 Gy) on the day of transplantation, and then received donor bone marrow cells. It was administered intravenously.

  Mouse KSL cells or whole bone marrow cells were used as donor bone marrow cells. Mouse KSL cells are c-Kit positive, Sca-1 positive, and lineage marker negative cells and are present in whole bone marrow cells.

First, whole bone marrow cells were obtained from the femur, tibia and pelvis of 8-12 week-old male mice of each strain by a conventional method. Next, mouse KSL cells were collected from whole bone marrow cells by the following method. C-Kit positive cells were selected using a MACS ™ LS column and c-Kit microbeads (Miltenyi Biotech, Bergisch Gladbach, Germany). Next, c-Kit positive cells were stained with CD4, CD8, B220, IL-7R, Gr-1, Mac-1 and Ter-119 as lineage markers using a biotinylated monoclonal antibody cocktail, and PE-Sca-1 Further stained with APC-c-Kit, APC-cyanine 7-streptavidin (Biolegend and e-Bioscience). After removing dead cells by propidium iodide staining, using FACS Aria II (manufactured by Becton Dickinson), KSL cells were placed on a 96-well plate using c-Kit positive, Sca-1 positive, and lineage marker negative as indices. It was collected.

  First, as shown in FIG. 1, 250, 500, 1000 or 2000 KSL cells derived from donor mouse B6 (CD45.1) were transplanted into irradiated mouse B6 (CD45.2). The survival rate of the recipient mice was confirmed.

  As a result, all the mice in the group transplanted with 500 or less KSL cells died, but the mice in the group transplanted with 2000 or more KSL cells showed 100% survival rate (FIG. 2). Therefore, in this experimental system, it was revealed that 2000 KSL cells are necessary for the survival of mice.

Next, whole bone marrow cells derived from donor mouse B6 (CD45.1) were transplanted into recipient mouse B6 (CD45.2) irradiated to confirm the survival rate of the recipient mouse. As a result, all the mice in the group transplanted with 1 × 10 ⁵ cells or less died, but the mice in the group transplanted with 2 × 10 ⁵ cells showed 100% survival rate (FIG. 3). . Therefore, in this experimental system, it was revealed that 2 × 10 ⁵ whole bone marrow cells are necessary for the survival of mice.

Furthermore, CBF1 (Balb / c × B6 F1, H2 ^{b / d} , CD45.2), B6C3F1 (B6 × C3HHe F1, H2 ^{b / k} , CD45.2), B6D1F1 (H2 ^{b / q} , CD45.2) ) 500 KSL cells obtained from the strain and the BDF1 (H2 ^{b / d} , CD45.1 / CD45.2) strain (all of which are allogeneic with the recipient mouse) were transplanted into the recipient mouse. All mice died in the groups transplanted with KSL cells of any strain (FIG. 4). In this experimental system, any line from which the donor cells are derived has the same MHC as the recipient, and thus does not cause GVHD to the recipient. On the other hand, because donor cells have MHC that the recipient does not have and can be attacked by the recipient, they can cause a host-to-graft response (HVGR). Therefore, this experimental system is a severe condition from the viewpoint of engraftment of transplanted donor cells.

In human umbilical cord blood transplantation, 2 × 10 ^{7 to} 3 × 10 ⁷ cells / kg body weight are required. Cells obtained from human umbilical cord blood are often about 7 × 10 ⁸ cells, but 70 k
When administered to an adult with a body weight of g, it is calculated to be about 1 × 10 ⁷ cells / kg body weight, which is less than the number of cells necessary for umbilical cord blood transplantation. That is, 2 to 3 units or more of cord blood is required for transplanting cord blood to an adult weighing 70 kg.

  According to this example, irradiated recipient mice were able to avoid lethality by transplanting 2000 or more KSL cells. Also, 500 KSLs were not sufficient for transplantation.

Similarly, according to this example, irradiated recipient mice were able to avoid lethality by transplanting 2 × 10 ⁵ whole bone marrow cells. In addition, 5 × 10 ⁴ whole bone marrow cells were not sufficient for transplantation.

By the way, transplantation of human umbilical cord blood requires umbilical cord blood containing 1 × 10 ⁹ or more nucleated cells for an adult having a body weight of 70 kg. However, in many cases, the amount of nucleated cells contained in human umbilical cord blood is about 1/4 to 1/3 thereof. In order to mimic this situation of human umbilical cord blood transplantation, subsequent experiments were conducted assuming that 1/4 of the required number of cells was regarded as a hematopoietic stem cell unit obtained from one person. That is, in the following examples, 500 KSL cells were used as one unit, and similarly, 5 × 10 ⁴ whole bone marrow cells were used as one unit.

