New composition for improvement of bone marrow reconstitution and peripheral recovery of neutrophil and platelet counts

Abstract

The present invention refers to a new use of a composition comprising a calcimimetic for enhancing engraftment and lineage reconstitution of hematopoietic stem and progenitor cells (HSPCs) in hematopoietic cell transplantation (HCT) recipients, wherein before transplantation, said HSPCs are pre-treated in vitro ("primed") with a prostacyclin analogue and an adenylate cyclase sensitizer, and the composition is administered shortly before, or at the time of transplantation of said HSPCs, and after transplantation of said HSPCs. The invention further refers to a therapeutic combination comprising HSPCs and a calcimimetic and a method for enhancing engraftment capabilities of HSPCs.

Description

[0001] NEW COMPOSITION FOR IMPROVEMENT OF BONE MARROW RECONSTITUTION

[0002] AND PERIPHERAL RECOVERY OF NEUTROPHIL AND PLATELET COUNTS

[0003] FIELD OF THE INVENTION

[0004] The present invention refers to a new use of a composition comprising a calcimimetic for enhancing engraftment and lineage reconstitution of hematopoietic stem and progenitor cells (HSPCs) in hematopoietic cell transplantation (HOT) recipients, wherein before transplantation, said HSPCs are pre-treated in vitro (“priming”) with a prostacyclin analogue and an adenylate cyclase sensitizer, and the composition is administered shortly before, or at the time of transplantation of said HSPCs, and after transplantation of said HSPCs. The invention further refers to a therapeutic combination comprising primed HSPCs and a calcimimetic and a method for enhancing engraftment capabilities of HSPCs.

[0005] BACKGROUND OF THE INVENTION

[0006] Transplantation of HSPCs is currently the standard of care in the treatment of many malignant and non-malignant hematopoietic diseases and inherited disorders. Several G protein-coupled receptors (GPCRs), which are expressed by HSPCs, have been implicated in the individual steps, which are required for reconstitution of hematopoiesis. In fact, activation of GPCRs, e.g., the E prostanoid receptors 2 and 4 (EP2R and EP4R), the calcium-sensing receptor (CaSR) or the chemokine-receptor 4 (which is the receptor for stromal cell-derived factor-1 ), or of the C3AR1 , the receptor for complement factor C3a, has been linked to enhanced engraftment.

[0007] EP2R and EP4R are known to be Gcts-coupled receptors, CXCR4 couples to Gi and C3AR1 to Gi / G12 / 13-proteins. The CaSR, however, is a multifaceted receptor, and signal transduction after activation of CaSR of HSPCs is not well understood yet.

[0008] Hoggatt etal. [1] and Goessling etal. [2] disclose that priming of HSPCs by in vitro preincubation with dimethyl-PGE2 enhances their ability to engraft and reconstitute the bone marrow in the recipient. Compared to dimethyl-PGE2, treprostinil is metabolically more stable and more selective. Additionally, Kazemi et al. [3] disclose that Treprostinil, when administered to recipient animals, further enhanced engraftment. These authors indeed could show that engraftment is further enhanced, if not only the HSPCs are primed but also the recipient animals are treated with treprostinil during the critical period of bone marrow reconstitution.

[0009] Efficient bone marrow reconstitution also depends on the production of stromal- derived-factor-1 (SDF-1) by stromal cells in the bone marrow niche. Broxmeyer et al. [4] disclose that DPP4-inhibitors do not suffice to efficiently prime HSPCs, but their therapeutic potential for HCT patients has been confirmed in first clinical trials [5].

[0010] The action of prostanoid agonists depends, at least in part, on CXCR4. Zebedin-Brandl et al. [6] and WO2016120310 disclose an enhancement of homing after a sequential treatment of HSPCs, which consisted in a pre-treatment with treprostinil and forskolin, and a further augmentation by vildagliptin.

[0011] Kazemi et al. and Hussain et a / .[3], [7] disclose the effect of treprostinil on stem cell transplantation.

[0012] Lam et al. [8] disclose that CaSR stimulation with cinacalcet increases the primitive hematopoietic cell activity, growth in stromal cell co-culture, adhesion to extracellular matrix molecules such as collagen I and fibronectin, migration toward SDF- 1 , augments the homing, CXCR4-mediated lodgment at the endosteal niche, and finally engraftment capabilities.

[0013] Umbilical cord blood (CB) is a source of HSPCs, but the low cell count is still a major limitation including the risk that HSPCs from the CB transplant may not engraft. Furthermore, using CB as a source takes longer for the CB HSPCs to engraft in the patient and thus to recover peripheral blood cells counts. Thus, the patient is put at high risk for infection, bleeding and overall mortality might increase.

[0014] It is a frequent observation in clinical practice that there are not enough blood- forming cells in a single CB unit for the size of the patient or to treat the particular indication. Therefore, a single CB unit is insufficient for transplantation and two CB units may be required. This in turn may increase the risks of graft-versus-host disease and failure to engraft and reduces the quality and utility of CB as a source of HSPCs.

[0015] Umbilical CB, however, has a less stringent human leukocyte antigen (HLA)- matching requirement when compared to other stem cell sources. This can be of particular interest for regions where the genetic diversity may be restrictive (e.g., sub- Saharan African population) [9].

[0016] Numerous approaches have been explored to expand the number of HSPCs in CB within isolated grafts in ex vivo settings

[0010] . The biggest caveat to ex vivo HSC expansion is to balance self-renewal and differentiation in culture. Accelerated selfrenewal or inadequate differentiation pose risks of leukemic transformation, while inadequate self-renewal or rapid differentiation led to HSC pool exhaustion

[0010] . Also, despite theoretically plausible, many of umbilical CB engineering techniques remain currently very challenging and expensive and thus cannot readily be translated into clinical practice.

[0017] Besides CB, bone marrow (BM) and peripheral blood (PB) are more often used as sources for HSPCs in clinical practice. Rapid engraftment enabling rapid recovery of peripheral blood cell counts is a critical determinant for successful transplantation and overall survival after the transplantation of HSPCs, independent from the source of HSPCs. And even if HCT is an established therapy, it is a fact that high numbers of stem and progenitor cells are needed for successful transplantation. If not enough cells with the potential to reconstitute peripheral blood are abundant in the bone marrow niche after transplantation, this extends the period of bone marrow aplasia, which results in a prolonged period of insufficient of mature blood cells and inefficient recovery of peripheral blood. In particular, neutrophil granulocytes and platelets are important determinants of successful peripheral recovery.

[0018] If cell numbers are below a threshold, this poses a significant problem. For example, it may be required to repeat the stem cell collection, to enforce the mobilization procedure or to find an alternate donor. This obviously delays the treatment and poses a significant risk to the patient. In addition, it may be required to postpone or adapt conditioning therapy. Overall, if cell numbers are below a threshold, this may result in transplant failure.

[0019] HCTs require prior harvesting of allogeneic or autologous HSPCs. HSPCs are usually present in bone marrow (BM) during the entire life, in CB at birth, or in peripheral blood (PB) after mobilization.

[0020] For harvesting of HSPCs, there are several technical advantages and risks, which limit the availability BM and PB donors: e.g., severe pain, bleeding, infection, long-lasting discomfort may occur during collection of bone marrow samples, thus repeated collections should be avoided. For PB donors, the use of mobilization agents and growth factors involves side effects and safety risks.

[0021] WO201 2095511A1 discloses a method for enhancing engraftment of hematopoietic stem cells (HSCs) by an ex vivo pretreatment of hematopoietic stem cells with a prostacyclin analogue and a composition comprising a prostacyclin analogue for use in the treatment of individuals undergoing HSC transplantation.

[0022] Adams et al.

[0011] disclose calcium-sensing receptor (CaSR) ability to specify bone marrow lodgment for HSCs but does not mention calcimimetics.

[0023] Guo et al.

[0012] relate to cord blood (CB) as an alternative source of HSCs for clinical transplantation and treatment and discloses methods to enhance the efficacy of CB HSC transplantation, particularly by ex vivo expansion or enhancing the homing efficiency of said HSCs, wherein, among other things, stimulation of HSCs with the CaSR cinacalcet enhances the homing and engraftment of transplanted HSCs by increasing their binding to bone marrow matrix molecules and the responsiveness of HSCs to CXCL12.

[0024] In summary, there is still a high and yet unmet need to provide methods and treatment regimens to enhance the potency and efficacy of the HSPCs to provide cell doses sufficient to shorten the aplastic phase and to accelerate the restoration of a functional (donor-derived) immune system. Enhancing the bone marrow reconstitution potential of HSPCs holds promise to rescue patients undergoing HCT, in particular those who are at high risk of transplant failure due to limited numbers of available HSCPs.

[0025] Furthermore, enhancing the potency of HSPCs might serve to fully realize the promise of restorative or regenerative hematopoietic stem cell therapies in the future and allow a broader clinical use in various clinical settings. This applies to all types of HSPCs, regardless of source of which they are obtained.

[0026] HLA-haploidentical hematopoietic stem cell transplantation (HSCT) is a therapeutic option for patients with high-risk leukemia, and without human leukocyte antigen (HLA)-matched donors, yet a mega-dose of positively selected CD34+cells is required. To provide improved means to therapeutically enhance the potency of HSPCs may also reduce or avoid the need of “mega-dose” collections of HSPCs, which are e.g., required for TCR-a|3 / CD19 — depleted haploidentical hematopoietic stem cell transplantation (HSCT) using CD34+HSPCs.

[0027] SUMMARY OF THE INVENTION

[0028] The problem is solved by the embodiments of the present invention.

[0029] Specifically, it has been shown by the inventors that a sequential regimen in which the HSPCs are primed ex vivo with a prostacyclin analogue and at least one adenylate cyclase sensitizer, followed by a further treatment of HCT recipients with a calcimimetic, favors efficient engraftment and rapid lineage reconstitution, and hence carries the potential to substantially reduce the time between transplantation and engraftment of said cells after transplantation of human HSPCs. As a consequence, such regimens have the potential to reduce the mortality and cut the number of hospitalization days, thus resulting in a decrease of resource utilization and reduction of health care burden.

[0030] By providing such specific combination therapy the functions of HSPCs are enhanced through multiple mechanisms, and the potency and efficacy of the treatment is increased in a synergistic manner.

[0031] According to the invention, there is provided a composition comprising a calcimimetic for use in enhancing the engraftment and lineage reconstitution of HSPCs in recipients transplanted with said HSPCs, wherein before transplantation, said HSPCs have been pre-treated in vitro (i.e. “primed”) with a prostacyclin analogue and an adenylate cyclase sensitizer, and said composition is administered shortly before or at the time of transplantation of said HSPCs, and said composition is administered after the transplantation of said HSPCs.

[0032] According a specific embodiment of the invention, there is provided a composition comprising cinacalcet for use in enhancing the engraftment and lineage reconstitution of HSPCs in recipients transplanted with said HSPCs, wherein before transplantation, said HSPCs have been pre-treated (“primed”) in vitro with treprostinil and forskolin, and said composition is administered shortly before or at the time of transplantation of said HSPCs, and said composition is administered after the transplantation of said HSPCs.

[0033] According to a further embodiment of the invention, the composition as described above is administered continuously.

[0034] According to a specific embodiment of the invention, the calcimimetic is administered at an effective dosage, which specifically ranges from 5 to 180 mg per day in adults.

[0035] According to a further embodiment of the invention, the HSPCs are derived from cord blood, peripheral blood, donor bone marrow, placenta, adipose tissue, urine, induced pluripotent stem cells (iPSCs), or ex vivo expanded CD34+HSPCs, specifically the HSPCs are present at an amount of 1 x 104to 5 * 105HSPCs per kg body weight. A further embodiment of the invention relates to the composition as described herein, wherein said calcimimetic is administered for a period of at least 10 days after hematopoietic stem cell transplantation, specifically for 10 to 28 days.

[0036] According to an alternative embodiment, said calcimimetic is administered until neutrophil counts reach at least 500-1.000 granulocytes per pL peripheral blood, specifically until platelet counts reach at least 20.000 platelets per pL.

[0037] According to a further embodiment there is provided a therapeutic combination comprising 1 x 104to 5 x 105HSPCs per kg body weight, wherein the HSPCs have been contacted in vitro with a prostacyclin analogue and an adenylate cyclase sensitizer, thereby stimulating migration of said HSPCs towards SDF-1 , and a calcimimetic is administered at an effective dosage, which specifically ranges from 5 to 180 mg, specifically 30-180 mg per day in adults, to stimulate adhesion and differentiation of the HSPCs.

[0038] A further embodiment of the invention provides a therapeutic combination, wherein the HSPCs are substantially free from the adenylate cyclase sensitizer.

[0039] According to a specific embodiment of the invention, the HSPCs are CD34+cells. Specifically, HSPCs are CD34+cells also in the case that they were expanded ex vivo before.

[0040] According to a further embodiment of the invention, the HSPCs were pre-treated in vitro with the prostacyclin analogue and the adenylate cyclase sensitizer for at least 1 hour, specifically for 1 to 2 hours.

[0041] According to a further embodiment of the invention, the recipient is an individual suffering from a myeloproliferative disorder, coronary artery disease, arteriosclerotic disease, diabetes mellitus type 1 and 2, myocardial infarction, cerebral infarction, or a bone marrow disease, which may be, but is not limited to, leukemia, bone marrow disease induced by chemotherapy or irradiation, hereditary and genetic diseases, which can be remedied by supplying genetically engineered HSPCs, or the bone marrow disease as a result of a defect of the blood cell compartment, wherein said defect is a hemoglobinopathy, a defect in neutrophil granulocyte function, or a defect in T- and / or B-lymphocytes.

[0042] A further embodiment of the invention relates to a the composition for use or use of the therapeutic combination as described herein for increasing HSPC engraftment in an individual suffering from any of, but not limited to, acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), juvenile myelomonocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, severe aplastic anemia, myeloproliferative neoplasm (MPN), Fanconi's anemia, congenital amegakaryocytic thrombocytopenia, dyskeratosis congenita, Diamond-Blackfan anemia, Shwachman- Diamond syndrome, congenital sideroblastic anemia, GATA2-associated marrow failure, SAMD9 or SMDL9 disorder, paroxysmal nocturnal hemoglobinuria (PNH), mucopolysaccharidoses I (Hurler syndrome), other mucopolysaccharidoses (II, III, VI), lysosomal metabolic diseases, globoid cell leukodystrophy, metachromatic leukodystrophy, cerebral X-linked adrenoleukodystrophy, pure red cell aplasia, severe combined immunodeficiency syndrome (SCID), Wiskott-Aldrich syndrome, betathalassemia major, sickle cell disease, adrenoleukodystrophy, myelodysplasia, refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloid metaplasia, familial hemophagocytic lymphohistiocytosis, , or solid tumors, or wherein the individual has breast cancer, ovarian cancer, brain cancer, prostate cancer, lung cancer, colon cancer, skin cancer, liver cancer, pancreatic cancer, or sarcoma, or wherein the subject has received bone marrow ablative or non-myeloablative chemotherapy or radiation therapy, or wherein the individual is a bone marrow donor a stem cell door, a cell donor or a donor of a transplantable cell type

[0043] According to a further embodiment, the calcimimetic may be, but is not limited to, cinacalcet, etelcalcetide, evocalcet, and pharmaceutically acceptable compounds thereof. Specifically, the calcimimetic is cinacalcet.

[0044] According to a further embodiment, the prostacyclin analogue may be, but is not limited to, treprostinil, iloprost, cicaprost, and beraprost or pharmaceutically acceptable salts thereof. Specifically, the prostacyclin analogue is treprostinil.

