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

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

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.

2. The culture support according to claim 1, wherein said calcium biomaterial comprises hydroxyapatite (HA) and tricalcium phosphate (TCP).

3. The culture support according to claim 1, wherein the calcium biomaterial comprises 55% to 75% hydroxyapatite and 25% to 45% tricalcium phosphate.

4. The culture support according to claim 1, wherein the calcium biomaterial comprises 60% to 70% hydroxyapatite and 30% to 40% tricalcium phosphate.

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.

6. The culture support according to claim 1, wherein the endothelial cells are Lin- CD144+ KDR+ cells.

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.

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.

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.

10. 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.

11. 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).

12. 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.

13. 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.

14. The method of claim 13, wherein the coloring agent is Hoechst-33342.

15. 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).

Description

SHORT DESCRIPTION OF THE FIGURES

(1) FIG. 1. Procedure for Preparing a 2D or 3D Culture Support According to the Invention

(2) The preparation of a culture support according to the invention requires the sharing of different stromas in a same well. In a first phase, the osteoclasts, the endothelial cells and the MSCs forming the niche may be seeded on one or several 2D or 3D biomaterials. The MSCs may subsequently be differentiated into osteoblasts and/or adipocytes. Hybrid BMs cellularized by the different types of stromal cells are thus obtained. The letters may then be gathered in a single well containing the whole of the hybrid BMs, in order to form the culture support according to the invention.

(3) FIG. 2. Expansion of the CD34.sup.+ Cells on a 2D Culture Support According to the Invention (*=p<0.05; **=p<0.01)

(4) A population of Lin.sup. cells from peripheral blood of non-mobilized healthy subjects, having an initial richness in CD34.sup.+ cells equivalent to 30%, was cultivated for 7 days on a 2D culture support according to the invention. At the end of this culture, the population of CD34.sup.+ represents about 96.25%+/1.71% (n=4) of the produced viable cells.

(5) FIG. 3. Evaluation of the Maintaining/Acquisition of the SP Functionality of Cells Cultivated on a Culture Support According to the Invention.

(6) In order to validate the capability of the culture support according to the invention of maintaining a pool of primitive hematopoietic cells, the maintaining of the SP functionality of the Lin.sup. medullary cells was evaluated after 3 to 7 days of culture. For this purpose, Lin.sup. medullary cells (2.10.sup.5) were cultured in the presence or in the absence of 2D cellularized culture support (n=3 4). In the presence of a cellularized culture support with MSCs and/or MSCs differentiated into osteoblasts, the percentage of SP cells (1.46+/0.33) is maintained relatively to the first day (before cultivation: 1.02+/0.03, NS), or even increased as compared with the condition without any cellularized culture support (0.05+/0.01, p<0.01) and with the transwell condition (0.17+/0.05, p<0.05).

(7) FIG. 4. Effect of the MSCs on the Acquisition of an SP Functionality

(8) Lin.sup. medullary cells were cultured in the presence or in the absence of 2D cellularized culture support with MSCs.

EXAMPLES

Example 1

Preparation of Various Cell Types Forming the Niche

(9) Isolation and Culture of Mesenchymatous Stem Cells (MSCs)

(10) Mononuclear cells of bone marrow are isolated from fragments of cancellous bones from operating residues of patients operated for a total hip prosthesis and seeded into 75 cm.sup.2 flasks at a concentration of 100,000 cellules/cm.sup.2 in a medium based on MEM (ATGC/biological Industries) containing 10% fetal calf serum (SVF, Hyclone) and 1% ciprofloxacin (Bayer). The culture flasks are incubated at 37 C. in an atmosphere containing 5% of CO.sub.2 and 20% of O.sub.2. The culture medium is renewed after three days of culture, the first week and then once a week until quasi-confluence (80% to 90%). When the cells have reached confluence, they are detached from the plastic support by enzymatic action of trypsin (trypsin 1: 250, Sigma) during 5 min at 37 C., numbered (trypan blue) and then frozen in a cryoprotective solution containing 5% human albumin and 10% DMSO. With view to their use, the MSCs are then thawed in a water bath at 37 C. and then diluted again and washed in a medium consisting of MEM, 1% ciprofloxacin and 20% albumin. After numbering and evaluating the viability by staining with trypan blue, the MSCs are then seeded under different experimental conditions described below in a culture medium based on MEM, 10% SVF, 1% ciprofloxacin. The culture medium is renewed once a week until the cells attain 80% confluence. They are then trypsinated and reseeded for a second passage.