Example 2: Effect of lethal dose of a mixture of KSL cells derived from different strains of mice on survival of irradiated mice In Example 1, the survival of lethal doses of irradiated mice is 4 units derived from a single strain. It was shown that (2,000) KSL cells were required. In this example, KSL cells derived from different strains of mice were mixed one unit at a time, and a total of 5 units (2,500 cells) were transplanted into lethal doses of irradiated mice to confirm their effects on survival.

Specifically, in this example, C57BL / 6 (B6, H2 ^b , CD45.1) system, CBF1 (Balb / c × B6 F1, H2 ^{b / d} , CD45.2) system, B6C3F1 (B6 × C3HHe F1, H2 ^{b / k} , CD45.2), B6D1F1 (H2 ^{b / q} , CD45.2) and BDF1 (H2 ^{b / d} , CD45.1 / CD45.2) KSL cells were mixed and transplanted into recipient mice obtained by the method described in Example 1. As a positive control, recipient and ^{C57BL / 6 (B6, H2 b} , CD45.1) with complete identical MHC antigen type recipe to 2,500 KSL cells lines obtained by the method described in Example 1 It transplanted to the ent mouse.

  As a result, surprisingly, the mice transplanted with the five-line mixed KSL cells obtained by mixing the five lines of KSL cells showed the same level of survival as the positive control (FIG. 5).

  Therefore, the recovery of leukocytes and platelets in recipient mice after transplantation was examined. The white blood cell count and platelet count of the mouse were determined by measuring peripheral blood obtained from the mouse by a conventional method using a fully automatic blood cell counter MEK-6450 Celltac α (manufactured by Nihon Kohden Co., Ltd.).

  As a result, KSL cells obtained by mixing KSL cells derived from five lines showed the same level of recovery as the positive control with respect to leukocytes and platelets (FIG. 6). That is, it was found that normally, 4 units of KSL cells are required, but the mouse hematopoietic recovery was observed even though the transplanted allogeneic syngeneic KSL cells were only 1 unit. This hematopoietic recovery is thought to be due to KSL cells derived from four lines that have an antigenic type completely inconsistent with the recipient's MHC serotype.

  In hematopoietic stem cell transplantation, it is generally required that the donor and recipient have the same HLA serotype. For example, bone marrow transplantation requires one antigen mismatch or complete antigen match. Blood transplantation requires two antigen mismatches, one antigen mismatch or complete antigen match. However, according to the present results, all strains of donor mice caused hematopoietic recovery in the recipients even though all of the MHC serotypes were inconsistent with the recipient mice (full allo). Hematopoietic stem cells have been shown to settle within the recipient.

Example 3: Contributing to recipients of KSL cells obtained by mixing KSL cells from five lines As described in Example 1, this experimental system can cause a host-to-graft response (HVGR) System, and allogeneic cells can be eliminated by the recipient. Therefore, when KSL cells derived from one allogeneic strain and four allogeneic strains are used in a mixture with the recipient mouse, the KSL cells derived from the four allogeneic strains are subject to host versus graft reaction. (HVGR) was excluded and it was thought that it could not contribute to the hematopoietic system. In order to confirm this, in this example, a chimerism analysis of transplanted hematopoietic stem cells engrafted in the recipient was performed.

KSL cells obtained by mixing KSL cells derived from 5 lines were introduced into the recipient as described in Example 4. To examine the contribution of each lineage to hematopoietic cells, peripheral blood was collected from the recipient and ACK lysis buffer (NH ₄ Cl 8,024 mg / L, KHCO ₃ 1,001 mg / L, disodium EDTA dihydrate). After treatment with product 3.722 mg / L), staining was performed with the following antibodies: Brilliant Violet 570-Ly-6G / 6C (Gr-1), PE-Cyanin5-CD45R / B220, Alexa Floor 700- CD4, Alexa Floor 700-CD8a, PE-cyanine 7-CD45.1, APC-cyanin7-CD45.2, Pacific Blue-H2Kd, FITC-H2Kk, PE-H2Kb, and Alexa Floor647-H2Kq (Biolegend). After staining cells of each lineage with different dyes in this way, analysis was performed using FACS Aria II (Becton Dickinson) and hematopoietic cells of each lineage using FlowJo software (TreeStar, Ashland, OR, USA). The contribution to was investigated.

  As a result, contrary to expectation, it became clear that KSL cells derived from all strains contribute to the recipient (FIG. 7). As described above, in this experimental system, it is considered that KSL cells derived from allogeneic strains can be eliminated by the host-versus-graft reaction (HVGR), and thus all strains of cells contributed equally to the recipient. That is very surprising.

  From the results of this example, even if KSL cells are derived from allogeneic strains, they can contribute to the recipient's hematopoietic cells by removing immune cells and transplanting them as a mixture of multiple strains. Became clear.