[0045] According to a further embodiment, the adenylate cyclase sensitizer may be, but is not limited to, forskolin, colforsin, de-acetyl forskolin, a water soluble forskolin derivative such as 6-(3-dimethylaminopropionyl) forskolin, 8,13-Epoxy-7|3-( / V- methylpiperazino-y-butyryloxy)-1a,6P,9a-trihydroxy-labd-14-en-11-one, 6-(4-dimethyl- aminobutyryl) forskolin, and pharmaceutical acceptable compounds thereof. Specifically, the adenylate cyclase sensitizer is forskolin. According to another embodiment of the invention, the therapeutic combination as described herein is part of a closed container system, such as, but not limited to a syringe, a bag, such as an IV bag, or a flask.

[0046] According to a further embodiment of the invention the engraftment capabilities of HSPCs can be enhanced by providing a sample comprising HSPCs, administering an effective amount of a prostacyclin analogue, or pharmaceutically acceptable salts thereof, and an adenylate cyclase sensitizer, or pharmaceutically acceptable versions thereof, to said cells. Subsequently, incubating said mixture for a period of time sufficient to sensitize adenylyl cyclases in said cells. Alternatively, said cells are isolated. Optionally, said prostacyclin analogue and said adenylate cyclase sensitizer are administered to said cells at the time of transplantation of said HSPCs. Finally, transplanting said cells into an individual in need thereof and administering to said individual an effective amount of a calcium-sensing receptor (CaSR) sensitizer, i.e. a calcimetic, is also encompassed herein. Said HSPCs may be derived from, but are not limited to, cord blood, peripheral blood, donor bone marrow, placenta, or ex vivo expanded CD34+HSPCs, or urine induced pluripotent stem cells (iPSCs) and adipose tissue. Specifically, the HSPCs are present at an amount of 1 x 104to 5 * 105HSPCs per kg body weight.

[0047] The sequential treatment i.e., ex vivo priming of HSPCs with a prostacyclin analogue (e.g., treprostinil) / adenylate cyclase sensitizer (e.g., forskolin) and a calcimimetic (e.g., cinacalcet) (Seq C) allows for highly efficient bone marrow reconstitution and enhances recovery of peripheral blood even in case of transplantation of low numbers of HSPCs.

[0048] In the present invention Seq C, or synonymously Seq TF-C as described above can be used as the first-ever, universal “rapid engraftment regimen”: Seq C does not need to be limited to the transplantation of cord-blood derived hematopoietic stem cells but can be applicable to all types of HCTs, independent of the source of HSPCs. The enhanced therapeutic efficacy of transplantable cells is of specific interest for all patients, who are at risk of engraftment failure due to limited numbers of HSPCs or delayed peripheral recovery, or who would benefit from an improved procedure. In one embodiment the enhanced therapeutic efficacy of transplantable cells is of specific interest for all patients, to reduce the risk of engraftment failure and to accelerate peripheral recovery. The present invention allows increased engraftment of hematopoietic stem and progenitor cells and may induce more rapid production of platelets and neutrophils in the peripheral circulation, especially in limited cell number transplants, but also in nonlimiting conditions.

[0049] Also, transplantation of autologous gene-corrected cells primarily depends on the collection and effective genetic modification of a sufficient number of stem cells. Poor harvesting of bone marrow stem cells limits the success of this procedure. It is conceivable that the herein described sequential treatment (Seq C) overcomes the need for two or more bone marrow aspirates to cure e.g., sickle cell disease, or to perform e.g. haploidentical age-adapted myeloablative transplant and regulatory and effector T cells for acute myeloid leukemia, and it is conceivable to enhance the potency of gene- corrected cells in general.

[0050] In summary, the method described herein (Seq C) is of interest in the following scenarios: i) for patients who are at high risk of engraftment failure due to limited numbers of HSPCs in one single cord blood unit; ii) to overcome the limitations of cord blood HCT in general and to “revive” the clinical utilization of cord blood HCT in clinical practice; iii) to allow a broader use of bone marrow (or peripheral blood) HSPC transplantations (e.g. for gene-therapy, front-line cancer therapy or regenerative medicine) in general. iv) to enhance the outcome of HCT independent of the source of HSPCs and in limiting or non-limiting conditions.

[0051] Considering that lower cell doses of HSPCs with enhanced potency are required for efficient engraftment and that an additional HSPC source is made applicable, it is proposed that HCT could be used in front-line therapy more often. Further, sequential treatment as described herein is even of interest for the reduction of HSPC donors’ burden and risks, because fewer cells need to be collected.

[0052] Further provided herein is a method of enhancing the engraftment and lineage reconstitution of transplanted hematopoietic stem and progenitor cells (HSPCs) in a subject, comprising:

[0053] (a) pre-treating said HSPCs in vitro with a prostacyclin analogue and an adenylate cyclase sensitizer, (b) administering a therapeutically effective amount of a composition comprising a calcimimetic to the subject shortly before or at the time of transplantation of said HSPCs into the subject,

[0054] (c) transplanting the HSPCs into the subject, and

[0055] (d) continuing to administer the calcimimetic composition to the subject after the transplantation of said HSPCs.

[0056] According to a further embodiment, herein provided is also a method of treating a subject who has received a hematopoietic stem transplant comprising HSPCs, comprising administering a composition containing a calcimimetic to the subject, wherein said HSPCs are treated in vitro before transplantation with a prostacyclin analogue and an adenylate cyclase sensitizer to enhance the engraftment and lineage reconstitution of said HSPCs, and wherein the composition containing the calcimimetic is administered beginning before or at the time of transplantation of said HSPCs and continuing after transplantation of said HSPCs.

[0057] FIGURES

[0058] Figure 1 : Concomitant stimulation of human HSPCs with treprostinil / forskolin and cinacalcet does not alter the cAMP response but exerts mutual antagonism on their migration and adhesion.

[0059] Figure 2: Synergism and mutual antagonism of treprostinil and cinacalcet in rescuing lethally irradiated recipient mice by transplantation of murine HSPCs.

[0060] Figure 3: Sequential regimen of murine HSPCs priming with treprostinil / forskolin and cinacalcet-administration (Seq C) to lethally irradiated HCT recipient mice accelerate recovery of peripheral blood in murine HCT.

[0061] Figure 4: Homing of HSPCs primed with treprostinil / forskolin is not differently enhanced by subsequent administration of treprostinil or cinacalcet.

[0062] Figure 5: After priming with treprostinil / forskolin, continued presence of cinacalcet synergistically promotes colony formation of human CD34+HSPCs.

[0063] Figure 6: Rescue of xeno-transplanted NSG recipient mice by sequential priming of human HSPCs with treprostinil / forskolin and cinacalcet administration.

[0064] Figure 7: Comparison of two calcimimetics, cinacalcet and etelcalcetide hydrochloride.

[0065] Figure 8: Seq C treatment fosters rare HSPC population. Figure 9: Rescue of xeno-transplanted NSG recipient mice by sequential priming of human HSPCs with treprostinil / forskolin and cinacalcet administration with transplantation of limiting cell numbers.

[0066] Figure 10: Rescue of xeno-transplanted NSG recipient mice by sequential priming of human HSPCs with treprostinil / forskolin and cinacalcet administration in nonlimiting conditions.

[0067] DETAILED DESCRIPTION

[0068] Providing improved methods and means to increase homing and engrafting of HSPCs to the bone marrow environment has strong biologic and medical implications.

[0069] Rapid engraftment enabling rapid recovery of peripheral blood cell counts is a critical determinant for a successful transplantation.

[0070] The rapid engraftment enabling rapid recovery of peripheral blood cell counts following HCT is highly important for clinical procedures as currently high numbers of HSPCs are required in clinical transplantation thus leading to the need of high amounts of donor cells. Such methods are also highly useful because often only limited numbers of HSPCs can be obtained from donors, for example in case of “poor mobilizers”. Specifically, as chemotherapy or radiation therapy have been applied to delete malignant hematologic cancer cells, also the patient’s normal blood cells and platelets are affected and reach a life-threatening nadir. Likewise, patients are often unable to find histocompatible donors, emphasizing the need for methods and compositions for reducing the number of HSPCs needed for successful transplantation. This can be, e.g., the case for patients with high-risk leukemia where an HLA-matched donor cannot be found. At present, HLA-haploidentical HSCT is at present applied to such patients, which successful transplantation is, however, limited by the need of mega-dose of positively selected CD34+cells. This applies for all types of HCT, because irrespective of the HSPCs’ source, there is unmet medical need to facilitate the clinical utility of HCT in general.

[0071] The ability to improve the potency of HSPCs (i.e. to improve the bone marrow reconstitution potential including homing and engrafting properties of HSPCs in vitro or ex vivo) implies two gains: i) it allows the collection of fewer cells from donors, thereby reducing risks and discomfort associated with collection of HSPCs from bone marrow / peripheral stem cell harvesting and increasing the pool of willing HSPC donors, and ii) it permits transplantations despite sub-threshold collection of HSPCs or may enhance the outcome in standard conditions.

[0072] The present invention provides a novel use of calcimimetics in the treatment of patients who undergo transplantation of HSPCs with the promise that said cells used for transplantation had been pre-incubated in vitro with at least one prostacyclin analogue for enhancing the engraftment of the cells, and in tandem with an adenylate cyclase sensitizer before administration or returning said cells to an individual’s body.

[0073] The compositions and methods described herein provide a solution to the need for an efficient and rapid engraftment that allows a rapid recovery of peripheral blood cell counts even in the case of the transplantation of the smallest amounts of HSPCs.

[0074] Unless indicated or defined otherwise, all terms used herein have their usual meaning in the art, which will be clear to the skilled person.

[0075] The terms “comprise”, “contain”, “have” and “include” as used herein can be used synonymously and shall be understood as an open definition, allowing further members or parts or elements. “Consisting” is considered as a closest definition without further elements of the consisting definition feature. Thus “comprising” is broader and contains the “consisting” definition.

[0076] The term “about” as used herein refers to the same value or a value differing by + / - 5 % of the given value.

[0077] As used herein and in the claims, the singular form, for example “a”, “an” and “the” includes the plural, unless the context clearly dictates otherwise.

[0078] The terms “individual”, “subject”, “patient”, or “recipient” are meant to broadly include any animal, specifically mammals, specifically humans who receive transplantation of HSPCs pretreated as described herein.

[0079] The terms “ex vivo" or “in vitro’’ refer to activities that take place outside an organism, such as experimentation, treatment, or measurements done in or on living tissue or cells, specifically HSPCs, in an artificial environment outside the organism, preferably with minimum alteration of the natural conditions. In certain embodiments, such tissues or cells can be collected and frozen, and later thawed for ex vivo treatment and / or ex vivo expansion. Tissue culture experiments or procedures lasting longer than a few days using living cells or tissue are typically considered to be "in vitro" though this term can be used interchangeably with ex vivo. The recitations “ex vivo administration”, “ex vivo treatment”, or “ex vivo therapeutic use”, relate generally to medical procedures in which one or more organs, cells, or tissues are obtained from a living or recently deceased subject, optionally purified / enriched, exposed to a treatment or procedure to treat the stem or progenitor cells (e.g., an ex vivo administration step that involves incubating the cells with compounds used for priming as described herein to enhance engrafting capabilities of hematopoietic stem cells). Said cells can then be administered to the same or different individual after that treatment or procedure.

[0080] As used herein, the terms “allograft”, “allotransplant”, “allogenic transplant”, or “homograft” are used to denote a cell transplant from one subject to a genetically nonidentical subject.

[0081] As used herein, the terms “autograft”, “autotransplant”, “autogenic transplant”, or “autogenous transplant” are used to denote a cell transplant that is transplanted from one part of the body of a subject to a different part within the same subject.

[0082] The terms “analogue” or “derivative” relate to a chemical molecule that is similar to another chemical substance in structure and function, often differing structurally by a single element or group, which may differ by modification of more than one group (e.g., 2, 3, or 4 groups) if it retains the same function as the parental chemical. Such modifications are routine to skilled persons, and include, for example, additional or substituted chemical moieties, such as esters or amides of an acid, protecting groups such as a benzyl group for an alcohol or thiol, and tert-butoxylcarbonyl groups for an amine. Also included are modifications to alkyl side chains, such as alkyl substitutions (e.g., methyl, dimethyl, ethyl, etc.), modifications to the level of saturation or unsaturation of side chains, and the addition of modified groups such as substituted phenyl and phenoxy. Derivatives can also include conjugates, such as biotin or avidin moieties, enzymes such as horseradish peroxidase and the like, and radio-labeled, bioluminescent, chemoluminescent, or fluorescent moieties. Further, moieties can be added to the agents described herein to alter their pharmacokinetic properties, such as to increase half-life ex vivo, or to increase their cell penetration properties, among other desirable properties. Also included are prodrugs, which are known to enhance numerous desirable qualities of pharmaceuticals (e.g., solubility, bioavailability, manufacturing, etc.).

[0083] The term “derivative” also includes, within its scope, alterations that have been made to a parent molecule including additions, deletions, and / or substitutions that provide for functionally equivalent or functionally improved molecules. A calcimimetic is a pharmaceutical drug that activates and / or modulates the CaSR and thereby mimics the action of calcium. Alternatively, a calcimimetic is a pharmaceutical drug that amplifies the sensitivity of the CaSR to extracellular Ca2+. It can act as positive allosteric modulators of the CaSR but can bind to different (allosteric) sites on the receptor. Specifically, calcimimetics are primarily approved to treat secondary hyperparathyroidism. Calcimimetics can enhance the homing, lodgment, and differentiation of HSPCs.

[0084] As used herein, the calcimimetic can be, but is not limited to cinacalcet, etelcalcetide, evocalcet, or pharmaceutically acceptable derivatives thereof or any combinations thereof. According to a specific embodiment, the calcimimetic is cinacalcet.

[0085] Cinacalcet (Cin) is a

[0086] A / -[( 1 R)-1 -(1 -naphthyl)ethyl]-3-[3-(trifluoromethyl)phenyl]propan-1 -amine. Cinacalcet is sold under the brand name Sensipar® or Mimpara®. Cinacalcet has the following structure:

[0087] Etelcalcetide is a (6R,9R,12R,15R,18R,21 R,24S,29R)-24-Acetamido-1 ,29- diamino-12,15,18-tris(3-carbamimidamidopropyl)-6-carbamoyl-1 -imino-9,21 -dimethyl- 8, 11 , 14, 17,20,23-hexaoxo-26,27-dithia-2,7, 10, 13, 16, 19,22-heptaazatria contan-30-oic acid.

[0088] Etelcalcetide has the following structure: Etelcalcetide is traded as “Parsabiv ®”.

[0089] Evocalcet is a 2-[4-[(3S)-3-[[(1 R)-1-Naphthalen-1-ylethyl]amino]pyrrolidin-1- yl]phenyl]acetic acid. Evocalcet is traded as “Orkedia ®”.

[0090] Evocalcet has the following structure:

[0091] According to the present invention the term “prostacyclin analogues” includes functional derivatives and functional analogues of said substances.

[0092] Prostacyclin is a prostaglandin member of the eicosanoid family of lipid molecules. It inhibits platelet activation and is also an effective vasodilator.

[0093] As used herein, the prostacyclin analogue or prostacyclin derivative can be, but is not limited to treprostinil, iloprost, beraprost, cicaprost, or pharmaceutically acceptable salts thereof. According to a specific embodiment of the present invention, (dimethylated) prostaglandin E2 is not encompassed within the definition of prostacyclin analogue.

[0094] Treprostinil (Trep) is a metabolically stable synthetic analogue of prostacyclin and is an agonist of the l-prostanoid (IP) receptor, of the E prostanoid 2 (EP2) and EP4- receptors. Treprostinil is marketed as Remodulin®, Trepulimix®, Orenitram®, Trisuva® or Tresuvi®.