(11) Osteoblast and Adipocyte Differentiation of MSCs Osteoblast differentiation: Osteoblast differentiations are induced from MSCs brought to confluence in a medium of MEM, 10% SVF, 1% ciprofloxacin enriched with 0.1 M of dexamethasone, 0.05 mM of L-ascorbic acid 2 phosphate and 10 mM of -glycerophosphate (Sigma). The induction media are renewed twice a week for three weeks. The quality of the differentiation is evaluated with a phase contrast microscope by appreciating the mineralization level, by immunohistochemistry by detecting the alkaline phosphatase activity (PAL) by a chemical reaction with naphthol AS-Biphosphate (Sigma), and by indirect immunofluorescence (IFI) with detection of osteocalcin, osteopontin and ALP. The IFI reactions are conducted after binding with 4% paraformaldehyde (Sigma) followed by saturation/permeabilization by PBS enriched with 3% bovine albumin and 0.1% of Triton X100 (Prolabo). The anti-osteocalcin, anti-osteopontin and anti-ALP antibodies are incubated in a PBS solution enriched with 1% bovine albumin and 0.05% Tween 20 (Biorad), and then the labelling are revealed by an antibody targeting the murine Igs coupled with phycoerythrin (goat antimouse-PE; Caltag). Finally, counter-staining with Hoechst allowing the viewing of the nuclei is performed (Hoechst 33258, Molecular Probes). Adipocyte differentiation: The adipocyte differentiations are induced from sub-confluent MSCs with three treatment cycles with 1 M of dexamethasone, 0.5 mM of 3-isobutyl-1-methylxanthin (IBMX), 0.2 mM of indomethacin and 0.01 mg/ml of insulin (Sigma). Each treatment cycle lasts for three days, the last cycle ending with three days of maintenance in a specific medium (10% FCS supplemented with 0.01 mg/ml of insulin). The adipocyte nature of the culture is confirmed by examination in a phase contrast microscope showing the presence of lipid vacuoles and by immunohistochemical staining with oil red O binding the lipid vacuoles.

(12) Osteoclast Differentiation from Hematopoietic Progenitors

(13) The mononuclear cells of human bone marrow obtained from fragments of cancellous bone from operating residues of patients operated for a total hip prosthesis are separated by a density gradient on a Ficoll cushion and then cultured for one night, in an incubator at 37 C., in a humid atmosphere containing 5% CO.sub.2. The next day, a Lin.sup. depletion is carried out on an affinity column in order to remove the differentiated cells expressing the following lines antigens: CD2, CD3, CD11b, CD14, CD15, CD16, CD19, CD56, CD123, GPA. The thereby obtained Lin.sup. cells (about 10.sup.7 cells) are cultured on HA/TCP biomaterials in a medium containing a cocktail of cytokines (SCF, Flt3 and TPO at 10 ng/ml). The Lin.sup. cells (2.10.sup.6 Lin.sup. cells) are plated on 2D or 3D biomaterials. After 5 days of cultivation, the culture medium is replaced with a myeloid differentiation medium containing the following cytokines: SCF, Flt3, TPO, IL-6 and GM-CSF at 10 ng/ml. After 15 days of cultivation, the cells are incubated in an osteoclast induction medium containing M-CSF, RANK-L and IL-6 at 20 ng/ml.

(14) The quality of the differentiation is controlled on a well not containing any biomaterial. The osteoclastic phenotype is determined by May Grumwald Giemsa (MGG) staining giving the possibility of showing the multi-nuclear aspect, resulting from the fusion of osteoclast cells, and by detection of the tartrate acid resistant phosphatase activity (TRAP).