Example 4: Hematopoietic recovery using 1 unit of allogeneic whole bone marrow and 4 units of allogeneic KSL cells In the above example, the transplanted allogeneic KSL cells were approximately equal in recipient mice. It has been shown to contribute to the hematopoietic system. In this example, recovery of the hematopoietic system was confirmed when one line was allogeneic and allogeneic bone marrow and the other was allogeneic KSL cells.

Whole bone marrow of syngeneic is, C57BL / 6 (B6, H2 b, CD45.1) according to the method described in Example 1 was prepared from strains of mice. Allogeneic KSL cells are CBF1 (Balb / c x B6 F1, H2 ^{b / d} , CD45.2), B6C3F1 (B6 x C3HHe F1, H2 ^{b / k} , CD45.2), B6D1F1 (H2 ^{b / q} , CD45.2) and BDF1 (H2 ^{b / d} , CD45.1 / CD45.2) strains were prepared by the method described in Example 1. 1 unit of whole bone marrow (the number of nucleated cells: 5 × 10 ⁴ cells) is mixed with 1 unit (500 cells) of the obtained 4 lines of allogeneic and allogenic bone marrow. A mixture of 4 units of KSL cells was obtained. The recipient mice, using ^{C57BL / 6 (B6, H2 b} , CD45.2) were irradiated as described in Example 1.

The obtained mixture of whole bone marrow and KSL cells was transplanted into recipient mice by intravenous administration to confirm the survival rate of the mice after transplantation. In the group administered with only 5 × 10 ⁴ whole bone marrow, Although mice died, all mice survived in the group that received a mixture of whole bone marrow and KSL cells (FIG. 8). In other words, hematopoietic stem cell transplantation required 4 units of whole bone marrow, and it was found that a mixture of 1 unit of whole bone marrow and allogeneic KSL cells showed hematopoietic recovery in mice. From this, it is clear that if whole bone marrow containing at least about 1/4 of the required number of cells is obtained, it can be used for hematopoietic stem cell transplantation by mixing it with hematopoietic stem cells such as KSL cells. It was. Moreover, the mixed KSL cells could be used regardless of the MHC suitability with the recipient.

  In addition, peripheral blood of surviving recipient mice was collected, and the contribution of cells derived from each strain to the hematopoietic system was examined by the method described in Example 3. Even though KSL cells derived from multiple strains were mixed with whole bone marrow and introduced into a recipient mouse, substantially only cells derived from whole bone marrow (about 94.7% ± 2.5%) entered the hematopoietic system. It became clear that it contributed (FIG. 9, B6 (CD45.1); H2b). In addition, almost no cells derived from recipient mice were observed (FIG. 9, B6 (CD45.2); H2b). This is because, immediately after transplantation, KSL cells can contribute to the recipient's hematopoietic system, but after recovery of hematopoiesis in the recipient, the KSL cells were eliminated by the immune system derived from the transplanted whole bone marrow. It seemed that. According to this example, it was shown that hematopoietic stem cells such as KSL cells have a cell engraftment promoting effect.

  In the above examples, bone marrow cell-derived hematopoietic stem cells were used as a model, but those skilled in the art will fully understand that the same applies when umbilical cord blood-derived hematopoietic stem cells are used. Umbilical cord blood enables more effective hematopoietic stem cell transplantation in that transplantation is possible even when the HLA compatibility is lower than that of bone marrow.

Example 5A: Cell components for assisting transplantation of hematopoietic stem cells In this example, it was attempted to use a culture of hematopoietic stem cells as an adjuvant for transplantation of hematopoietic stem cells.

The design of the experiment is as shown in FIG. 10A, and a culture obtained by culturing hematopoietic stem cells (CD34 ⁻ KSL cells) in the presence of a specific hematopoietic factor under conditions of competition with whole bone marrow is given to the recipient. After transplantation, the contribution of cells to the hematopoietic system after transplantation was examined.

Mouse whole bone marrow was collected from the femur, tibia and pelvis of 8-12 weeks old male mice. In this example, CD34 ⁻ KSL cells were obtained by a conventional method as hematopoietic stem cells, and each well was present in the presence of 50 ng / mL thrombopoietin (TPO, manufactured by Peprotech) and 50 ng / mL stem cell factor (SCF, manufactured by Peprotech). 50 hematopoietic stem cells were cultured. As the anti-CD34 antibody, FITC-anti-CD34 antibody (manufactured by Biolegend) was used. The medium used was s-clone medium (Eidia Co., Ltd.) supplemented with 10% bovine serum albumin (SigmaAldrich, A4503) and 1% penicillin-streptomycin-glutamine (Gibco, 10378).

