# US 9,752,121 B2 · In vitro modelling of haematopoietic stem cell medullary nests: a tool for studying the regulation of haematopoiesis, evaluating the nesting potential of a haematopoietic graft and testing the pharmacotoxicology of medicaments > Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt - Number: US 9,752,121 B2 - Document ID: US9752121B2 - Status: Granted - Kind: B2 - Application number: 14/238,898 - Filed: 2012-08-14 - Published: 2014-11-27 - Granted: 2017-09-05 - Earliest claimed: 2011-08-16 - Assignees: Etat Francais (Ministere De La Defense), Service De Sante Des Armees; Institut National De La Sante Et De La Recherche Medicale (Inserm) - Applicants: Jean-Jacques Lataillade; Marie-Caroline Le Bousse-Kerdiles - Inventors: Jean-Jacques Lataillade; Marie-Caroline Le Bousse-Kerdiles - Web page: https://patentgrep.com/patent/US9752121B2 ## CPC - C12N 5/0647: {Haematopoietic stem cells; Uncommitted or multipotent progenitors} - C12N 5/0662: {Stem cells} - C12N 5/0697: {Artificial constructs associating cells of different lineages, e.g. tissue equivalents (blood vessels C12N5/0691)} - C12N 2500/02: Atmosphere, e.g. low oxygen conditions - C12N 2501/125: Stem cell factor [SCF], c-kit ligand [KL] - C12N 2501/145: Thrombopoietin [TPO] - C12N 2501/26: Flt-3 ligand (CD135L, flk-2 ligand) - C12N 2502/1142: Osteoclasts - C12N 2502/1171: Haematopoietic stem cells - C12N 2502/1305: Adipocytes - C12N 2502/1311: Osteocytes, osteoblasts, odontoblasts - C12N 2502/1352: Mesenchymal stem cells - C12N 2502/1358: Bone marrow mesenchymal stem cells (BM-MSC) - C12N 2502/28: Vascular endothelial cells - C12N 2533/18: Calcium salts, e.g. apatite, Mineral components from bones, teeth, shells - C12N 2533/70: Polysaccharides - C12N 2533/90: Substrates of biological origin, e.g. extracellular matrix, decellularised tissue - C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00) ## Abstract The present invention relates to a culture support for cultivating hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs), comprising a calcium biomaterial, osteoclasts, endothelial cells and mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes. The present invention also relates to a method for preparing such a culture support, and an in vitro HSC and/or HP cultivation method. The use of such a culture support for studying cellular mechanisms involved in hematopoiesis and/or differentiation of HSC/HPs and/or for studying the efficacy and/or the toxicity of a medicament candidate is also described. ## Claims ### Claim 1 A culture support for hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs), consisting of: a. a calcium biomaterial, which is a decellularized bone fragment or a synthetic calcium biomaterial; b. osteoclasts; c. endothelial cells; d. mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes; and e. optionally one or more component(s) of extracellular matrix (ECM), wherein the osteoclasts, the endothelial cells and the mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes are grafted on the calcium biomaterial, and wherein the culture support is capable of in vitro or ex vivo supporting proliferation of HSCs and/or HPs. ### Claim 2 (depends on 1) The culture support according to claim 1, wherein said calcium biomaterial comprises hydroxyapatite (HA) and tricalcium phosphate (TCP). ### Claim 3 (depends on 1) The culture support according to claim 1, wherein the calcium biomaterial comprises 55% to 75% hydroxyapatite and 25% to 45% tricalcium phosphate. ### Claim 4 (depends on 1) The culture support according to claim 1, wherein the calcium biomaterial comprises 60% to 70% hydroxyapatite and 30% to 40% tricalcium phosphate. ### Claim 5 A culture support for hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs), consisting of: a. a calcium biomaterial, which is a decellularized bone fragment or a synthetic calcium biomaterial; b. osteoclasts; c. endothelial cells; d. one or more components of extracellular matrix (ECM) grafted on the calcium biomaterial; e. mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes; and f. optionally, one or more component(s) of extracellular matrix (ECM), wherein the osteoclasts, the endothelial cells and the mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes are grafted on the calcium biomaterial, and wherein the culture support is capable of in vitro or ex vivo supporting proliferation of HSCs and/or HPs. ### Claim 6 (depends on 1) The culture support according to claim 1, wherein the endothelial cells are Lin- CD144+ KDR+ cells. ### Claim 7 A culture support for hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs), consisting of: a. a calcium biomaterial, which is a decellularized bone fragment or a synthetic calcium biomaterial; b. osteoclasts; c. endothelial cells; d. mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes; e. hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs); and f. optionally one or more component(s) of extracellular matrix (ECM), wherein the osteoclasts, the endothelial cells, the MSCs and/or osteoblasts and/or adipocytes, and the HSCs and/or HPs are grafted on the calcium biomaterial, and wherein the culture support is capable of in vitro or ex vivo supporting proliferation of the HSCs and/or the HPs. ### Claim 8 A culture support for hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs), consisting of: a. calcium biomaterial, which is a decellularized bone fragment or a synthetic calcium biomaterial; b. osteoclasts; c. endothelial cells; d. one or more component(s) of extracellular matrix (ECM) grafted on the calcium biomaterial; and e. mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes; and f. optionally, one or more component(s) of the ECM, wherein the osteoclasts, the endothelial cells and the mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes are grafted on the calcium biomaterial, and wherein the culture support is capable of in vitro or ex vivo supporting proliferation of HSCs and/or HPs. ### Claim 9 A culture support for hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs), consisting of: a. a calcium biomaterial, which is a decellularized bone fragment or a synthetic calcium biomaterial; b. osteoclasts; c. endothelial cells; d. a biological glue; e. mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes; and f. optionally, one or more component(s) of extracellular matrix (ECM), wherein the osteoclasts, the endothelial cells and the mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes are grafted on the calcium biomaterial, and wherein the culture support is capable of in vitro or ex vivo supporting proliferation of HSCs and/or HPs. ### Claim 10 (depends on 1) A method for preparing a culture support as defined in claim 1, comprising the steps of grafting the calcium biomaterial with osteoclasts, endothelial cells and mesenchymatous stem cells (MSCs) and/or osteoblasts and/or adipocytes and optionally one or more component(s) of the ECM. ### Claim 11 (depends on 1) An in vitro method for culturing hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs), comprising the steps of: a. seeding at least one culture support as defined in claim 1 with hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs); and b. culturing said hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs). ### Claim 12 (depends on 1) A method for studying the cellular mechanism involved in hematopoiesis and/or differentiation of hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs), said method comprising the steps of: a. culturing hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs) on a culture support as defined in claim 1, and b. determining the ALDH enzymatic activity and/or the SP functionality and/or the expression level of the differentiation antigens (Lin-) and/or the expression level of surface CD34. ### Claim 13 (depends on 1) A method for evaluating the nesting potential of a hematopoietic graft, said method comprising the steps of: (a) culturing hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs) on a culture support as defined in claim 1; and (b) evaluating the capability of the cells cultured at step (a) of causing effluence of a fluorescent coloring agent, wherein level of effluence of a fluorescent coloring agent corresponds to nesting potential. ### Claim 14 (depends on 13) The method of claim 13, wherein the coloring agent is Hoechst-33342. ### Claim 15 (depends on 1) A method for studying the efficiency and/or the toxicity of a medicament candidate, said method comprising the steps of: (a) culturing hematopoietic stem cells (HSCs) and/or hematopoietic progenitors (HPs) on a culture support as defined in claim 1 in the presence or in the absence of the medicament candidate, (b) comparing the phenotypes of the cells present in the cultures obtained at step (a) in the presence or in the absence of the medicament candidate, and/or evaluating the amount or the proportion of apoptotic or necrotic cells present in the cultures obtained at step (a) in the presence or in the absence of the medicament candidate, and (c) deducing the efficiency and/or the toxicity of the medicament candidate from the results obtained at step (b).