[0095] Treprostinil is a (1 R,2R,3aS,9aS)-[[2,3,3a,4,9,9a-hexahydro-2-hydroxy-1-[(3S)-3- hydroxyoctyl]-1 / 7-benz[f]inden-5-yl] oxy]acetic acid monosodium salt.

[0096] Iloprost is marketed as llomedin® and is a 5-{(E)-(1 S,5S,6R,7R)-7-hydroxy-6[(E)- (3S, 4RS)-3-hydroxy-4-methyl-1 -octen-6-inyl]-bi-cyclo[3.3.0]octan-3-ylidene} pentanoic acid.

[0097] Beraprost is a 2,3,3a,8b-tetrahydro-2-hydroxy-1-(3-hydroxy-4-methyl-1-octen-6- ynyl)-1 / 7-cyclopenta(b)benzofuran-5-butanoic acid.

[0098] Cicaprost is a 2-[(2E)-2-[(3aS,4S,5R,6aS)-5-hydroxy-4-[(3S,4S)-3-hydroxy- 4-methylnona-1 ,6-diynyl]-3,3a,4,5,6,6a-hexahydro-1 / 7-pentalen-2-ylidene] ethoxy]acetic acid.

[0099] According to a specific embodiment of the invention, the treprostinil derivative is selected from the group of isomers of treprostinil; acid derivatives of treprostinil; prodrugs of treprostinil, such as treprostinil palmitil, polymorphs of treprostinil; anhydrous polymorphs of treprostinil; glycosidic derivatives of treprostinil, such as, but not limited to a glycoside derivative of treprostinil of general formula I, wherein R1, R2and R3are independently from one another H or a carbohydrate, and wherein at least one of R1, R2and R3is not H; specifically, any one of

[0100]

[0101] Similarly, iloprost, cicaprost, or beraprost can be derivatives from the group of acid derivatives, prodrugs, polymorphs, or isomers therefrom.

[0102] The GPCRs EP2R, EP4R, CXCR4 and CaSR can be addressed with drugs already approved for human use, albeit in different indications. Treprostinil is an agonist at the EP2R, EP4R and IPR, the I prostanoid receptor. The additional action on IPR is useful because human (and murine) HSPCs express IPR. The activity of the CaSR can be modulated by the allosteric activator cinacalcet. Expression and affinity of CXCR4 is enhanced by treprostinil and cinacalcet, thereby CXCR-4 mediated migration, adhesion and engraftment is enhanced. According to a specific inventive embodiment, two, specifically three or more different prostacyclin analogues can be used in the inventive method. Alternatively, four, five or six or even more different prostacyclin analogues can be used.

[0103] According to the embodiment of this invention, the adenylate cyclase sensitizer is used in combination with a prostacyclin sensitizer for pre-treatment of the HSPCs.

[0104] According to the invention, the term “cyclic AMP (cAMP) enhancer” can be used for any compounds which increase the intracellular cAMP levels by activating adenylyl cyclases. As an unlimited example, forskolin is a cAMP enhancer and an adenylate cyclase sensitizer.

[0105] The “adenylate cyclase sensitizers” of the present invention typically increase or maintain the intracellular levels and / or activity of cAMP. Most generally, cyclic adenosine monophosphate (cAMP, cyclic AMP, or 3’-5’-cyclic adenosine monophosphate) acts as an important secondary messenger in many biological processes. Secondary messenger systems relate to methods of cellular signaling, whereby a diffusible signaling molecule is rapidly produced / secreted upon a certain activation signal, which can then activate effector proteins within the cell to exert a cellular response. For instance, among other responses, cAMP signaling transfers the effects of prostaglandins, which otherwise cannot pass through the cell membrane. cAMP also regulates the passage of Ca2+through ion channels.

[0106] The adenylate cyclase sensitizers described herein are typically capable of stimulating adenylate cyclases (also commonly known as adenyl cyclases and adenylyl cyclases), which catalyze the conversion of adenosine triphosphate to cAMP and thereby, increase the cAMP levels.

[0107] Examples of adenylate cyclase sensitizers include, but are not limited to, phorbol ester, forskolin, colforsin, de-acetyl forskolin, water soluble forskolin derivatives, sclareline, 8-bromo-cAMP (8-Br-cAMP), cholera toxin (CT), aminophylline, 2,4 dinitrophenol (DNP), norepinephrine, epinephrine, isoproterenol, isobutylmethylxanthine (IBMX), caffeine, theophylline (dimethylxanthine), dopamine, rolipram, prostaglandin Ei , prostaglandin E2, pituitary adenylate cyclase activating polypeptide (PACAP), and vasoactive intestinal polypeptide (VIP), among others known in the art.

[0108] Phorbol esters are a class of chemical compounds, more specifically they are ester derivatives of the tetracyclic diterpenoid phorbol. Examples are phorbol 12- myristate 13-acetate and phorbol 12,13-dibutyrate. Forskolin (Fsk) is a (3R, 4aR, 5S, 6S,6aS,1 OS, 10aR,10bS)-3-ethenyl-6,10,10b- tri hydroxy-3, 4a, 7, 7, 10a-pentamethyl-1 -oxododecahydro-1 / 7-benzo[ / ]chromen-5-yl acetate.

[0109] Water soluble derivatives of forskolin may be selected from the group consisting of NKH 477 (i.e., 6-(3-dimethylaminopropionyl) forskolin, HCI), L-858051 (i.e., 8,13- Epoxy-7P-(N-methylpiperazino-y-butyryloxy)-1 a,6P,9a-trihydroxy-labd-14-en-11 -one, 2HCI), or 6-(4-dimethylaminobutyryl)forskolin, or pharmaceutically acceptable compounds thereof.

[0110] Forskolin is specifically preferred to be comprised in the therapeutic priming described herein.

[0111] Very specifically, for the inventive application, the effective concentration, or alternatively, the effective amount of treprostinil is in the range of 0.1 pM to 100 pM, specifically 1 pM to 50 pM, specifically 5 pM to 25 pM, specifically about 10 pM, specifically 0.1 , 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 pM.

[0112] According to a further specific embodiment of the invention, the optimum concentration range, or effective amount for the prostacyclin analogue corresponds to 10 to 30 times of its ECso for stimulation of cAMP accumulation in HSPCs.

[0113] According to a specific embodiment of the invention, the ratio of prostacyclin analogue and the adenylate cyclase sensitizer, e.g. forskolin, may be about 1 :3. The HSPCs treated with the adenylate cyclase sensitizer, e.g. forskolin, and prostacyclin analogues may be washed before being injected, however, as an alternative, the HSPCs may also be injected without further purification or washing steps as low amounts of forskolin may be present but may not cause any negative side effects.

[0114] According to a specific aspect, the concentration of the adenylate cyclase sensitizer, specifically of forskolin used for incubating the HSPCs may range from 1- 100 pM, specifically between 10-50 pM, specifically about 30 pM.

[0115] For the inventive application, the effective concentration (i.e., effective amount or dosage) of cinacalcet is in the range of 0.1-3.5 mg per kg body weight per day, specifically 1.5-3.5 mg per kg body weight per day, specifically 2-3 mg per kg body weight per day, specifically 0.1 , 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1 .0, 1 .5, 2.0, 2.5, 3.0, or 3.5 mg per kg body weight per day, In a preferred embodiment the effective dosage of cinacalcet is 2.43 mg per kg body weight per day. The effective concentration of etelcalcetide and evocalcet is in the range of 1-12 mg per kg body weight per day, specifically 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 10.5, 11.0, 11 .5, or 12.0 mg per kg body weight per day.

[0116] As used herein, “hematopoietic stem and progenitor cells” (HSPCs), are a population of cells comprising stem cells (HSCs) and progenitor cells (HPCs). Specifically murine and human HSPCs express several prostanoid receptors (i.e., EPi, EP2, EP3, EP4, IP and DP1). As used herein, HSPCs generally relate to populations of pluripotent or multipotent stem cells and progenitor cells that give rise to the blood cell types, including myeloid (e.g., monocytes and macrophages, neutrophils, basophils, eosinophils, erythrocytes, megakaryocytes / platelets, dendritic cells), and lymphoid lineages (e.g., T-cells, B- cells, NK-cells), and others known in the art.

[0117] For example, HSPCs may be found in the bone marrow of adults, including femurs, hip, ribs, sternum, and other bones. HSPCs may be obtained directly by removal from the hip using a needle and syringe, or from the blood, often following pre-treatment or mobilization of HSCs into peripheral blood with e.g., cytokines, such as G-CSF (granulocyte colony-stimulating factors), or plerixafor or mavorixafor that induce cells to be released from the bone marrow compartment.

[0118] HSPCs may be identified according to certain phenotypic or genotypic markers. For example, HPSCs may be identified by their small size, lack of lineage (lin) markers, low staining (side population) with vital dyes such as rhodamine 123 (rho10) or Hoechst 33342, and presence of various antigenic markers on their surface, many of which belong to the cluster of differentiation series (e.g., CD5, CD11 b, CD34, CD38, CD90, CD133, CD105, CD45, GR-1 (=Ly-6G / C), 7-4, Ter-119 and c-kit). HPSCs are mainly negative for the markers that are typically used to detect lineage commitment, and, thus, are often referred to as lin- cells. Most human HPSCs may be characterized as CD5+, CD45R (B220+), CD11+, GR-1+, CD34+, CD59+, Thyl / CD90+, CD38l0 / ~, C-kit / CD117+, and lin-. However, not all stem cells are covered by these groups, as certain HPSCs are CD347+and CD38+. Also, some studies suggest that earliest stem cells may lack c-kit on the cell surface. However, within the stem cell pool, particularly enhancing the CD49f+HSC subsets is of interest, which support long-term engraftment. Treprostinil is known to specifically activate all Gs-coupled receptors, i.e., EP2, EP4, IP and DP1 receptors. Treprostinil has high affinity for the DP1, EP2 and IP receptors, low affinity for EP1 and EP4 receptors and even lower affinity for EP3, FP and TP receptors

[0011] . In contrast, the prostaglandin E2 (PGE2) analog 16,16-dimethyl-PGE2 (dmPGE2) also stimulates EPs receptors. It was shown that treprostinil stimulates cAMP in HSPCs. In human HSPCs, the concentration-response curve for treprostinil was more than two orders of magnitude between 10 and 90 % of the response. This is consistent with the activation of several stimulatory receptors. Treprostinil-induced cAMP-accumulation can be enhanced upon combination with an adenylate cyclase sensitizer such as, but not limited to forskolin, a direct activator of adenylyl cyclase.

[0119] HSPCs can be exposed to the combination of a prostacyclin analogue, e.g., treprostinil, and an adenylate cyclase sensitizer, e.g., forskolin, without any detectable effect on their viability and their ability to subsequently undergo asymmetric cell division and differentiation into the erythroid and granulocyte / monocyte lineage. Thus, the number of cells needed for transplantation in view of a subsequent in vivo calcimimetic, e.g., cinacalcet, treatment is significantly less compared to the cells needed for transplantation without pre-treatment with a prostacyclin analogue and an adenylate cyclase sensitizer.

[0120] Although the demarcation between stem and progenitor cells may sometimes not be clearly defined

[0013] , according to the present invention, the term „progenitor cell" includes a biological cell that has a tendency to differentiate into a specific type of cell, but is already more specific than a stem cell and is pushed to differentiate into its “target cell”. Progenitor cells are early descendants of stem cells that can differentiate to form one or more kinds of cells. The most important difference between stem cells and progenitor cells is that stem cells can replicate indefinitely, whereas progenitor cells can divide only a limited number of times. Most progenitors are described as oligopotent, they may be compared to stem cells. Progenitors are said to be in a further stage of cell differentiation. They are in the “center” between stem cells and fully differentiated cells. Progenitor cells can move through the body and migrate towards the tissue where they are needed. Many properties are shared by stem cells and progenitor cells.

[0121] As used herein, the term “hematopoiesis” refers generally to the process of cellular differentiation or formation of specialized blood cells from an HPSC. During development, hematopoiesis translocates from the fetal liver to the bone marrow, which then remains the site of hematopoiesis throughout adulthood. Once established in the bone marrow, HSPCs are not distributed randomly throughout the bone cavity. Rather, HSPCs are typically found in close proximity to the endosteal surfaces. The more mature stem cells increase in number as the distance from the bone surface increases.

[0122] Hematopoietic tissues contain cells with long-term and short-term regeneration capacities, as well as committed multipotent, oligopotent, and unipotent progenitors.

[0123] The sample containing HSPCs specifically can be bone marrow.

[0124] HSPCs can be obtained by known techniques from any source known to contain populations of stem and / or progenitor cells, specifically from peripheral blood, umbilical cord or cord blood, placenta, and bone marrow. HSPCs from human origin are preferred for the methods and compositions of the invention.

[0125] In some embodiments, the population of HSPCs is HLA haplotyped.

[0126] For purification of lin- HSPCs by flow cytometry, magnetic-activated cell sorting (MACS), or fluorescence-activated cell sorting (FACS), an array of mature blood-lineage marker antibodies may be used to deplete the lin+cells or late multipotent progenitors (MPP), including, for example, antibodies to CD3epsilon, CD5, CD45R, CD11 b, CD16, GR-1 , 7-4 and Ter-119, CD 13, CD32 and CD33, CD71 , CD 19, CD61 , Mac-1 (CDI lb / CD18), Gr-I, 117Ra, CD3, CD4, CD5, and CD8 among others known in the art. Additional purification methods are known in the art, for example, methods that use the particular signature of the 'signaling lymphocyte activation molecules' (SLAM) family of cell surface molecules.

[0127] HSPCs, whether from cord blood, bone marrow, peripheral blood, placenta, ex vivo expanded CD34+HSPCs or other source, may be grown or expanded in any suitable, commercially available, or custom defined medium, with or without serum. HSPCs from human source are preferred embodiments of the invention.

[0128] Specifically, the HSPCs are present at an amount of 1 x 104to 5 * 105HSPCs per kg body weight.

[0129] The composition and methods described herein, however, allow also the transplantation of less than 1 x 104to 5 x 105HSPCs per kg body weight.

[0130] In one embodiment, the composition and methods described herein also allow the transplantation of more than 5 x 105HSPCs per kg body weight, specifically the composition and methods described herein allow the transplantation 1 x 103to 5 x 106HSPCs per kg body weight.

[0131] For instance, in certain embodiments, serum free medium may utilize albumin and / or transferrin. Further, cytokines may be included, such as Flt-3 ligand, stem cell factor (SCF), and thrombopoietin (TPO), among others. HPSCs may also be grown in vessels such as bioreactors. A suitable medium for ex vivo expansion of HPSCs may also comprise HPSC supporting cells, such as stromal cells (e.g., lymphoreticular stromal cells), which can be derived, for instance, from the disaggregation of lymphoid tissue, and which have been shown to support the in vitro, ex vivo, and in vivo maintenance, growth, and differentiation of HPSCs, as well as their progeny. Such media known in the art e.g., Stemline® (Pluripotent Stem Cell Culture Medium, Sigma- Aldrich Co. LLC), or StemSpan™ (serum-free hematopoietic cell expansion medium, Stemcell Technologies).

[0132] As used herein, the term “cord blood” or “umbilical cord blood” relates generally to a relatively small amount of blood from a newborn baby that returns to the neonatal circulation. Cord blood is rich in HPSCs and can be retrieved from the placenta and the umbilical cord after birth. There are different ways of collecting cord blood and isolating HSPCs for direct or later use according to techniques known in the art. For later use, HSPCs can be stored according to techniques known in the art.

[0133] As used herein, the term “lineage reconstitution” refers to the production or substitution of all members of a given blood cell type lineage as described above.

[0134] As used herein, CD34 refers to the transmembrane phosphoglycoprotein encoded by the CD34 gene in humans and mice. CD34 is a cell surface glycoprotein and functions as a cell-cell adhesion factor. CD34 is also involved in the attachment of hematopoietic stem cells to bone marrow extracellular matrix or directly to stromal cells.