  Cells after 7 days, 12 days or 14 days of culture were as shown in the photograph of FIG. 10B.

  Expression of c-kit, Lin, Sca1 and CD48 in the obtained culture was confirmed. The surface antigen was stained with biotinylated lineage marker, streptavidin APC-Cy7, PE-CD48, APC-c-kit, PE-cyanin 7-Sca1- (manufactured by Biolegend), and analyzed with FACS Aria II. The number of cells after the culture was measured with FACS Aria II using Flow-Count (trademark) Fluorospheres (manufactured by Beckman Coulter).

  The results are as shown in FIG. 11, and the cell population of the Lin− and c-kit + fractions was selectively amplified after 7 days of culture.

  A competition experiment was conducted by transplanting cells cultured (hereinafter also referred to as “induction”) in the presence of the above hematopoietic factors or cells that were not cultured to designated several recipient mice.

The competitive hematopoietic reconstitution assay is an experimental system that utilizes the mouse CD45 subtype to quantify the hematopoietic potential of the graft. A lethal dose (9.8 Gy) of B6-CD45.1-derived CD34 ⁻ KSL cells and cultured hematopoietic cells together with whole bone marrow cells (cell count: 1 × 10 ⁶ cells) of B6-CD45.1 / 45.2 mice After transplantation to irradiated B6-CD45.2 recipient mice, the subsequent peripheral blood of the recipients was analyzed over time. The peripheral blood of the recipient was treated with ACK hemolyzing agent, and then FITC-Ly5.2, PE-cyanin 7-Ly5.1, PE-Mac1, PE-Gr1, APC-cyanin 7-CD45R / B220, APC- Stained with CD4 and APC-CD8a antibodies and analyzed using FACS Aria II. By measuring the proportion of CD45.1 cells in each blood cell lineage, the frequency of donor-derived cells in myeloid cells was detected.

  The result was as shown in FIG. Cells cultured for 7 days showed a remarkably high chimera rate one week after transplantation, and were found to have the ability to contribute to hematopoiesis of neutrophils at the initial stage after transplantation. On the other hand, the chimera rate of cells induced for 7 days greatly decreased at 4 weeks, and cells derived from whole bone marrow came to dominate. This means that the cells induced for 7 days have the ability to help the early hematopoietic system after hematopoietic transplantation and disappear after the hematopoietic system has started up. Since the hematopoietic system is established, it disappears, and this induced cell has the great advantage that it can be used as an allograft for the recipient.

Example 6A: Examination of red blood cell and platelet hematopoietic ability of TPO and SCF-induced hematopoietic stem cell cultures In this example, the contribution of hematopoietic stem cell cultures obtained by the method of Example 5A to red blood cells and platelets was confirmed in more detail. .

First, FIG. 13 shows an outline of the design of the experiment. As the donor mouse, a mouse in which blood cells are labeled with wedge orange. 500 KSL cells obtained from bone marrow, 50 hematopoietic stem cells (CD34 ⁻ KSL cells), and 50 hematopoietic stem cells (CD34 ⁻ KSL cells) induced for 7 days under the conditions of Example 5A And transplanted into recipient mice irradiated with 9.8 Gy together with whole bone marrow (1 × 10 ⁶ cells) of B6 (CD45.1 / 45.2) mice. Peripheral blood was collected 14 days after transplantation, and the contribution rate of cell culture in erythrocytes and platelets was confirmed using KuO fluorescence as an index.

  As shown in S. Hamanaka et al., Biochem Biophys Res Commun 435, 586 (2013), a mouse made of wedge orange (KuO) was backcrossed with B6-CD45.1 mice 7 times. The obtained mouse was used for the experiment.

  The results were as shown in FIGS. 13B-D. As shown in FIGS. 13B-D, hematopoietic stem cells induced with TPO and SCF for 7 days were particularly effective against bone marrow, erythrocytes and platelets. An excellent contribution rate was shown in the early period after transplantation. This means that hematopoietic stem cells induced with TPO and SCF have a high ability to assist early hematopoietic recovery of the three blood cell lines (neutrophils, erythrocytes and platelets).

Example 7A: Factors Determining Final Cell Engraftment In this example, factors that ultimately removed induced cell cultures were examined.

  For this purpose, the chimera rate in recipients of KDF cells 2000 cells of BDF1 (45.1 / 45.2) mice was examined under competitive conditions.

The competing cells
No. 1, WT-B6 (45.1) whole bone marrow 2.0 x 10 ⁵ cells,
No. 2, WT-B6 (45.1) KSL 2,000 cells,
No. 3, Rag2KO-B6 (45.1) whole bone marrow 2.0 x 10 ⁵ cells,
No. 4, Rag2KO-B6 (45.1) whole bone marrow 2.0 x 10 ⁵ cells plus 4,000 T cells,
No. 5, Rag2KO-B6 (45.1) total bone marrow 2.0 × 10 ⁵ cells plus 40,000 B cells.
Note that Rag2 knockout (Rag2KO) mice lack mature B cells, mature T cells, and mature NKT cells.