(15) Obtaining Endothelial Cells

(16) The endothelial cells may be obtained from two sources: 1) blood-circulating endothelial progenitors (ECPs) from peripheral blood. For this, the colonies derived from the ECPs are generated from mononuclear cells obtained from 20 mL of blood. Each colony produces about 10.sup.6 cells after 2 passages. Thus, after 3 or 4 passages, it is possible to obtain up to 10.sup.8 functional endothelial cells. 2) mononuclear cells of the bone marrow. The first step consists in immunomagnetic depletion of the positive lineage cells allowing removal of the majority population of engaged hematopoietic cells. On the Lin population, CD144/KDR sorting is then performed, and the sorted cells are cultivated in an endothelial medium. The isolated medullary endothelial cells then rapidly proliferate in this medium.

(17) Isolation of Hematopoietic Stem Cells (HSCs)/Hematopoietic Progenitors (HPs)

(18) Lin.sup.: The medullary or blood mononuclear hematopoietic cells are obtained after centrifugation on a Ficoll Hypaque density gradient. Then the Lin.sup. cells (negative lineage) are sorted after labelling with a cocktail of antibodies targeted against various differentiation antigens (Lineage cell depletion kit, Miltenyi Biotec). After washing in a buffer medium, sorting is performed by depletion on an automaton (Automacs, Miltenyi Biotech).

(19) SP: The Lin cells are incubated in the presence of Hoechst-33342 (5 g/ml), an intercalator of DNA, in an amount of 10.sup.6 cells/ml in DMEM containing 2% FCS. The cells are then incubated for 90 mins at 37 C.; the remainder of the experiment is conducted under cold conditions in order to avoid passive effluence of Ho by cells not expressing the pumps. The cells are then centrifuged for 15 mins at 4 C. and at 1,500 rpm and the cell pellet is re-suspended in HBSS without Ca.sup.2+, or Mg.sup.2+, under cold conditions at a concentration from 2 to 4.10.sup.6 cells/ml.

(20) ALDH.sup.strong: ALDEFLUOR technology uses the Bodipy-AminoAcetAldehyde Diethyl Acetal (BAAA-DA substrate) of the ALDH-A1 enzyme. This substrate is dissolved in DMSO and exposed to the action of HCl so as to be converted into BAAA, a fluorescent substrate of the enzyme. The cells are incubated in the presence of BAAAA (1.5 M) at 37 C. which diffuses through the plasma membranes of the viable cells. The ALDH enzyme converts this substrate into a fluorescent product (BAA) which is then retained inside the cells due to its negative charge and to the polarity of the cell membranes. The ALDEFLUOR incubation buffer contains an inhibitor of MDR effluence pumps. Therefore, the cells which express a high level of ALDH are those which retain the BAA within their cytoplasm, but also those which have a high fluorescence level. This fluorescence may be measured in the FL1 channel (FITC fluorescence) in flow cytometry.

(21) CD34.sup.+: The CD34.sup.+ cells are purified from mononuclear cells stemming from an allogenic marrow sample. The mononuclear cells are separated according to a Ficoll gradient and the pellet is diluted in a PBS buffer enriched with 2% human albumin (Vialebex, LFB) and 0.5% of human polyvalent immunoglobulins (Tegeline, LFB) and then incubated for 5 minutes so as to saturate the receptors non-specific to immunoglobulins (Ig) present at the surface of the CD34.sup.+ cells. The cells are then incubated in the presence of an anti-CD34 antibody coupled with magnetic beads (20 l/10.sup.8 cells; Clinimacs, Miltenyi Biotech) for 30 minutes and then washed in PBS-2% albumin (PA) before being loaded on an immunomagnetic column (Automacs, Miltenyi Biotech). The cells may then be frozen in 5% albumin, 10% dimethylsulfoxide (DMSO, Sigma). The quality of the sorting is controlled by flow cytometry so as to evaluate the CD34.sup.+ cell purity in the final cell suspension.