[0135] As used herein, CD34+refers to cells expressing CD34.

[0136] As used herein, the term “engraftment” relates to the process by which hematopoietic stem cells make their way (homing) to free bone marrow niches where they can settle down (lodgment) and find optimal conditions to survive and differentiate. Once they have reached the bone marrow microenvironment, said cells have to differentiate to generate all hematopoietic progeny subsets. A fundamental goal for successful engraftment is that the transplanted hematopoietic cells are capable of lineage reconstitution and sustaining long-term effective hematopoiesis - production of red blood cells, white blood cells, and platelets - and their release to peripheral blood. Engraftment is the most important variable for a better overall survival after stem cell transplant and is the prerequisite for successful bone marrow reconstitution. Pre-treating HSPCs (“priming”) with the combination of a prostacyclin analogue, specifically treprostinil, and an adenylate cyclase sensitizer, specifically forskolin, enhances bone marrow engraftment in irradiated organisms.

[0137] The additional and sequential treatment of a recipient with the calcimimetic in vivo further enhances bone marrow engraftment and accelerates the recovery of peripheral blood.

[0138] According to the invention, the terms “treating”, “pre-treating”, “incubation”, or “priming” can be used interchangeably. The terms are used with regard to isolated HSPCs that are brought into contact with a prostacyclin analogue and an adenylate cyclase sensitizer.

[0139] More specifically, it means that a sample containing HSPCs is admixed with at least one prostacyclin analogue and at least one adenylate cyclase sensitizer to obtain a mixture, incubating said mixture for a period of time sufficient to stimulate Gcts-signaling in said cells.

[0140] The inventive method advantageously provides stimulated stem cells with enhanced potency, which can directly be administered to individuals and further stimulates homing and engraftment and lineage reconstitution of said cells.

[0141] The period of time which is needed to stimulate the Gcts-signaling in said cells can be measured according to known methods, for example by using cAMP measurements of which there are many variations: radioimmune assay (RIA), Fluorescence Resonance Energy Transfer (FRET) with EPAC (epad), radiochemical methods etc. Stimulated cells wherein G alphas-signaling is occurring can be selected or discriminated or isolated from unstimulated cells by methods known in the art like a FRET -based cAMP reporter.

[0142] According to a further embodiment, the in vitro incubation time for the pretreatment of the HSPCs with the prostacyclin analogue and the adenylate cyclase sensitizer is in the range of about 10 to 60 minutes, at least 10 minutes, specifically at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, at least 60 minutes, and at about 37 °C in medium such as serum-free culture or expansion medium (SFEM) [3]

[0143] The phrases “shortly before” or “at the time of’ as used herein define a time window of up to a few minutes, specifically up to 60 minutes, specifically up to 30 minutes, specifically up to 15 minutes. According to a further aspect of the invention, at least 1 x 105donor cells / mL are incubated with the prostacyclin analogue and the adenylate cyclase sensitizer at about 37 °C. In a specific embodiment, due to the advantageous compositions and methods described herein, the number of donor cells can be less than 1 x 105donor cells / mL

[0144] The cAMP-dependent pathway is an essential pathway for promoting engraftment of HSPCs. A prostacyclin analogue can trigger cAMP elevation in HSPCs. It does so by activating multiple receptors, i.e., IP- and EP-receptors thus leading to increased Gas- signaling. Accordingly, prostacyclin analogues like treprostinil, iloprost, cicaprost, or beraprost are effectively raising cAMP levels.

[0145] Also, the CaSR-pathway is known to be central to efficient homing, lodgment, and engraftment. The CaSR is a multi-faceted receptor. A calcimimetic can activate or modulate the CaSR thus leading to alterations of intracellular calcium levels and impinging on several other signaling pathways as well.

[0146] The individuals who receive stem cell transplantation can suffer from hereditary, genetic or other diseases, such as, but not limited to any bone marrow disease, which may be any in the group consisting of leukemia, bone marrow disease induced by chemotherapy or irradiation, hereditary and genetic diseases, which can be remedied by supplying genetically engineered HSPCs, a disease wherein the normal bone marrow architecture is displaced by malignancies, sickle cell disease, myelodysplastic syndrome, myeloproliferative disorders, coronary artery diseases, arteriosclerotic diseases, aplastic anemia, or infections leading to a decrease in the production of blood cells and blood platelets, or the bone marrow disease as a result of a defect of the blood cell compartment, wherein said defect is a hemoglobinopathy (e.g., thalassemia), or a defect in neutrophil granulocyte function, a defect in T- and / or B-lymphocytes (e.g., severe combined immunodeficiency, Bruton’s agammaglobulinemia).

[0147] In an alternative embodiment, individuals who receive stem cell transplantation suffer from diabetes mellitus type 1 and 2, myocardial infarction, or cerebral infarction. Stroke remains one of the leading causes of death and disability worldwide. With the composition and method described herein, HSCT may also be highly advantageous on stroke patients and patients suffering from myocardial infarction. Co-transplantation with various types of cells, including bone marrow mononuclear cells, bone marrow / adipose- derived stem / stromal cells, umbilical cord blood stem cells, neural stem cells, and olfactory ensheathing cells (OECs) can enhance the neurological outcomes. The use of e.g., mesenchymal stem cells (MSCs) in the treatment of diabetes does not involve tumorigenic risks. The herein described composition can also be used for treating patients suffering from diabetes mellitus type 1 and type 2, specifically to treat T 1 DM by reconstitution of immunotolerance and preservation of islet 0-cell function.

[0148] The composition as described herein can be administered by any method known in the art, specifically subcutaneously, intravenously, or orally; orally being the preferred method of administration.

[0149] As used herein, the term “continuous administration” refers to the continuous delivery of a liquid or oral formulation to an individual over a certain period of time which leads to slow and sustained absorption rates of said liquid or oral formulation and steady state plasma levels. Herein, continuous administration can be for a period of 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12,13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, or 28 days, specifically for 10 to 28 days or even longer. Specifically, neutrophil counts are determined, and administration is continued until neutrophil counts in the recipient reach at least 500-1 .000 granulocytes per pL peripheral blood, specifically 500, 600, 700, 800, 900, 1.000 or even more granulocytes per pL peripheral blood. Specifically, the delivery can be a continuous constant administration. Specifically, platelet counts are determined, and administration is continued until platelet counts in the recipient reach at least 20.000-50.000 platelets per pL peripheral blood, specifically 20.000, 25.000, 30.000, 35.000, 40.000, 45.000, 50.000, or even more platelets per pL peripheral blood. Specifically, the continuous administration referred herein may refer to the continuous delivery of a liquid or oral formulation carrying a pharmaceutical drug, specifically the pharmaceutical drug is cinacalcet, to an individual over a certain period of time which leads to slow and sustained absorption rates of said pharmaceutical drug to ensure an effective level of said pharmaceutical drug, specifically cinacalcet, within the individual. Specifically, the continuous administration referred herein may be performed with a timerelease formulation, which releases its contents after a time lag, or a little at a time, or in some other predetermined way. Advantages of time-release formulations include, but are not limited to, reduced local adverse effects such as in the gastrointestinal tract, reduced adverse effects associated with peak blood levels, artificially extended half-life, convenience of dosing, improved compliance, and less fluctuation in blood levels across the course of the day. Specifically, the continuous administration referred herein may be performed with a modified-release (MR) formulation, extended-release formulation (ER, XR), controlled release formulation, long-acting release formulation (LAR), delayed- release formulation (DR), prolonged-release formulation, or sustained-release (SR) formulation.

[0150] The term “constant” with regard to the administration of a liquid as used herein means that the dose is not changed during the respective time period, thus there is no steady increase or decrease of dosage during said time interval.

[0151] The term “constant” with regard to the oral administration of a treatment as also used herein means that said treatment is administered orally as multiple single identical doses, specifically at least two doses, such that the time interval between two subsequent doses is sufficiently long to ensure a constant absorption of the treatment by the body.

[0152] The effective dosage of the calcimimetic as used herein refers to the dosage of about 10 to 360 mg / day, specifically 30 to 360 mg / day, specifically 5 to 180 mg / day, more specifically 30 to 180 mg / day with reference to an adult. Specifically, it is 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 180, 200, 250, 300, 350, 360 mg / day. Specifically, the dosage of the calcimimetic can be determined based on the serum calcium level of the subject.

[0153] Herein, the serum calcium level refers to the concentration of total and / or free calcium in peripheral blood and is determined via methods known in the art. As used herein, serum calcium levels in adults range from 6 to 13 mg / dL. Specifically, the dosage of the calcimimetic is adapted to the serum calcium levels, specifically if the subject's serum calcium level drops below 8.4 mg / dL.

[0154] According to a specific embodiment, serum calcium level can be increased, specifically by administration of calcium containing phosphate binding agents such as, but not limited to calcium carbonate, calcium acetate, vitamin D, hydrochlorothiazide, or other thiazide diuretics.

[0155] As described herein, the therapeutic combination comprises HSPC in range of availability, at minimum 5 * 105to 4 x 109, 1 x 106to 4 x 109, 1 x 107to 4 x 109, 1 x 107to 1 x io9HSPCs, specifically 1 x 108to 4 x io8HSPCs. More specifically the combination comprises about 5 x 105, 6 x 105, 7 x 1Q5, 8 x 1Q5, 9 x 105, 1 x 106, 2 x 106,

[0156] 3 x 1 o6, 4 x 106, 5 x 106, 6 x 106, 7 x 1Q6, 8 x 1Q6, 9 x 106, 1 x 1Q7, 2 x 1Q7, 3 x 107,

[0157] 4 x 107, 5 x 107, 6 x 1Q7, 7 x 107, 8 x 1Q7, 9 x 107, 1 x 1Q8, 2 x 1Q8, 3 x 1 Q8, 4 x 1Q8HSPCs. Alternatively, in the case that cell number are not limiting (non-limiting conditions), the number of cells in the therapeutic combination can be adjusted to the respective body weight, specifically it can be in the range of 1 x 103to 5 * 106, 2 x 103to 4 x 106, 3 x 103to 3 x 106, 4 x 1Q3to 2 x 1 o6, 5 x 103to 1 x 106, 6 x 103to 9 x 105, 7 x 1Q3to 8 x 1 o5, 8 x 103to 7 x 105, 9 x 103to 6 x 105, 1 x 1Q4to 5 x 105, 2 x 1Q4to 4 x 1Q5HSPCs per kg body weight, more specifically 1 x 104to 5 x 105HSPCs per kg body weight. Specifically, the number of cells in the therapeutic combination is 1 x 104,

[0158] 2 x 104, 3 x 1Q4, 4 x 104, 5 x 104, 6 x 1Q4, 7 x 1Q4, 8 x 104, 9 x 1 Q4, 1 x 1Q5, 2 x 105,

[0159] 3 x 1Q5, 4 x 1Q5, or 5 x 105HSPCs per kg body weight.

[0160] According to one embodiment, the term “limiting conditions” refers to a cell number of up to 1 x 105HSPCs per kg body weight.

[0161] According to one embodiment, the term “non-limiting conditions” refers to a cell number of at least 2.5 x 105HSPCs per kg body weight.

[0162] As described herein, the HSPCs are pretreated in vitro for 10, 20, 30, 60, 70, 80, 90, 100, 110, 120 minutes.

[0163] As used herein, a composition or therapeutic combination is said to be “substantially free from” a compound when said composition or therapeutic combination only contains trance amounts of said compound, preferably less than 1 wt%, preferably less than 0.5 wt%, more preferably less than 0.2 wt%, and most preferably less than 0.1 wt% of said compound.

[0164] The period of time which is needed to sufficiently sensitize adenylyl cyclases in said HSPCs can be measured according to known methods, specifically by measuring cAMP accumulation by specified assays known in the art, e.g. but not limited to radioactive cAMP measurement assays, or fluorescence energy transfer (TR-FRET) immunoassay (LANCE® cAMP kit, Perkin Elmer, Waltham, MA).

[0165] The therapeutic priming as described herein may also be used to generate HSPCs for ex vivo genetic manipulations (e.g., for vector-based gene therapy), to enhance the fitness of HSPCs.

[0166] In one embodiment of the invention, the therapeutic priming described herein may also be used in cases wherein the recipient is an individual suffering from a non- hematopoietic disease such as an ischemic stroke, or diabetes mellitus type 1 or type 2. For unmanipulated HSPCs derived from umbilical cord blood (CB) a successful neutrophil engraftment takes around 24 days and platelet engraftment around 49 days. The herein described embodiments allow successful engraftment which takes place within 21 days, specifically 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7 days after transplantation of primed HSPCS as described herein.

[0167] In one embodiment, the therapeutic priming described herein may also be used to enhance homing of CD24+cells to the bone marrow niche, whereas Seq C specifically enhances the subsets of CD49F+HSC, and thereby also fostering a long-term engraftment potential.

[0168] In one embodiment, the therapeutic priming followed by cinacalcet application as described herein (i.e., the composition as described herein: Seq C) enhances the longterm repopulation capacity of HSPCs, supports their differentiation potential, and, to some extent, may also prevent transplanted HSPCs from exhaustion. iPSCs may also be used for transplantation according to the invention. Such cells can be derived from many sources, an unlimiting example herein is urine comprising huge amounts of somatic cells that can be transformed into iPSCs. Somatic cells can be transformed into iPSCs by means of exogenous factors (this method has also been termed “nuclear reprogramming by exogenous factors”

[0014] ). Remarkably, iPSCs are similar to ESCs and have the potential to be used in patient-specific treatments, thus avoiding the risk of immune rejection. So far, human iPSCs have been generated using donor cells from skin (fibroblasts and keratinocytes), amniotic fluid, extra-embryonic tissues (placenta and umbilical cord;

[0015] cord blood, periosteal membrane, dental tissue, adipose tissue, neural stem cells, hepatocytes, amnion-derived mesenchymal stem cells and peripheral blood cells

[0015] ,

[0016] . In search for reprogramming of new tissues, iPSCs have also been produced from mouse meningeal membrane

[0017] and mammary epithelial cells

[0018]

[0019] , and in humans from periosteum and adipose stem cells

[0019] , umbilical cord matrix and placenta

[0015] .

[0169] In one embodiment of the invention, the pre-treated HSPCs are co-transplanted with iPSCs or mesenchymal stem cells (MSCs) to exploit the potent immunomodulatory properties of said iPSCs and said MSCs.

[0170] The present invention also encompasses the following embodiments:

[0171] 1. A composition comprising a calcimimetic for use in enhancing the engraftment and lineage reconstitution of hematopoietic stem and progenitor cells (HSPCs) in recipients transplanted with said HSPCs, wherein (a) before transplantation, said HSPCs have been pre-treated in vitro with a prostacyclin analogue and an adenylate cyclase sensitizer, and

[0172] (b) the composition is administered shortly before, or at the time of transplantation of said HSPCs, and

[0173] (c) the composition is administered after the transplantation of said HSPCs.

[0174] 2. The composition for use according to embodiment 1 , wherein the composition is administered continuously.

[0175] 3. The composition for use according to embodiment 1 or 2, wherein said calcimimetic is administered at an effective dosage, which, depending on the serum calcium levels, ranges from 5 to 180 mg per day in adults.

[0176] 4. The composition for use according to any one of embodiments 1 to 3, wherein the HSPCs are from cord blood, peripheral blood, donor bone marrow, placenta, adipose tissue, or ex vivo expanded CD34+HSPCs, specifically the HSPCs are present at an amount of 1 x 104to 5 * 105HSPCs per kg body weight.

[0177] 5. The composition for use according to any one of embodiments 1 to 4, wherein said calcimimetic is administered for a period of at least 10 days after hematopoietic stem cell transplantation, specifically for 10 to 28 days, specifically until neutrophil counts in the recipient reach at least 500-1.000 granulocytes / pl peripheral blood.