  The result was as shown in FIG. That is, no. 3, when competed with whole bone marrow of Rag2KO mice, BDF1 (45.1 / 45.2) mouse KSL cells contributed to the recipient. When competing with whole bone marrow of Rag2KO mice containing T cells at 4, BDF1 (45.1 / 45.2) mouse KSL cells failed to contribute to the recipient. No. Even when competing with whole bone marrow of Rag2KO mice containing B cells at 5, BDF1 (45.1 / 45.2) mouse KSL cells contributed to the recipient. This suggests that T cells are a determinant of hematopoietic chimerism in recipients.

Example 8A: Donor hematopoietic stem cell transplantation experiment using a culture of allo-hematopoietic stem cells In this example, hematopoietic stem cells induced with TPO and SCF were obtained from multiple strains of mice and mixed to obtain all the cells. Transplantation into 9.8 Gy irradiated mice with donors containing bone marrow confirmed subsequent survival.

Control group 1 is a group in which only B6 (Ly45.1 / 45.2) H2b whole bone marrow 1x10 ⁵ cells are transplanted,
Test group 2 is a group in which whole bone marrow is transplanted with a mixture of 80 cells of culture obtained by inducing hematopoietic stem cells derived from B6 (Ly45.1) H2b mice with TPO and SCF,
Test group 3 is a group in which the whole bone marrow is transplanted with a mixture of hematopoietic stem cells derived from B6 (Ly45.1) H2b mice and cultured 200 cells obtained by induction with TPO and SCF.
Test group 4 is a group in which whole bone marrow is transplanted with a mixture of 50 cells of culture obtained by inducing hematopoietic stem cells derived from KuO-B6C3F1 (Ly45.1 / 45.2) H2b / k mice with TPO and SCF,
The test group 5 is a group in which the whole bone marrow is transplanted with a mixture of 20 cells of culture obtained by inducing hematopoietic stem cells derived from 4 allo lineages with TPO and SCF,
Test group 6 is a group in which whole bone marrow is transplanted with a mixture of 50 cells of culture obtained by inducing hematopoietic stem cells derived from 4 allo lineages with TPO and SCF,
It was. The four lines used were KuO-B6C3F1 (Ly45.1 / 45.2) H2b / k, B6D1F1 (Ly45.2) H2b / q, BDF1 (Ly45.1 / 45.2) H2b / d and CBF1 (Ly 45.2) H2b / d. KuO-B6C3F1 (Ly45.1 / 45.2) H2b / k strain was prepared by crossing C3HHeJ (C3H, H2k, CD45.2) mice purchased from Japan SLC with KuO-CD45.1 mice.

  Then, in the control group 1 which does not use cells that assist early hematopoietic recovery, most mice died within a few days. On the other hand, in the test groups 2 to 6 in which hematopoietic stem cells induced with TPO and SCF for 7 days were mixed, all achieved a survival rate of 100%. Here, there was no difference in results between the group in which the cultures derived from allo mice were mixed (test groups 4 to 6) and the group in which the cultures derived from congenic mice were mixed (test groups 2 and 3). .

  Changes in chimera rate in erythrocytes after transplantation were examined up to 8 weeks after transplantation. The result was as shown in FIG. That is, as shown in FIG. 16, the allo-mouse-derived graft can contribute to early hematopoiesis for 2 to 3 weeks after transplantation, while it contributes to hematopoiesis 8 weeks after transplantation. lost. In addition, the change in chimera rate in platelets was examined in the same manner, but it was possible to contribute to early hematopoiesis almost 2 to 3 weeks after transplantation, but no contribution to hematopoiesis was seen 8 weeks after transplantation. (FIG. 17). As for the chimera rate in neutrophils, allo-mouse-derived grafts can also contribute to early hematopoiesis in 2 to 3 weeks after transplantation, but include whole bone marrow about 30 days after transplantation. The contribution of donor-derived cells to hematopoiesis was almost 100% (FIG. 18).

Example 9A: Transplantation experiment in the presence of both GVH and HVG reactions In the previous transplantation experiments, experiments were conducted in a system in which only a host-to-graft (HVG) reaction occurred. Specifically, experiments were conducted in a system in which cells derived from recipient mice and F1 mice of other strains were used as transplanted cells. In this example, this experiment system was modified, and a similar transplantation experiment was conducted using a donor that simultaneously generated a graft-versus-host (GVH) reaction.

  Mice from DBA / 1JJmsSlc (DBA / 1, H2q, CD45.2), DBA / 2CrSlc (DBA / 2, H2d, CD45.2) and C3HHeJ (C3H, H2k, CD45.2) were purchased from Japan SLC. Used in this example. The design of the experiment is as shown in FIG. 19A. Hematopoietic stem cells obtained from the mouse bone marrow were cultured for 7 days in the same manner as in Example 5A to obtain a culture, which was mixed with donor whole bone marrow (B6 (Ly45.1 / 45.2) H2b). . Transplanted into recipients (B6, CD45.2) irradiated with 8 Gy.

Control group 11 is a group transplanted with whole bone marrow 1 × 10 ⁵ cells of B6 (Ly45.1 / 45.2) H2b mice,
The test group 12 was a group in which 60 cells obtained by inducing hematopoietic stem cells obtained from B6 (Ly45.1) H2b mice were transplanted in addition to the whole bone marrow.
Test group 13 was a group in which 150 cells obtained by inducing hematopoietic stem cells obtained from B6 (Ly45.1) H2b mice in addition to the whole bone marrow were transplanted,
In the test group 14, in addition to the whole bone marrow, 50 cells obtained by inducing hematopoietic stem cells obtained from DBA2 (Ly45.2) H2d mice were transplanted,
Test group 15 was obtained by inducing hematopoietic stem cells obtained from DBA1 (Ly45.2) H2q mice, DBA2 (Ly45.2) H2d mice and C3H (Ly45.2) H2k mice in addition to the whole bone marrow. A group of 20 transplanted cells,
Test group 16 was obtained by inducing hematopoietic stem cells obtained from DBA1 (Ly45.2) H2q mice, DBA2 (Ly45.2) H2d mice and C3H (Ly45.2) H2k mice in addition to the whole bone marrow. Each group was transplanted with 50 cells.

  The survival curve after transplantation was as shown in FIG. 19B. In control group 11 transplanted only with whole bone marrow, most mice died within 7 to 14 days. On the other hand, in the test groups 14 to 16 capable of producing both GVH and HVG responses, the survival rate of the mice was improved depending on the number of transplanted cells.

  Next, the contribution of cells in neutrophils in the recipient body after transplantation was observed over time. Then, as shown in FIG. 20, on day 7 after transplantation, the cells obtained by induction contributed to the hematopoietic system (Nos. 3 to 5). Contribution of the obtained cells to the hematopoietic system was not recognized (No. 3 to 5).

  Thus, the cells obtained by culturing hematopoietic stem cells for 7 days with TPO and SCF very well support hematopoiesis at an early stage until donor-derived hematopoiesis including all bone marrow is sufficiently established, and include whole bone marrow. Once donor-derived hematopoiesis started, it disappeared quickly and it had the property of not contributing to the recipient's hematopoiesis. This means that cells obtained by culturing hematopoietic stem cells with TPO and SCF for 7 days may have a complete allo relationship with each donor or recipient. Even a mixture of allografts from each other could assist in hematopoiesis from donors, each containing whole bone marrow.

Example 10B: Transplantation experiment using human umbilical cord blood In this example, it was confirmed that the results of the above examples were similarly established in humans using human umbilical cord blood.

(Human umbilical cord blood)
HLA donated from Kanto Koshinetsu Block Blood Center (Japan Red Cross Kanto Koshinetsu Cord Blood Bank) and frozen cord blood with known blood type were used. The HLA type and blood type of each of the four types of frozen cord blood used were as shown in Table 1.

(Purification of hematopoietic stem / progenitor cells)
Frozen umbilical cord blood was thawed in a 37 ° C. warm bath on the day of transplantation, and mononuclear cells were separated using CliniMACS Prodigy (Miltenyi Biotech) in a sterile state. During separation, Ficoll Paque Premium 1.084 (GE Healthcare) and DNase I (Roche Applied Science) were allowed to act in the same machine. In an experiment using multiple units of frozen umbilical cord blood, mixing was performed immediately before the operation of CliniMACS Prodigy, and the same machine was used. The obtained mononuclear cells were purified using CD34 Microbeads Kit and CD3 Microbeads Kit (both Miltenyi Biotech), respectively. CD34 positive cells and CD3 positive cells were stained with CD45-APCCy7 or Pacific Blue, CD3-PE-Cyanine 7, CD4-PE, CD8a-APC, Lineage-FITC, and CD34-Pacific Blue or APC (Biolegend) It was analyzed by Canto (BD, Bioscience) and confirmed to be purified.

  The frozen cord blood used contained only a small amount of anti-A IgM antibody and anti-B IgM antibody, and the contained T cells were mainly naïve T cells. From this, when using a plurality of umbilical cord blood with different blood types and HLA, a mixture of a plurality of umbilical cord blood is prepared immediately before the purification step, and agglutination between units is not caused. Hematopoietic stem / stem progenitor cells were purified.