[0178] 6. A therapeutic combination comprising 1 x 104to 5 x 105HSPCs per kg body weight, wherein

[0179] (a) the HSPCs have been contacted in vitro with a prostacyclin analogue and an adenylate cyclase sensitizer, thereby stimulating migration of said HSPCs towards stromal-derived-factor-1 (SDF-1), and

[0180] (b) a calcimimetic is administered at an effective dosage, which depending on the serum calcium levels ranges from 5 to 180 mg per day in adults, to stimulate adhesion and differentiation of the HSPCs.

[0181] 7. The therapeutic combination of embodiment 6, wherein said HSPCs are substantially free from the adenylate cyclase sensitizer.

[0182] 8. The composition for use or the therapeutic combination according to any one of embodiments 1 to 7, wherein the HSPCs are CD34+, specifically ex vivo expanded CD34+ HSPCs. 9. The composition for use or the therapeutic combination according to any one of embodiments 1 to 8, wherein the HSPCs were expanded ex vivo.

[0183] 10. The composition for use or the therapeutic combination according to any one of embodiments 1 to 9, wherein the HSPCs are pre-treated in vitro for at least 1 hour, specifically for 1 to 2 hours.

[0184] 11 . The composition for use or the therapeutic combination or the therapeutic combination according to any one of embodiments 1 to 10, wherein the recipient is an individual suffering from any medical condition of the group consisting of bone marrow disease, myeloproliferative disorder, coronary artery disease, arteriosclerotic disease, diabetes mellitus type 1 and 2, myocardial infarction, or cerebral infarction

[0185] 12. The composition for use or use of the therapeutic combination or the therapeutic combination according to any one of embodiments 1 to 11 , wherein the bone marrow disease is leukemia, a defect of the blood cell compartment, bone marrow disease induced by chemotherapy or irradiation, hereditary and genetic diseases, which can be remedied by supplying genetically engineered HSPCs.

[0186] 13. The composition for use or use of the therapeutic combination according to any one of embodiments 1 to 12, wherein said defect of the blood cell compartment is a hemoglobinopathy, a defect in neutrophil granulocyte function or a defect in T- and / or B-lymphocytes.

[0187] 14. The composition for use or use of the therapeutic combination according to any one of embodiments 1 to 13, in a method of increasing HSPC engraftment in an individual, wherein the individual has acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), juvenile myelomonocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, severe aplastic anemia, Fanconi's anemia, paroxysmal nocturnal hemoglobinuria (PNH), pure red cell aplasia, amegakaryo- cytosis / congenital thrombocytopenia, severe combined immunodeficiency syndrome (SCID), Wiskott-Aldrich syndrome, beta-thalassemia major, sickle cell disease, Hurler's syndrome, adrenoleukodystrophy, metachromatic leukodystrophy, myelodysplasia, refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloid metaplasia, familial erythrophagocytic lymphohistiocytosis, or solid tumors, or wherein the individual has breast cancer, ovarian cancer, brain cancer, prostate cancer, lung cancer, colon cancer, skin cancer, liver cancer, pancreatic cancer, or sarcoma, or wherein the subject has received bone marrow ablative or non-myeloablative chemotherapy or radiation therapy, or wherein the individual is a bone marrow donor.

[0188] 15. The composition for use, or the therapeutic combination, according to any one of embodiments 1 to 14, wherein the calcimimetic is selected from the group consisting of cinacalcet, etelcalcetide, evocalcet, or pharmaceutically acceptable compounds thereof.

[0189] 16. The composition for use, or the therapeutic combination according to any one of embodiments 1 to 15, wherein the prostacyclin analogue is selected from the group consisting of treprostinil, iloprost, cicaprost, and beraprost, or pharmaceutically acceptable salts thereof.

[0190] 17. The composition for use, or the therapeutic combination, according to any one of embodiments 1 to 16, wherein the adenylate cyclase sensitizer is selected from the group consisting of forskolin, colforsin, de-acetyl forskolin, and a water soluble forskolin derivative such as 6-(3-dimethylaminopropionyl) forskolin, 8,13-Epoxy-7|3-( / V- methylpiperazino-y-butyryloxy)-1a,6P,9a-trihydroxy-labd-14-en-11-one, or 6-(4- dimethylaminobutyryl) forskolin, or pharmaceutical acceptable compounds thereof.

[0191] 18. A closed container system containing the therapeutic combination according to any of embodiments 6 to 17.

[0192] 19. A method for enhancing the engraftment capabilities of HSPCs comprising the sequential steps of,

[0193] (a) providing a sample comprising HSPCs,

[0194] (b) administering an effective amount of a prostacyclin analogue, or pharmaceutically acceptable salts thereof, and an adenylate cyclase sensitizer, or pharmaceutically acceptable versions thereof, to said cells,

[0195] (c) incubating said mixture for a period of time sufficient to sensitize adenylyl cyclases in said cells,

[0196] (d) isolating said cells,

[0197] (e) optionally, the composition is administered at the time of transplantation of said HSPCs,

[0198] (f) transplanting said cells into an individual in need thereof, and

[0199] (g) administering to said individual an effective amount of a calcium-sensing receptor (CaSR) sensitizer. 20. The method according to embodiment 19, wherein said HSPCs are derived from cord blood, peripheral blood, donor bone marrow, placenta, adipose tissue, and ex vivo expanded CD34+HSPCs.

[0200] The examples described herein are illustrative of the present invention and are not intended to be limitations thereon. Many modifications and variations may be made to the techniques described and illustrated herein without departing from the scope of the invention.

[0201] EXAMPLES

[0202] Example 1 :

[0203] Methods

[0204] Isolation of murine and human HSPCs

[0205] Murine bone marrow and human cord blood HSPCs were harvested as previously reported [3], [6]: briefly, bone marrow cells were flushed from the femora and tibiae of donor mice. After osmotically induced lysis of erythrocytes, lineage-negative (lin-) and lineage-positive (lin+) cells were separated with antibody-coated magnetic beads (Lineage Cell Depletion Kit, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Human HSPCs were isolated from donated umbilical cord blood: mononuclear cells were enriched using Lymphoprep (StemCell Technologies, Vancouver, Canada), contaminating erythrocytes were removed by osmotic cell lysis and CD34+cells were retrieved by sorting with antibody-coated magnetic beads (CD34 MicroBead Kit Ultra Pure, Miltenyi Biotec GmbH).

[0206] Culture and priming of murine and human HSPCs

[0207] Murine HSPCs were cultured in StemSpan™ serum-free expansion medium (SFEM) containing benzylpenicillin and streptomycin (0.5 mg L-1each), murine stem cell factor, human FLT3, interleukin (IL)-11 (50 pg L-1each), and murine IL-3 (150 pg L-1) (PeproTech, Vienna, Austria) [3], [6]. Human HSPCs were cultured in IMDM medium containing benzylpenicillin and streptomycin (0.5 mg L-1each), human FLT3, thrombopoietin, and stem cell factor (50 ng mL-1each; R&D Systems, Inc., Minneapolis, MN). For in vitro priming, murine and human cells were incubated for 1 h at 37 °C in their respective culture medium in the absence (untreated controls) and in the presence of 10 pM treprostinil and 30 pM forskolin [3].

[0208] Analysis of gene expression by quantitative PCR. Total mRNA from human HSPCs was isolated using the RNeasy Micro Kit

[0209] (QIAGEN, Hilden, Germany) and was reverse-transcribed into complementary DNA (cDNA) using RevertAid First Strand cDNA Synthesis Kit (ThermoFisher Scientific, Vienna, Austria). Quantitative PCR (qPCR) was performed with Maxima SYBR Green / ROX qPCR Master Mix (ThermoFisher Scientific, Vienna, Austria) in 41 cycles (15 second denaturation at 95 °C, annealing at 60 °C for 30 seconds, extension 72 °C for 30 seconds) to assess the levels of human transcripts encoding adenylyl cyclases (AC1 to AC9). Human hypoxanthine phosphoribosyl transferase (HPRT) was used as a reference gene for qPCR. Primer efficiency was verified by serial dilution of the template. Each reaction was performed in triplicate. Relative abundance of transcripts was calculated using the 2ACtmethod (gene-specific expression level relative to that of the reference gene). The primers (sequences listed in Table 1) were purchased from Microsynth AG (Balgach, Switzerland).

[0210]

[0211] Table 1 Sequences pf primers used for amplification of AC subtypes in human RNA samples pH]cAMP accumulation. Accumulation of cAMP was determined as previous reported [3]. Briefly, human

[0212] HSPCs were incubated with medium containing [3H]adenine (1 pCi mL-1) (PerkinElmer, Boston, MA) for 16 h and were subsequently resuspended in medium containing phosphodiesterase inhibitors Ro 20-1724 and isobutylmethylxanthine (Calbiochem / EMD Millipore, Darmstadt, Germany) at 100 pM and 125 pM, respectively. In parallel, cyclic AMP levels were also assessed in cells, which had been pre-incubated for 24 h with pertussis toxin (100 ng mL-1) (Sigma-Aldrich, St. Louis, MO).

[0213] Cyclic AMP formation was stimulated by the addition of 10 pM treprostinil and 30 pM forskolin. After an incubation for 30 min at 37 °C, the cells were lysed in ice-cold 2.5 % perchloric acid containing 0.1 mM cAMP for 30 min at 4 °C; the solution was neutralized with 4.2 M KOH. ATP and cAMP were separated by sequential chromatography on columns containing Dowex 50-X4 (Sigma- Aldrich) and neutral alumina [3]. The accumulated [3H]cAMP was quantified by liquid scintillation counting.

[0214] Chemotaxis assay

[0215] After in vitro priming, human HSPCs (2 x 106cells) were resuspended in StemSpan SFEM. The cell suspension (0.1 mL containing about 1 x 105cells) was added to the upper chamber of a two-chamber transwell system (Corning Life Sciences, Tewksbury MA). The lower chamber contained the same medium and 100 ng mL-1SDF- 1 (stromal cell-derived factor-1 = CXCL12). In parallel incubations, the CXCR-4 antagonist AMD3100 (= plerixafor; Abeam, Cambridge, UK) was added to a final concentration of 10 pM to document that chemotaxis was dependent on activation of CXCR4. After incubating for 4 h at 37 °C, migrated HSPCs were counted in a Luna automated cell counter (Logos Biosystems, Annandale, VA). Cell count was expressed as percentage of the total cells originally added to the upper chamber.

[0216] Cell adhesion assay

[0217] Cell adhesion was assessed as described in [8] with the following modifications: after in vitro priming, human HSPCs (5 x 102) were added to 96-well plates (Sarstedt AG & Co. KG, Numbrecht, Germany) pre-coated with collagen type I (Corning Inc., NY) and incubated for 3 h at 37 °C per 5 % CO2 in a humidified atmosphere. Adhesion to 1 % bovine serum albumin (Sigma-Aldrich) was quantified to control for nonspecific adhesion. Non-adherent cells were removed with PBS, and adherent cells were counted manually.

[0218] Time lapse imaging

[0219] Central channels of p-slide Chemotaxis ibiTreat (ibidi) were coated with 0.41 mg mL-1collagen type I for one hour at room temperature, washed with PBS and incubated over night at 37 °C with 5 % CO2 together with the respective medium for gas equilibration

[0020] . On the next day, primed human HSPCs (1.8 x 105) were stained with 0.5 pM CellTracker™ Green CMFDA (Molecular Probes, Vienna, Austria) for 15 min, washed and seeded on the p-slide. Reservoirs were filled with chemoattractant-free medium, and cells were allowed to adhere for 2 h. After adherence of HSPCs, the p- slide was placed into the ibidi Stage Top Incubation (allowing for an atmosphere of constant gas and humidity), which was mounted on an Olympus X-171 fluorescence microscope equipped with a 20x objective (UPlanSApo, NA: 0.75; Olympus, Vienna). A gradient of SDF-1 (100 ng mL-1) was generated, and cell movement was imaged for 7 h on a fluorescence microscopy system (ibidi). Movement was quantified with the Imaged plugin “TrackMate”, and “Chemotaxis and Migration Tool” provided by ibidi.

[0220] Colony Formation Assay

[0221] Human HSPCs (1 x 103per assay) were primed for 1 h as outlined above and resuspended in MethoCult Enriched (StemCell Technologies, Vancouver, Kanada) containing cytokines and supplements, which supported the proliferation and differentiation of CFU-GM, BFU-E and CFU-GEMM. For sequential treatment, cinacalcet (2.5 pM) was added to the methylcellulose for 7-10 days. The cell suspension was plated on 35 mm dishes using a 10 mL syringe with a 21 G needle and was incubated at 37 °C in an atmosphere containing 5 % CO2. Individual types of colonies were determined visually according to their morphology. Colonies were counted under a Nikon Elipse TS100 microscope and 4x / 0.13 objective using a scoring grid.

[0222] Mice

[0223] BALB / c, C57BL / 6J (CD45.2+), B6.SJL-PtrcAPep3B / BoyJ (CD45.1+) and NOD.Cg-Prkdcscid H2rgtm1Wjl / SzJ (NSG) mice were purchased from Charles River Germany (Sulzfeld, Germany) or bred in-house. Eight-week-old mice were used for all experiments. Animal housing and husbandry was in accordance with the recommendations and requirements as defined by the Federation of Laboratory Animal Science Associations (FELASA) in Europe. Animal technicians monitored animal welfare and health status under the supervision of a veterinarian.

[0224] Engraftment of murine and human HSPCs.

[0225] Murine HSPCs and human cord blood-derived HSPCs were obtained as described above. The transplantation was performed as described in [6]: recipient mice were lethally irradiated (Siemens Primus, 6MV, Siemens Austria). After 24 h, 1.5 or 2.5 x 105murine HSPCs or 1.5 x 105human HSPCs (after one hour priming, as described above) were injected via the tail vein into recipient mice. Irradiated recipient mice, which were not administered murine HSPCs, were used to control efficient irradiation. Prior to transplantation and for the following 10 days, recipient mice were injected subcutaneously (s.c.) with cinacalcet (30 mg kg-124 h-1 ;Seq C), or the concomitant combination of cinacalcet and treprostinil (0.15 mg kg-18 h-1; Con) in a total volume of 0.1 mL. In the sequential regimen, recipient mice received HSPCs, which had been primed with treprostinil / forskolin, and were administered cinacalcet (30 mg kg-1 24 h-1, Seq C) in vivo. Control mice received unprimed cells and sham injections of PBS. The in vivo doses were selected on the basis of the approved clinical indications of treprostinil and cinacalcet: human doses were converted to their murine equivalent by allometric correction

[0021] , Untreated control mice received sham-injections of equal volume. The experiment was performed in a blind fashion: a humane endpoint was determined using score sheets. The people determining the score did not know which treatment the mice had been subjected to. Sick mice were sacrificed by cervical dislocation. Recovery of peripheral blood cell counts of surviving recipient mice was assessed by quantifying platelets (PLT), granulocytes (GRA) and red blood cells (RBC) using a Hemavet 950FS.

[0226] Homing of murine HSPCs.

[0227] For homing assays, murine HSPCs were isolated from CD45.1+B6.SJL- PtrcAPep3B / BoyJ donor mice as described in [3]. Briefly, CD45.2+C57BL6 / J recipient mice were lethally (9.5 Gy) irradiated. After 24 h, they were administered subcutaneously treprostinil (0.15 mg kg-1), cinacalcet (30 mg kg-1), or the combination thereof in a total volume of 0.1 mL. Subsequently primed 2 x 105CD45.1+HSPCs were injected via the tail vein. Untreated control mice received sham-injections of equal volume. After 16 h, bone marrow cells of recipient mice were isolated, and homing was quantified by flow cytometry according to CD45.1 and CD45.2 surface expression.