(Transplantation experiment)
The outline of the transplantation experiment is shown in FIG. FIG. 21 shows that frozen cord blood is treated with CliniMACS Prodigy to isolate mononuclear cells, and then CD34 positive cells or CD3 positive cells are purified using CD34 Microbeads Kit or CD3 Microbeads Kit. It is the schematic which shows a mode that it transplanted intravenously to the irradiated immunodeficient mouse (NOG mouse).

  Specifically, the recipient is infused with acidic water (pH 2.5) for at least 7 days prior to the transplantation date, irradiated with 1.2 Gy on the day of transplantation, and transplanted donor bone marrow cells intravenously. did. A mixture of 340 μL of enrofloxacin (Baitril ™ 10% injection) per 200 mL of acidic water was ingested from the day of transplantation.

(FACS analysis)
To analyze the chimerism of hematopoietic cells in recipient mice, bone marrow was collected from recipients and treated with ACK lysis buffer (NH4Cl 8,024 mg / l, KHCO3 1,001 mg / l, EDTA.Na2.2H2O 3.722 mg / l). Later, staining was performed with the following antibodies: HLA-A2-PE, human CD45-PB, mouse CD45-PE-Cyanin7, CD19-FITC, CD3-APC, Streptoavidin-APCCy7 (Biolegend), HLA-A30A31-biotin ( One Lambda) and CD33-PerCP5.5 (BD, Bioscience). These cells were analyzed using FACS Aria (Becton Dickinson, San Jose, Calif.), And the results were processed using FlowJo software (TreeStar, Ashland, OR, USA).

(result)
The experimental results are shown in FIG. 22. In recipient mice transplanted with a total of 1.28 × 10 ⁵ mixed CD34 positive cells consisting of 4 units of cord blood, CD34 + cells collected from 1 unit of cord blood 3.2 × Higher hematopoietic cell chimerism was observed compared to 10 ⁴ . Furthermore, in this group of CD34 positive cells mixed with 4 units, when CD3 positive T cells were extracted from one unit and added, units containing CD3 positive T cells selectively survived and converged to a single chimerism.

In addition, the upper left end of FIG. 22 is a group transplanted with 3.2 × 10 ⁴ CD34 positive cells from Unit A. In the second row from the upper left, CD34 positive cells in which 3 units of Unit B, C and D are mixed are added to 3.2 × 10 ⁴ of unit A-derived CD34 positive cells to give a total of 1.28 × 10 ⁵ cells. Is a group transplanted. The third row from the upper left is a group transplanted with 1.28 × 10 ⁵ CD34 positive cells derived from Unit A. In the fourth row from the upper left, CD34 positive cells in which 3 units of Unit B, C and D are mixed with 3.2 × 10 ⁴ of unit A-derived CD34 positive cells are added, and a total of 1.28 × 10 ⁵ cells are obtained. Then, 3.2 × 10 ⁵ of A3 CD3 positive T cells were added. When human hematopoietic cell chimerism in mouse bone marrow was compared, the mixed umbilical cord blood unit exhibited a level of chimerism equivalent to the group transplanted with a sufficient number (1.28 × 10 ⁵ ) of human hematopoietic stem cells derived from one unit. did.

  Furthermore, when the contribution rate of each donor cord blood to the hematopoietic cells in these groups was examined, at the time of 21 days after transplantation, a group in which CD34 positive cells derived from a plurality of cord blood were mixed and transplanted (FIG. 22). In the second column from the left), cells from a plurality of donors contributed to hematopoiesis, but in a group in which 1 unit-derived CD3-positive T cells were added thereto, cells derived from a single donor containing T cells Only contributed to hematopoiesis (fourth column from the left in FIG. 22).

  FIG. 23 shows the results of tracing the chimerism of human hematopoietic cells in the recipient over time using HLA-A2 as Merckmar. As shown in FIG. 23, a mixed chimerism was formed in the group transplanted by mixing only CD34 positive cells (second row from the left), and a cord blood unit positive for HLA-A2 contributed to hematopoiesis. It is recognized that On the other hand, the HLA-A2 positive rate in the group to which CD3 positive T cells derived from 1 unit of HLA-A2 negative were added was recognized at a constant rate on the 15th day after transplantation and formed a mixed chimerism. Although it was considered, it decreased after 21 days after transplantation and finally converged to a single chimerism (fourth column from the left).

  In this transplantation model, as in our previous reports, mixing of the cord blood-derived T cells into one type of cord blood unit (Unit A) that would ultimately contribute to hematopoiesis A single donor chimerism (that is, only one umbilical cord blood-derived cell contributes to hematopoiesis) while chimerism (that is, a state where cells derived from multiple umbilical cord blood contribute to hematopoiesis) is formed transiently Converged).