[0228] Results

[0229] Ca2+sensitive adenylyl cyclases are expressed in human HSPCs, but cinacalcet does not inhibit cAMP accumulation in treprostinil / forskolin-treated human HSPCs.

[0230] The interplay between Ca2+and cAMP signaling is fundamental to numerous aspects of cellular functions, including cell migration and cytokine release. The nine isoforms of membrane-bound adenylyl cyclase are expressed in a cell type-specific manner. It is, however, not known which isoforms are expressed in human HSPCs. It was therefore catalogued human HSPCs for the mRNA expression of AC isoforms by quantitative real-time polymerase chain reaction (qPCR). AC3, AC6, AC7 and AC9 were identified as the isoforms, which are most abundant in human HSPCs (Figure 1A).

[0231] Figure 1 shows that the concomitant stimulation of human HSPCs with treprostinil / forskolin and cinacalcet does not alter the cAMP response but exerts mutual antagonism on their migration and adhesion. Figure 1A shows the expression of AC isoforms by human HSPCs assessed by qPCR relative to HPRT. Data are shown as means ± standard error of the mean (n = 7 donors)

[0232] AC3 and AC6 are activated and inhibited, respectively, by Ca2+and protein kinase C; AC6 and AC7 are subject to direct inhibition by Gai and AC3 to that by GPy-subunits. Thus, based on the isoforms present in HSPCs, it is conceivable that the cAMP response may be affected by different mechanisms of cross-talk resulting from concomitant stimulation of Gs-coupled prostanoid receptors and of the Gi / Gq-coupled CaSR by treprostinil and cinacalcet, respectively. A possible crosstalk was examined by determining intracellular cAMP accumulation in human HPSCs by comparing the effect of treprostinil and cinacalcet and their combination. Forskolin was used to amplify the cAMP response to Treprostinil. In addition, HSPCs were preincubated in the presence of pertussis toxin to disrupt coupling to Gi / Go. Figure 1 B shows the cAMP response of human HSPCs with and without pre-incubation with PTX (100 ng mL1) for 16 h (Ctrl; control). The statistical comparison was done by ordinary one-way ANOVA followed by Tukey’s multiple comparison test (n = 3 donors). As illustrated in Figure 1 B, cinacalcet per se did neither alter basal cAMP levels nor did it affect the cAMP response to the combination of treprostinil and forskolin. In addition, pretreatment with pertussis toxin (PTX) did not result in any appreciable change in cAMP levels (right handset of bars in Figure 1 B). Thus, in human HSPCs there wasn’t any evidence for a synergistic or antagonistic modulation by the CaSR of cAMP formation, which had been induced by treprostinil-activated Gas-coupled prostanoid receptors.

[0233] Treprostinil and cinacalcet are mutually antagonistic on migration, adhesion, and movement of human HSPCs

[0234] Treprostinil and cinacalcet enhance migration and / or adhesion of murine and human HSPCs in a CXCR4 dependent manner. Accordingly, the effects of treprostinil / forskolin, cinacalcet and the combination thereof were compared on migration (Figure 1C) and adhesion (Figure 1 D) of human and murine HSPCs: treprostinil was more effective in stimulating the migration of human HPSCs towards SDF-1 (right hand set of bars in Figure 1C). Figure 1C shows the migration of human HPSCs with and without AMD3100. The statistical comparison was done by ordinary one-way ANOVA followed by Tukey’s multiple comparison test (n = 3 donors). Figure 1 D shows the adhesion of human HSPCs to collagen type I. The statistical comparison was done by ordinary one-way ANOVA followed by Tukey’s multiple comparison test (n = 6 donors).

[0235] It was verified that the chemotactic response was dependent on CXCR4, because migration was substantially reduced in the presence of the CXCR4 antagonist AMD3100 (=plerixafor) (right handset of bars in Figure 1C). Unexpectedly, the concomitant stimulation with treprostinil / forskolin and cinacalcet resulted in mutual antagonism (fourth bar in Figure 1C). Conversely, cinacalcet was more effective than treprostinil in promoting adhesion to collagen type 1 , but combined application of both drugs again resulted in mutual antagonism (Figure 1 D). Cell migration in time-lapse movies was investigated to visualize the effect of the combination of treprostinil / forskolin and cinacalcet. Tracking the movement of individual human CD34+HSPCs allowed for generating a two-dimensional map of the distance covered over the incubation time: the trajectories illustrated in the upper panel of Figure 1 E show that control (i.e. , untreated) HSPCs underwent large random migration. Figure 1 E shows single-cell movement of human HSPCs on collagen type I. The tracking diagrams are based on time-lapse imaging (images captured every minute for 7h and every minute for 5h for Ctrl) and depict the trajectories of human HSPCs towards human SDF-1. The arrows point to where the cells moved.

[0236] HSPCs, which had been exposed to the combination of treprostinil / forskolin and cinacalcet, were strikingly different: their random migration was essentially abolished (lower panel in Figure 1 E). Taken together, these observations unequivocally showed that simultaneous stimulation of HSPCs by treprostinil and cinacalcet resulted in mutual antagonism.

[0237] Sequential application of treprostinil and cinacalcet enhances survival

[0238] During engraftment HSPCs must migrate into and adhere to the bone marrow niche. Thus, their enhanced migration and adherence in response to treprostinil or cinacalcet is likely linked to their benefit for reconstitution of the bone marrow. Conversely, the mutual antagonism, which was observed in vitro, predicts that the signaling pathways stimulated by treprostinil and cinacalcet must not be simultaneously activated during engraftment. Accordingly, it was explored, if the sequential application of these drugs improved the outcome of HCT. A murine model, which was previously established to mimic engraftment failure after transplantation of insufficient numbers of HSPCs was used. HSPCs were primed in vitro with treprostinil / forskolin and recipient animals were then administered cinacalcet (regimen referred to as Seq C). The outcome after this regimen was compared to that observed with HSPCs primed either with treprostinil / forskolin in vitro and recipient animals administered concomitantly the combination of treprostinil and cinacalcet (regimen Con), with (ji) HSPCs and recipient animals exposed solely to each single agent (regimen Mono 1 and Mono 2) and (iii) to that seen in recipient animals receiving untreated HSPCs (Ctrl). The limiting number of murine bone marrow derived HSPCs (1.5 x 105) did not suffice to rescue the control group of lethally (Figure 2A, solid lines Ctrl). Figure 2 shows the synergism and mutual antagonism of treprostinil and cinacalcet in rescuing lethally irradiated recipient mice by transplantation of murine HSPCs. Figure 2A shows the Kaplan-Meier curves of survival. Statistical comparison was done by a log-rank test and is shown in Table 2. Illustrated are significantly different results (*, P < 0.05; **, P < 0. 01 ; *** P < 0.001) (n = 7 mice / group). Treatment with single agents (Mono 1 and Mono 2) afforded partial rescue (Figure 2A). In line with the mutual antagonism observed in vitro, the survival rate of recipient mice was not improved, if they were concomitantly administered treprostinil and cinacalcet in vivo (Figure 2A, dashed line Con). Most importantly, the sequential regimen substantially improved the survival of recipient mice (Figure 2A, dotted line Seq C).

[0239] In line with improved survival, the recovery of peripheral blood cells was significantly better in mice receiving the Seq C regimen: 7 days after transplantation GRA and PLT counts in these mice were significantly higher than those seen in control mice and in mice allocated to the concomitant regimen Con group (Figure 2B). Figure 2B shows GRA, PLT and red blood cell (RBC) counts of surviving recipient mice assessed on day 7 after transplantation. The statistical comparison was done by one-way ANOVA followed by Tukey’s multiple comparison test multiple comparison post-hoc test (*, P < 0.05; **, P < 0. 01 ; ***, P < 0.001 ) (n = 3-4 / per group).

[0240] Treatment P-value (log-rank) Summary

[0241] Mono 1 vs Seq C 0.0221 *

[0242] Mono 1 vs Ctrl 0.0476 *

[0243] Seq C vs Ctrl 0.0002 ***

[0244] Seq C vs Mono 2 0.0064 **

[0245] Con vs Mono 1 0.0002 Seq C vs Con 0.0002 ***

[0246] Ctrl vs Con 0.0002

[0247] Table 2 Statistical comparison of data in Figure 2A. Statistical comparison was done by a log-rank test.

[0248] GRA recovery was also enhanced in mice solely treated with cinacalcet (Mono 2), but not in mice subjected to the other regimen (Mono 1 , Con). Hence, monotherapy with cinacalcet sufficed to accelerate GRA recovery, but the sequential combination was significantly superior because of the additional enhancement in PTL recovery. The mutual antagonism of concomitant cinacalcet and treprostinil was also evident from the impaired recovery of granulocyte and platelet counts, which presumably reflected the reduced capacity of transplanted HSPCs to repopulate the bone marrow.

[0249] Regimen Seq C promotes early onset of and rapid increase in granulocyte and platelet recovery in peripheral blood

[0250] Because of the low number of surviving animals, it was not possible to assess the time course of GRA and PLT recovery in a comparative manner. Accordingly, the experiment was repeated with a higher cell dose (2.5 x 1 o5HSPCs per recipient animal) and PTL and GRAs counts for up to 30 days after transplantation were monitored. The focus was on regimen Seq C, i.e., in vitro priming of HSPCs with treprostinil / forskolin and administration of cinacalcet in vivo, because this was the most effective regimen (Figure 2A). The mutual antagonism of treprostinil and cinacalcet was evident: all recipient mice subjected to the Seq C regimen survived (Figure 3A, dotted line Seq C), but the majority of recipient mice, which had been administered the combination of cinacalcet and treprostinil (Con) succumbed to bone marrow failure (Figure 3A, dashed line, Con). Figure 3 shows that the sequential regimen of murine HSPCs priming with treprostinil / forskolin and cinacalcet-administration (Seq C) accelerates recovery of peripheral blood in murine HCT. Figure 3A shows the Kaplan-Meier curves of survival. Statistical comparison was done by a log-rank test and is shown in Table 3. (*, P < 0.05; **, P < 0. 01 ; *** P < 0.001 ; (n = 5 mice / Ctrl and Seq C, n = 6 mice / Con). In fact, survival of mice subjected to the Con regimen was only marginally improved over that of control animals, which had been injected un-primed HSPCs and which were subsequently administered vehicle (Figure 3A, solid line, Ctrl). Treatment P-value (log-rank) Summary

[0251] Ctrl vs Seq C 0.0128 *

[0252] Seq C vs Con 0.031 *

[0253] Table 3 Statistical comparison of data in Figure 3A. Statistical comparison was done by a log-rank test.

[0254] PLTs and GRAs were counted in peripheral blood obtained from the surviving mice. In recipient mice subjected to the Seq C regimen (Figure 3B, squares, Seq C), recovery of GRAs and PTLs was significantly more rapid than in mice, which were concomitantly administered treprostinil and cinacalcet up to day 16 (Figure 3B, upward pointing triangles, Con). Similarly, recovery of GRAs and PTLs was delayed in the single surviving mouse of the control group (Figure 3B, circles, Ctrl). Figure 3B shows GRA and PLT counts of recipient mice assessed over time as indicated. The dotted and dashed lines indicate 50 % and 100 %, respectively, of average cell counts seen in healthy control animals. An unpaired two-tailed t-test was used to assess the difference between animals subjected to the Seq C and the Con regimen for statistical significance. Error bars represent means ± SEM. It should be noted that there was only one surviving mouse in the control group after 7 days. On day 30, all surviving animals had comparable counts of PTLs and GRAs. In fact, they were within the normal range indicating effective reconstitution of hematopoiesis. These findings show that a regimen based on the sequential application of treprostinil and cinacalcet does have translational potential in preventing bone marrow failure, because it promotes the rapid recovery of GRAs and PTLs after HCT.

[0255] Priming with treprostinil / forskolin promotes HSPC homing

[0256] After intravenous injection, homing of HSPCs is the first step required in bone marrow reconstitution. The effects of the different treatment regimen on homing of murine HSPCs were assessed by priming CD45.1+HSPCs and subsequently injecting them into CD45.2+recipient mice. Recipient mice were also administered treprostinil and / or cinacalcet. The control was the absence of any treatment, i.e., injection of unprimed CD45.1+HSPCs and subsequent vehicle administration in vivo. After 16 h, HSPCs were retrieved from the bone marrow and quantified by flow cytometry. It is evident from Figure 4 that the highest number of CD45.1+HSPCs was retrieved from the bone marrow of recipient mice, which had been subjected to regimen Seq C and Mono 1. Figure 4 shows that the homing of HSPCs primed with treprostinil / forskolin is not enhanced by subsequent administration of treprostinil or cinacalcet. 16 h after HSPC transplantation, bone marrow cells were harvested from femora of recipients, immunostained for CD45.1 and CD45.2 and their relative proportion quantified by flow cytometry. The statistical comparison was done by one-way ANOVA followed by Tukey’s multiple comparison test. Significantly different results are illustrated as: *, P < 0.05; **, P < 0. 0021 ; *** P < 0.0002, **** P<0.0001.

[0257] In contrast, the bone marrow of animals subjected to the regimen Mono 2 (i.e. , priming with cinacalcet and subsequent administration of cinacalcet) contained less CD45.1+HSPCs (Figure 4, third bar). Finally, concomitant administration of treprostinil and cinacalcet again resulted in mutual antagonism, because the number of CD45.1+HSPCs was lower in the regimen Con than in Seq C (Figure 4). Taken together, these observations indicated that treprostinil was more effective in supporting homing than cinacalcet, but that homing did not perse suffice to account for enhanced survival in the regimen, because survival in the regimen Mono 1 was lower than in the regimen Seq C (Figure 2A).

[0258] Cinacalcet synergizes with priming of HSPCs by treprostinil / forskolin in promoting outgrowth of CFU-GEMMs

[0259] After reaching the bone marrow niche, HSPCs must undergo differentiation to reconstitute hematopoiesis. It was posited that the efficacy of regimen Seq C resulted from synergism, where the subsequent action of cinacalcet promoted differentiation of HSPCs primed by treprostinil / forskolin. This hypothesis was examined in colony forming unit assays (CFU) in methylcellulose: because of their translational relevance, the focus was on human CD34+HSPCs and the effect of cinacalcet on un-primed HSPCs and on HSPCs, which had been primed with treprostinil / forskolin was compared. Colonies comprising all lineages including those representing the earliest progenitors (i.e., multilineage CFU-GEMMs (granulocyte erythrocyte macrophage megakaryocyte), BFU-Es (burst forming unit erythroid) and CFU-GM (granulocyte macrophage) were counted. After ten days, priming with treprostinil / forskolin and subsequent culture in cinacalcet- containing methylcellulose (equivalent to the regimen Seq C) was most effective in enhancing total colony formation (Figure 5A and 5B). Figure 5 shows that the priming by treprostinil / forskolin and propagation with cinacalcet synergistically promotes colony formation by human CD34+HSPCs. Figure 5A shows representative photographs of colonies taken on day 10. The colonies were identified according to size and morphology and counted. Figure 5B shows the total number of all colonies,

[0260] When examining individual types of colonies, it was observed that, under this condition, the number of multi-lineage CFU-GEMMs was higher than under all other conditions (Figure 5C, middle panel). Figure 5C shows the number of CFU-GEMM, BFU-E and CFU-GM colonies counted on day 10. The statistical comparison was done by one-way ANOVA followed by T ukey’s multiple comparison (*, P < 0.03; **, P < 0. 002; ***, P < 0.0002; ****, P<0.00001 , n = 4 independent donors). This difference was already evident on days 8 and 9 (Figure 5D). Figure 5D shows the colony numbers counted on days 8, 9 and 10 (depicted are all 4 donors over time, ± SD). Interestingly, priming with treprostinil / forskolin impaired formation of BFU-Es; the number of BFU-Es was also lower in un-primed HSPCs maintained in the presence of cinacalcet than in the un-primed control HSPCs. Formation of BFU-Es was, however, restored under the culture condition corresponding to Seq C (middle panels in Figure 5C and 5D). This indicated that treprostinil, in particular, had an inhibitory action on the erythroid lineage, a finding consistent with the reduction in red blood cell content 10 days after transplantation (cf. regimen Mono 1 in Figure 2B (continued)). Sole priming of HSPCs with treprostinil / forskolin did not affect the differentiation into the granulocyte / macrophage lineage (Figure 5C and 5D). However, both priming of HSPCs with cinacalcet or their maintenance in cinacalcet stimulated the outgrowth of CFU-GM to essentially the same levels seen in the condition corresponding to Seq C (Figure 5C and 5D).

[0261] Thus, taken together the observations summarized in Figures 4 and 5 indicate that both, homing promoted by treprostinil / forskolin and the synergistic action of cinacalcet on HPSC primed with treprostinil / forskolin on their differentiation contribute to improved survival of recipient animals subjected to the sequential regimen.

[0262] Seq C promotes engraftment of xeno-transplanted human HSPCs

[0263] The regimen Seq C is of potential relevance to overcome limitations in human patients undergoing cord blood HCT or to allow the collection and transplantation of fewer HSPCs, independent from the HSPC source.

[0264] The translational implication by xenotransplantation of human cord blood-derived CD34+HSPCs into (immunodeficient) NSG recipient mice was further verified. A limiting dose of HSPCs was selected. Accordingly, lethally irradiated recipient mice, which received un-primed HSPCs, succumbed as rapidly to bone marrow failure as the irradiation control animals (black and grey lines, respectively, in Figure 6A). Figure 6 shows the rescue of xeno-transplanted NSG recipient mice by sequential priming of human HSPCs with treprostinil / forskolin and cinacalcet administration. Figure 6A shows the Kaplan-Meier curves of survival. The statistical comparison was done with a log-rank test and is shown in Table 4 (*** P < 0.001 ; n = 5 mice / Ctrl and Con, n = 6 mice / Seq C and Mono 1). In contrast, all mice undergoing the regimen Seq C were rescued (Figure 6A, dotted line, Seq C). In fact, these animals survived for more than 200 days after transplantation. The regimen Mono 1 (i.e., injection of HSPCs primed with treprostinil / forskolin and subsequent administration of treprostinil) achieved a survival rate of 87.5 % (Figure 6A, dash-dotted line, Mono 1). Finally, mutual antagonism was again observed: recipient animals subjected to the Con regimen (i.e., injection of HSPCs primed with treprostinil / forskolin and subsequent concomitant administration of cinacalcet and treprostinil) were as likely to die of bone marrow failure as the irradiation control (Figure 6A, dashed line, Con). Figure 6B shows the schematic representation (drawn with BioRender) summarizing the key findings and outlining an application of the Seq C as a rescue regimen for patients undergoing cord blood HCT.

[0265] Treatment P-value (log-rank) Summary

[0266] Seq C vs Con 0.0002 ***

[0267] Seq C vs Ctrl 0.0003 ***

[0268] Seq C vs Irradiation Ctrl 0.0002 ***

[0269] Table 4 Statistical comparison of data in Figure 6A. Statistical comparison was done by a log-rank test.

[0270] Human dose calculation

[0271] Dose translation from animal to human is performed according to Reagan-Shaw S, Nihal M, and Ahmad N (2008)

[0021] and USFDA Guidance for Industry: Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers (July 2005).

[0272] The formula for dose translation of a human equivalent dose (HED) from a mouse dose as used herein is as follows:

[0273] HED (mg / kg) = Animal dose (mg / kg) x (3 / 37). In other words, to convert a mouse dose in mg / mg to a HED in mg / kg either divide a mouse dose by 12.3 or multiply the animal dose by 0.081.

[0274] The conversion of some of the doses as used herein is shown below:

[0275] Conclusion

[0276] The sequential treatment with treprostinil / forskolin and cinacalcet (Seq C) allows for bone marrow reconstitution and enhances recovery of peripheral blood despite the transplantation of low numbers of HSPCs.

[0277] The results of the present example 1 were obtained with human cord blood and murine bone marrow HSPCs. It was posited that Seq C can be used as the first-ever, universal “rapid engraftment regimen”: it need not be confined to the transplantation of cord-blood derived hematopoietic stem cells but ought to be applicable to all types of HCTs, independent of the source of HSPCs. The enhanced therapeutic efficacy of transplantable cells is of interest for all patients, who are at risk of engraftment failure due to limited numbers of HSPCs or delayed peripheral recovery.

[0278] Also, successful transplantation of autologous gene-corrected cells primarily depends on the collection and effective genetic modification of a sufficient number of stem cells. Poor harvesting of bone marrow stem cells limits the success of this procedure. It could be conceivable that Seq C overcomes the need for two or more bone marrow aspirates to cure, e.g., sickle cell disease or to enhance the potency of gene- corrected cells in general.

[0279] In summary,

[0280] I) Seq C is of interest for patients who are at high risk of engraftment failure due to limited numbers of HSPCs in one single cord blood unit. Overcoming the limitations of cord blood HCT could “revive” the clinical utilization of cord blood HCT in general.

[0281] II) Seq C is of interest to allow a broader use of bone marrow (or peripheral blood) HSPC transplantations in general. Considering that lower cell doses of HSPCs with enhanced potency are required for efficient engraftment and that an additional HSPC source is made applicable, HCT could be used in front-line therapy more often.

[0282] III) Seq C is of interest for the reduction of HSPC donors’ burden and risks, because fewer cells need to be collected.

[0283] Example 2:

[0284] Methods

[0285] Human HSPCs were cultured in IMDM medium containing 10 % FBS, 0.5 mg L-1benzylpenicillin and streptomycin, supplemented with 50 ng mr1each of human FLT3, thrombopoietin X, and stem cell factor X (R&D Systems, Inc., Minneapolis, MN). For priming, cells were incubated in culture medium for one hour at 37 °C in the presence of 10 pM treprostinil / 30 pM forskolin. Afterwards they were resuspended in MethoCult Enriched (StemCell Technologies, Vancouver, Kanada) containing cytokines and supplements, which supported the proliferation and differentiation of CFU-GM and CFU- GEMM. For sequential treatment, cinacalcet (Seq C, 2.5 pM) or etelcalcetide hydrochloride (MedChemExpress, Seq Ete (synonymously Seq TF-E for etelcalcetide, 25 pM) was added to the methylcellulose. The cell suspension was plated on 35 mm dishes using a 10 mL syringe with a 21G needle and was incubated at 37 °C in an atmosphere containing 5 % CO2 for 10 days.

[0286] Individual types of colonies were determined visually according to their morphology. Colonies were counted under a Nikon Elipse TS100 microscope and 4x / 0.13 objective using a scoring grid.

[0287] Results

[0288] Figure 7 shows a head-to-head comparison of the two calcimimetics, cinacalcet and etelcalcetide hydrochloride. Both calcimimetics show an increase in CFU-GM and CFU-GEMM. Example 3:

[0289] Methods

[0290] Isolation of murine and human HSPCs

[0291] Murine bone marrow and human cord blood HSPCs were harvested as previously reported [3], [6]: briefly, bone marrow cells were flushed from the femora and tibiae of donor mice. After osmotically induced lysis of erythrocytes, lineage-negative (lin-) and lineage-positive (lin+) cells were separated with antibody-coated magnetic beads (Lineage Cell Depletion Kit, Miltenyi Biotec GmbH, Bergisch Gladbach, Germany). Human HSPCs were isolated from donated umbilical cord blood: mononuclear cells were enriched using Lymphoprep (StemCell Technologies, Vancouver, Canada), contaminating erythrocytes were removed by osmotic cell lysis and CD34+cells were retrieved by sorting with antibody-coated magnetic beads (CD34 MicroBead Kit Ultra Pure, Miltenyi Biotec GmbH). After collection, cells were stored in freezing medium (90 % FBS, 10 % DMSO) in liquid nitrogen.

[0292] Culture and priming of murine and human HSPCs

[0293] Murine HSPCs were cultured in StemSpan SFEM medium containing benzylpenicillin and streptomycin (0.5 mg L-1each), murine stem cell factor, human FLT3, interleukin (IL)-11 (50 pg L-1each), and murine IL-3 (150 pg L-1) (PeproTech, Vienna, Austria) [3], [6]. Human HSPCs were cultured in IMDM medium containing benzylpenicillin and streptomycin (0.5 mg L-1each), human FLT3, thrombopoietin, and stem cell factor (50 ng mL each; R&D Systems, Inc., Minneapolis, MN). For in vitro priming, murine and human cells were incubated for 1 hour at 37 °C in their respective culture medium in the absence (untreated controls) or in the presence of 2.5 pM cinacalcet [8], or 10 pM treprostinil and 30 pM forskolin [3], or the combination of 2.5 pM cinacalcet, 10 pM treprostinil and 30 pM forskolin.

[0294] Mice

[0295] Eight-week-old BALB / c, C57BL / 6J (CD45.2+), B6.SJL-PtrcAPep3B / BoyJ (CD45.1+) and NOD.Cg-Prkdcscid H2rgtm1Wjl / SzJ (NSG) mice were purchased from Charles River Germany (Sulzfeld, Germany) or bred in-house. Housing and husbandry were in accordance with the current recommendations and requirements as defined by the Federation of Laboratory Animal Science Associations (FELASA) in Europe. All recipient mice were randomly allocated to the treatment groups (variation in cell numbers and drug regimen). The allocation was concealed from the person carrying out the tagging. All experiments were performed according to the ARRIVE guideline.

[0296] In vivo Engraftment of murine or human HSPCs.

[0297] Murine HSPCs and human CB HSPCs were obtained as described above. The transplantation was performed as described in [6]: BALB / c or NSG recipient mice were lethally or sub-lethally irradiated (9, 4.5 or 2,4 Gy, split doses, Siemens Primus, 6MV, Siemens Austria). 24 hours after irradiation, 1.5 or 2.5 x 105murine HSPCs or 1 x 105(limiting conditions) or 2.5 x 105(non-limiting conditions) human HSPCs (after in vitro priming) or total BM (2.5 x 106) of xeno-transplanted mice were injected via the tail vein into recipient mice. Prior to transplantation and for the following 10 days, recipient mice were injected subcutaneously (s.c.) with treprostinil (0.15 mg kg-18 h-1), cinacalcet (30 mg kg-124 h-1), or the combination thereof in a total volume of 0.1 mL. In the sequential regimen, recipient mice received HSPCs that had been primed with treprostinil / forskolin and were administered cinacalcet (s.c. or orally; 30 or 10 mg kg-124 h’1, Seq C), in vivo. Control mice received unprimed cells and sham-injections of PBS or orally placebo treatment. The in vivo doses were selected on the basis of the approved clinical indications of treprostinil and cinacalcet: human doses were converted to their murine equivalent by allometric correction

[0021] , Untreated control mice received sham-injections of equal volume.

[0298] The experiment was performed in a blinded fashion: the person who injected the drugs or vehicle did not prepare the syringes and was blinded to the content. All animals were monitored daily, and pain and severity assessments were conducted via score sheets according to FEI.ASA recommendations for mice. The overall score defined the intervals of health checks, ranging from daily to twice a day to three times a day, and finally to the humane end point. Briefly, scores were collected by monitoring changes in grimace scales, activity, grooming, food and water intake, breathing, and weight loss. Mice meeting end point criteria were immediately sacrificed by cervical dislocation.

[0299] Recovery of peripheral blood cell counts of surviving recipient mice was assessed by quantifying platelets (PLT), granulocytes (GRA) and red blood cells (RBC) using a Hemavet 950FS. For blood collection the “Recommendations for blood sampling in laboratory animals, especially small laboratory animals” by the specialist information from the Committee for Animal Welfare Officers (GV-SOI.AS) and Working Group 4 in the TVT; July 2017 were followed. In brief, the vena facialis was punctured using a 4- 5.5 mm lancet. The puncture was performed 3-4 mm dorsocaudal to the whorl of hair at the mandible. For repeated blood sampling we followed the rule to not exceed a volume of 1 % of the total blood volume per day. To reduce distress, the group size was doubled to bleed each mouse only once a week to obtain blood results of the same experimental group twice a week, respectively.

[0300] [3]Platelet activation

[0301] 16 weeks post transplantation, heparinized blood was collected, and platelet activation was measured by incubating for 15 minutes with either PBS (control) or ADP (concentration as indicated in Figure 10). In brief, samples were FACS stained according to manufacturer’s guide using anti-human CD61-PB450, anti-mouse CD41-APC, antihuman CD62P-Violet-610, and anti-mouse CD62P-PC7, respectively. Samples were fixed with 2 % paraformaldehyde. After erythrocyte lysis, samples were resuspended in 200 pL PBS, and 30000 PLTs were measured using a CytoFLEX S (Beckman Coulter®).

[0302] Flow cytometry

[0303] Harvested cells (mBM, spleen, blood, CB) were resuspended in FACS buffer. Cell concentration was determined. Cells were washed and stained according to manufacturer’s recommendations. The following antibodies were used and all were purchased from BioLegends: anti-mouse panCD45 (PB), anti-human lineage cocktail (FITC), anti-human panCD45 (FITC) , anti-human CD56 (APC), anti-human CD3 (PE), anti-human CD11c (PE-Cy7), anti-human CD19 (BV605), anti-human CD11 b (APC- Cy7), anti-human CD34 (APC), anti-human CD38 (PE-Cy7), anti-human CD45RA (BV605), anti-human CD90 (PE), anti-human CD10 (APC-Cy7), anti-human CD49f (PerCP-Cy5.5). Data were acquired using a CytoFLEX S (Beckman Coulter®) and analyzed with CytExpert.

[0304] Statistical analysis

[0305] All data were analyzed using GraphPad Prism® or SigmaPlot® software. Samplesize calculations were made using R and GINGER Tool. It was calculated within a power of 90 % to compare all groups with each other, including a control group or against a control group. Differences in survival are visualized by Kaplan-Meier plots and analyzed using the Mantel-Cox log-rank test. Statistical significance of differences between two samples was tested using an unpaired t-test. If more than two data sets were compared, statistical significance was assessed by one-way ANOVA followed by the appropriate post hoc-tests (Bonferroni, Dunnett’s or Tukey's, as indicated in the figure legend). Results

[0306] Seq C treatment fosters rare HSPC populations

[0307] Rare Hematopoietic stem cells (HSCs) isolated from umbilical cord blood have been characterized by distinct surface marker profiles, including CD45+CD34+CD38_CD45RA-CD90+CD49t (HSC1 subset) and CD45+CD34+CD38-CD45RA- CD90+CD49f+(HSC2 subset). RNA sequencing (RNA-Seq) analysis revealed that not only the CD90+CD49f+subset but also the CD90“CD49f+subset harbors self-renewing HSCs. Both CD49f+subpopulations exhibit the highest repopulation potential when xeno-transplanted into NSG mice. To investigate whether Seq C treatment modulates the composition of rare HSC subsets, CD34+cells were primed with treprostinil / forskolin following cinacalcet treatment for various time points and the stem cell populations were characterized using flow cytometry (Figure 8). After one hour of treatment, no significant differences were observed compared to the control group, except for an increase in multipotent progenitors (MPPs). However, after 5 hours of treatment, all HSC populations, including HSC1 and HSC2, showed a significant increase. Notably, the single CD49f+HSC subset was markedly elevated 24 hours post-treatment (Figure 8).