  From the above results, it was possible to verify the effectiveness of the technique of mixing and administering hematopoietic stem cells derived from allogeneic umbilical cord blood using human umbilical cord blood cells. Furthermore, in this model as well, it was found that CD34 positive cells derived from multiple cord blood contribute to human hematopoietic cell chimerism in a cooperative manner in the early stage (21st day) after transplantation. Even in this case, it was observed that mixed chimerism was formed early after transplantation of multiple cord blood, and finally converged to a single chimerism derived from a donor containing a small amount of T cells.

Claims (19)

A pharmaceutical composition for use in assisting hematopoietic stem cell transplantation, comprising:
A pharmaceutical composition comprising cells obtained by culturing hematopoietic stem cells in the presence of thrombopoietin (TPO) and stem cell factor (SCF).   The pharmaceutical composition according to claim 1, comprising cells obtained by culturing hematopoietic stem cells in the presence of TPO and SCF for 5 to 10 days.   The pharmaceutical composition according to claim 2, comprising cells obtained by culturing hematopoietic stem cells in the presence of TPO and SCF for 7 days. With cord blood or bone marrow,
An implant material comprising the pharmaceutical composition according to any one of claims 1 to 3. A combined cell preparation for hematopoietic stem cell transplantation, comprising:
Umbilical cord of a donor whose MHC fitness with the recipient is any of 0-2 antigen mismatch,
Combination cells comprising cells obtained by culturing a cell fraction comprising hematopoietic stem cells obtained from cord blood of at least one individual in the presence of thrombopoietin (TPO) and stem cell factor (SCF) Formulation.   The combined cell preparation according to claim 5, wherein the cells obtained by the culture are any of 3 to 6 antigen mismatches with donor umbilical cord blood.   The combined cell preparation according to claim 5 or 6, wherein the MHC fitness between any two individuals is independently any of 3 to 6 antigen mismatches.   The cell fraction comprising hematopoietic stem cells is obtained by collecting CD34-positive cells or CD34-positive and CD38-negative cells from cord blood, according to any one of claims 5 to 7. Combination cell preparation.   The combined cell preparation according to any one of claims 5 to 8, wherein the individual consists of 2 to 5 individuals.   The combined cell preparation according to any one of claims 5 to 9, wherein all umbilical cord blood is human umbilical cord blood. A method for producing the pharmaceutical composition according to any one of claims 1 to 3,
Obtaining a hematopoietic stem cell fraction from bone marrow or cord blood;
Culturing the hematopoietic stem cell fraction in the presence of TPO and SCF to obtain cells;
Comprising a method. A method for producing a combined cell preparation for hematopoietic stem cell transplantation, comprising:
(A) selecting a plurality of umbilical cord blood from a stock of umbilical cord blood (except that the MHC fitness of all umbilical cord blood and the recipient is 0-2 antigen mismatch);
(B) substantially removing immune cells from each cord blood of all individuals selected in (a) and culturing in the presence of thrombopoietin (TPO) and stem cell factor (SCF);
(C) selecting one donor's umbilical cord blood from umbilical cord blood whose MHC fitness with the recipient is 0-2 antigen mismatch. Step (b) collects CD34-positive cells or CD34-positive and CD38-negative cells from each cord blood of all individuals selected in (a), after which the resulting cells are thrombopoietin (TPO) and The method according to claim 12, which is performed by culturing in the presence of stem cell factor (SCF).   14. The method of claim 12 or 13, wherein the umbilical cord blood is human umbilical cord blood. A cell preparation for hematopoietic stem cell transplantation in humans,
Umbilical cord of a donor whose MHC fitness with the recipient is any of 0-2 antigen mismatch,
A cell preparation comprising a combination of at least one umbilical cord blood with at least immune cells substantially removed from the umbilical cord blood. The cell preparation according to claim 15,
A cell preparation, which is at least one umbilical cord blood, wherein the umbilical cord blood from which at least immune cells are substantially removed is a hematopoietic stem cell fraction obtained from the umbilical cord blood.   The cell preparation according to claim 15 or 16, wherein only donor umbilical cord blood whose MHC compatibility with the recipient is any of 0 to 2 antigen mismatches contributes to the long-term hematopoiesis of the recipient.   A combination of at least two umbilical cord blood with at least immune cells substantially removed to facilitate engraftment of the donor umbilical cord blood to the recipient during hematopoietic stem cell transplantation A combination for use in administering to a recipient.   19. The combination according to claim 18, wherein the cord blood of at least two people, wherein at least immune cells are substantially removed is a hematopoietic stem cell fraction.