[0308] Figure 8 shows the percentage of positive cells in CD34-purified CB HSPCs of a set of HSC subset markers (n=3; technical triplicates). The statistical comparison was done by one-way ANOVA followed by Dunnett’s multiple comparison test. Significantly different results are illustrated as: *, P < 0.05; **, P < 0. 01 ; *** P < 0.001.

[0309] Collectively, these findings suggest that in vitro pretreatment with treprostinil / forskolin primarily promotes rapid homing, whereas cinacalcet modulates the stem cell pool, particularly enhancing the CD49f+HSC subsets, which support long-term engraftment.

[0310] In conclusion, the findings illustrated in Figure 8 and Figure 5 indicate that treprostinil, forskolin, and cinacalcet operate through distinct mechanisms. Specifically, treprostinil promotes homing efficiency, whereas Seq C selectively amplifies rare HSC populations, which are essential for successful engraftment and differentiation. To translate these insights into clinical applications, the synergistic effects on recipient survival in xenotransplantation models were evaluated. Seq C promotes long-term repopulation capacity of xeno-transplanted human HSPCs

[0311] To evaluate the long-term repopulation potential of Seq C treated xenografted CD34+human HSPC in a more clinical setting, non-limiting cell numbers (2.5 x 105primed cells) were transplanted into sub-lethally irradiated NSG mice.

[0312] Then, the human lineage compartment was assessed 16 weeks posttransplantation, followed by a secondary transplantation. Human HSPCs were primed with treprostinil and forskolin for 1 hour, followed by the injection of 2.5 x 105primed HSPCs into sub-lethally irradiated NSG mice. The mice were then treated in vivo with cinacalcet for 10 days (oral, 10 mg kg-124-1). After 16 weeks, spleen, bone marrow, and blood samples were collected and analyzed. Subsequently, 1 .25 x 1 o7bone marrow cells of the collected bone marrow cells were transplanted into two additional sub-lethally irradiated NSG mice for a secondary transplant, and blood analysis was performed 6 weeks later. Control mice received primed HSPCs followed by placebo treatment. Mice were randomized into treatment groups for the first (n=3 in control group, n=4 in treatment group, 7 mice in total) and second (n=6 in control group, n=8 in treatment group, 14 mice total) transplant. Treprostinil / forskolin-primed cells served as controls. Seq C treatment significantly enhanced engraftment. Also, pan-hCD45+cell infiltration in blood, bone marrow, and spleen was better when compared to control animals (Figure 9B and Figure 10).

[0313] Figure 9 shows the analysis of two subsequent transplants of xenotransplanted human HSPCs. Figure 9A FACS analysis of blood samples following the first transplant, showing the percentage of human CD45+cells within the lymphocyte population. Statistic comparisons were performed using an unpaired t-test. Figure 9B shows Kaplan- Meier survival curves of the second transplant. Statistical comparisons were performed using a log-rank test. Figure 9C shows FACS analysis of blood samples following the second transplant, showing the percentage of human CD45+cells within the lymphocyte population. Statistic comparisons were performed using an unpaired t-test.

[0314] Lineage differentiation and platelet functionality was also analyzed. While platelet dysfunctions have been documented in various disease states, Seq C treatment did not impair platelet function (Figure 10A). Importantly, CD34+HSPCs differentiated into all major lineages, including T cells, NK cells, B cells, and myeloid cells (Figure 10B-D). Figure 10 shows the rescue of xeno-transplanted NSG recipient mice by sequential priming of human HSPCs with treprostinil / forskolin and cinacalcet administration. Figure 10A shows a FACS analysis of blood samples following the first transplant, showing the percentage (left) and the MFI (right) of human CD62P+cells after adenosine diphosphate (ADP) activation of platelets. Figure 10B-D show FACS analysis of (B) blood, (C) spleen, and (D) bone marrow samples, respectively, following the first transplant, showing the percentage of human CD3+T-cells, CD56+NKCells, CD19+B-cells and CD11 b+and / or CD11c+myeloid cells within the human CD45+lymphocyte population. Figure 10E shows a FACS analysis of blood samples following the second transplant, showing the percentage of human CD3+T-cells, CD56+NK-Cells, CD19+B-cells and CD11 b+and / or CD11c+myeloid cells within the human CD45+lymphocyte population. Statistic comparisons were performed using an unpaired t-test.

[0315] In the secondary transplantation, Seq C treated total bone marrow cells showed enhanced repopulation capacity, resulting in a greater number of rescued mice and elevated levels of pan-hCD45+cells (Figure 9). Six weeks post-transplantation, cells of the human immune compartment were differentiated (Figure 10E).

[0316] In summary, the Seq C treatment regimen not only promotes survival but also enhances the long-term repopulation capacity of HSPCs, supports their differentiation potential, and, to some extent, prevents transplanted HSPCs from exhaustion.

[0317] Conclusion

[0318] Here, settings that correspond to clinical practice in non-limiting transplantation conditions demonstrating better human cell engraftment accompanied by elevated immune cell differentiation in Seq C recipient mice have been shown. Moreover, the success of the transplantation regimen underscores long-term repopulation capacity. And, Seq C treatment prevents HSCs from exhaustion.

[0319] From a clinical perspective, it is important to note that the administration of treprostinil to patients undergoing HCT presents a significant limitation: treprostinil effectively inhibits platelet aggregation. This increased risk of bleeding is a major concern in myeloablated patients. Consequently, treprostinil may not be directly applied to patients in Seq C. Treprostinil may be used for in vitro priming only. Nevertheless, it could be successfully demonstrated that platelet functions in the presented settings are unaltered. REFERENCES

[0320] [1] J. Hoggatt etal., Blood, 2009, vol. 113, no. 22, pp. 5444-5455

[0321] [2] W. Goessling et al., Cell Stem Cell, 2011 , vol. 8, no. 4, pp. 445-458

[0322] [3] Z. Kazemi et al., Mol Pharmacol, 2016, vol. 89, no. 6, pp. 630-644

[0323] [4] H. E. Broxmeyer et al., Blood Cells Mol Dis, 2014, vol. 53, no. 1-2, pp. 34-38

[0324] [5] S. S. Farag et al., Oncotarget, 2017, vol. 8, no. 66, pp. 110350-110357

[0325] [6] E. Zebedin-Brandl et al., J Mol Med, 2020, vol. 98, no. 2, pp. 233-243

[0326] [7] F. Hussain et al., BMC Pharmacol, 2011 , vol. 11 , no. S2, p. A6

[0327] [8] B. S. Lam et al., Blood, 2011 , vol. 117, no. 4, pp. 1167-1175

[0328] [9] J. Mellet et al., Stem Cell Res Ther, 2024, vol. 15, no. 1 , p. 317

[0329]

[0010] J. Li et al., Acta Pharm Sin B, 2022, vol. 12, no. 6, pp. 2808-2831

[0330]

[0011] G. B. Adams et al., Nature, 2006, vol. 439, no. 7076, pp. 599-603

[0331]

[0012] B. Guo et al., “Ex Vivo Expansion and Homing of Human Cord Blood Hematopoietic Stem Cells,” 2023, pp. 85-104. doi: 10.1007 / 978-981-99-7471- 9_6.

[0332]

[0013] E. Laurenti et al., Nature, 2018, vol. 553, no. 7689, pp. 418-426

[0333]

[0014] K. Takahashi et al., Cell, 2006, vol. 126, no. 4, pp. 663-676

[0334]

[0015] J. Cai et al., Journal of Biological Chemistry, 2010, vol. 285, no. 15, pp. 11227— 11234

[0335]

[0016] Z. Ye et al., Regenerative Med, 2010, vol. 5, no. 4, pp. 521-530

[0336]

[0017] D. Qin et al., Journal of Biological Chemistry, 2008, vol. 283, no. 48, pp. 33730- 33735

[0337]

[0018] R. Li et al., Cell Stem Cell, 2010, vol. 7, no. 1, pp. 51-63

[0338]

[0019] M. A. Esteban et al., Cell Stem Cell, 2010, vol. 6, no. 1 , pp. 71-79

[0339]

[0020] A.-V. Fonseca et al., Journal of Biological Chemistry, 2010, vol. 285, no. 41, pp. 31661-31671

[0340]

[0021] S. Reagan-Shaw et al., The FASEB Journal, 2008, vol. 22, no. 3, pp. 659-661

Claims

CLAIMS1. A composition comprising a calcimimetic for use in enhancing the engraftment and lineage reconstitution of hematopoietic stem and progenitor cells (HSPCs) in recipients transplanted with said HSPCs, wherein(a) before transplantation, said HSPCs have been pre-treated in vitro with a prostacyclin analogue and an adenylate cyclase sensitizer, and(b) the composition is administered shortly before, or at the time of transplantation of said HSPCs, and(c) the composition is administered after the transplantation of said HSPCs.

2. The composition for use according to claim 1 , wherein the composition is administered continuously.

3. The composition for use according to claim 1 or 2, wherein said calcimimetic is administered at an effective dosage, which, depending on the serum calcium levels, ranges from 5 to 180 mg per day in adults, specifically 30 to 180 mg per day in adults.

4. The composition for use according to any one of claims 1 to 3, wherein the HSPCs are from cord blood, peripheral blood, donor bone marrow, placenta, adipose tissue, or ex vivo expanded CD34+HSPCs, specifically the HSPCs are present at an amount of 1 x 104to 5 * 105HSPCs per kg body weight.

5. The composition for use according to any one of claims 1 to 4, wherein said calcimimetic is administered for a period of at least 10 days after hematopoietic stem cell transplantation, specifically for 10 to 28 days, specifically until neutrophil counts in the recipient reach at least 500-1000 granulocytes / pl peripheral blood.

6. A therapeutic combination comprising 1 x 104to 5 x 105HSPCs per kg body weight, wherein(a) the HSPCs have been contacted in vitro with a prostacyclin analogue and an adenylate cyclase sensitizer, thereby stimulating migration of said HSPCs towards stromal-derived-factor-1 (SDF-1), and(b) a calcimimetic is administered at an effective dosage, which specifically ranges from 5 to 180 mg per day in adults, to stimulate adhesion and differentiation of the HSPCs.

7. The therapeutic combination of claim 6, wherein said HSPCs are substantially free from the adenylate cyclase sensitizer.

8. The composition for use or the therapeutic combination according to any one of claims 1 to 7, wherein the HSPCs are CD34+, specifically ex vivo expanded CD34+HSPCs.

9. The composition for use or the therapeutic combination according to any one of claims 1 to 8, wherein the HSPCs were expanded ex vivo.

10. The composition for use or the therapeutic combination according to any one of claims 1 to 9, wherein the HSPCs are pre-treated in vitro for at least 1 hour, specifically for 1 to 2 hours.11 . The composition for use or the therapeutic combination or the therapeutic combination according to any one of claims 1 to 10, wherein the recipient is an individual suffering from any medical condition of the group consisting of bone marrow disease, myeloproliferative disorder, coronary artery disease, arteriosclerotic disease, diabetes mellitus type 1 and 2, myocardial infarction, or cerebral infarction.

12. The composition for use or use of the therapeutic combination or the therapeutic combination according to any one of claims 1 to 11 , wherein the bone marrow disease is leukemia, a defect of the blood cell compartment, bone marrow disease induced by chemotherapy or irradiation, hereditary and genetic diseases, which can be remedied by supplying genetically engineered HSPCs.

13. The composition for use or use of the therapeutic combination according to any one of claims 1 to 12, wherein said defect of the blood cell compartment is ahemoglobinopathy, a defect in neutrophil granulocyte function or a defect in T- and / or B- lymphocytes.

14. The composition for use or use of the therapeutic combination according to any one of claims 1 to 13, in a method of increasing HSPC engraftment in an individual, wherein the individual has acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myelogenous leukemia (CML), chronic lymphocytic leukemia (CLL), juvenile myelomonocytic leukemia, Hodgkin's lymphoma, non-Hodgkin's lymphoma, multiple myeloma, severe aplastic anemia, myeloproliferative neoplasm (MPN), Fanconi's anemia, paroxysmal nocturnal hemoglobinuria (PNH), pure red cell aplasia, amegakaryocytosis / congenital thrombocytopenia, severe combined immunodeficiency syndrome (SCID), Wiskott-Aldrich syndrome, beta-thalassemia major, sickle cell disease, Hurler's syndrome, adrenoleukodystrophy, metachromatic leukodystrophy, myelodysplasia, refractory anemia, chronic myelomonocytic leukemia, agnogenic myeloid metaplasia, familial erythrophagocytic lymphohistiocytosis, or solid tumors, or wherein the individual has breast cancer, ovarian cancer, brain cancer, prostate cancer, lung cancer, colon cancer, skin cancer, liver cancer, pancreatic cancer, or sarcoma, or wherein the subject has received bone marrow ablative or non- myeloablative chemotherapy or radiation therapy, or wherein the individual is a bone marrow donor, a stem cell donor, cell donor, or a donor of a transplantable cell type.

15. The composition for use, or the therapeutic combination, according to any one of claims 1 to 14, wherein the calcimimetic is selected from the group consisting of cinacalcet, etelcalcetide, evocalcet, or pharmaceutically acceptable compounds thereof.

16. The composition for use, or the therapeutic combination according to any one of claims 1 to 15, wherein the prostacyclin analogue is selected from the group consisting of treprostinil, iloprost, cicaprost, and beraprost, or pharmaceutically acceptable salts thereof.

17. The composition for use, or the therapeutic combination, according to any one of claims 1 to 16, wherein the adenylate cyclase sensitizer is selected from the group consisting of forskolin, colforsin, de-acetyl forskolin, and a water soluble forskolinderivative such as 6-(3-dimethylaminopropionyl) forskolin, 8,13-Epoxy-7|3-(N- methylpiperazino-y-butyryloxy)-1a,6P,9a-trihydroxy-labd-14-en-11-one, or 6-(4- dimethylaminobutyryl) forskolin, or pharmaceutical acceptable compounds thereof.

18. A closed container system containing the therapeutic combination according to any of claims 6 to 17.

19. A method for enhancing the engraftment capabilities of HSPCs comprising the sequential steps of,(a) providing a sample comprising HSPCs,(b) administering an effective amount of a prostacyclin analogue, or pharmaceutically acceptable salts thereof, and an adenylate cyclase sensitizer, or pharmaceutically acceptable versions thereof, to said cells,(c) incubating said mixture for a period of time sufficient to sensitize adenylyl cyclases in said cells,(d) isolating said cells,(e) optionally, the composition is administered at the time of transplantation of said HSPCs,(f) transplanting said cells into an individual in need thereof, and(g) administering to said individual an effective amount of a calcimimetic.

20. The method according to claim 19, wherein said HSPCs are derived from cord blood, peripheral blood, donor bone marrow, placenta, adipose tissue, and ex vivo expanded CD34+ HSPCs.

21. A method of enhancing the engraftment and lineage reconstitution of transplanted hematopoietic stem and progenitor cells (HSPCs) in a subject, comprising:(a) pre-treating said HSPCs in vitro with a prostacyclin analogue and an adenylate cyclase sensitizer,(b) administering a therapeutically effective amount of a composition comprising a calcimimetic to the subject shortly before or at the time of transplantation of said HSPCs into the subject,(c) transplanting the HSPCs into the subject, and(d) continuing to administer the calcimimetic composition to the subject after the transplantation of said HSPCs.

22. A method of treating a subject who has received a hematopoietic stem transplant comprising haematopoietic stem cells and progenitor cells (HSPCs), comprising administering a composition containing a calcimimetic to the subject, wherein said HSPCs are treated in vitro before transplantation with a prostacyclin analogue and an adenylate cyclase sensitizer to enhance the engraftment and lineage reconstitution of said HSPCs, and wherein the composition containing the calcimimetic is administered beginning before or at the time of transplantation of said HSPCs and continuing after transplantation of said HSPCs.

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