Example 2

Selection of the Biomaterials

(22) Modeling the niche requires the sharing of different stromas on a same osteoconductive 2D or 3D support. These biomaterials (BMs) may comprise in particular hydroxyapatite (HA) and of tricalcium phosphate (TCP) in various proportions and have variable porosities. From among the latter, let us mention B2D and B3D (BD Biocoat Osteologic Bone Cell Culture System) and Calciresorb 35 (Ceraver) which is a biphasic ceramic comprising 65% of HA and 35% of TCP. The results shown in Example 5 were obtained with Calciresorb 35 (Ceraver).

Example 3

Selection of the Extracellular Matrix

(23) Natural glycosaminoglycans (GAG) or their mimetics (which have the advantage of not being degraded by glycanases) may be associated with the biomaterial. The preferentially used mimetic GAGs are marketed by OTR3 (for Organ, Tissue, Regeneration, Repair and Replacement) under the name of RGTAs for <<ReGeneraTing Agents>> and are ideally used at a concentration from 10 to 50 mg/ml. The results shown in Example 5 were obtained with OTR4131.

Example 4

Modeling of the Hematopoietic Niche in 2D or 3D

(24) The modeling of the niche requiring the sharing of the various stromas in a same well, the MSCs, the osteoclasts and the endothelial cells are ideally seeded on a same BM or on different 2D or 3D BMs (one per cell type) (FIG. 1). The results shown in Example 5 were obtained by seeding on various 3D BMs of the Calciresorb 35 type. The MSCs are induced towards osteoblast and adipocyte differentiations. Hybrid BMs cellularized by the different types of stromal cells are thereby obtained. The latter are then collected in a single well containing the whole of the hybrid BMs in which HSCs/HPs are then added at variable concentrations in order to produce co-cultures. The results shown in Example 5 were obtained after adding Lin.sup. cells to the concentration of 1.10.sup.5 cells/ml/wells. An alternative to this procedure consists of producing cultures of each differentiated cell type in the form of balls (by preventing them from adhering to the support during their differentiation). The different cell types are then mixed and seeded on a single and same biomaterial (De Barros et al., PLoS One. 2010; 5(2), page 11, 3rd paragraph).

(25) In detail, in the case of the preparation of various types of stromal cells on independent BMs, the BMs are individually seeded in microtubes at a concentration of 5.10.sup.4 to 2.10.sup.5 cells per ml and per BM. After 3 hours of incubation at 37 C. in atmosphere enriched with 5% CO.sub.2, the cellularized BMs are transferred into a microplate (four well) in a MEM medium, 10% FCS, 1% ciprofloxacin in order to achieve the osteoblast and adipocyte inductions, 2 to 5 days later. The osteoblast induction period is limited to fourteen days since the BMs induce per se self-induction of the MSCs towards the osteoblast line. Several BM <<controls>> (one per cell type) are analyzed in immunohistochemistry by reaction with naphthol AS-Biposphate and by staining with oil red O so as to confirm the positive nature of the osteoblast and adipocyte differentiations.

(26) The HSCs/PHs are thawed on the day of their being put into a co-culture and diluted in the SYNH medium (95L01HSA, AbCys) containing 1% ciprofloxacin, enriched with hematopoietic growth factors (FCH, 10 ng/mL): thrombopoietin (Tpo, Peprotech), stem cell factor (SCF, Peprotech) and Flt3-ligand (RetD System). The quality of the thawed samples is evaluated by flow cytometry (expression of CD34) and by culture in a semi-solid medium. The thawed cells are then seeded (5.10.sup.4 to 2.10.sup.5 cells per well) in the presence of different hybrid BMs to form a niche and then cultured for 1 to 2 weeks in the previously defined SYNH medium.

(27) These cultures are achieved either in a relative oxygen concentration of 20%, or in a hypoxic condition closer to the physiological situation in bone marrow, i.e. at concentrations between 1 and 5%. The results shown in Example 5 were obtained under conditions of a relative oxygen concentration of 3%.

Example 5

Biological Characterization of the Niche

(28) Characterization of the Different Cell Types of the Niche

(29) The evaluation of the osteoblast, adipocyte and osteoclast differentiation with tests of specific induction is carried out by staining, as described in Example 1.

(30) Functional Evaluation of the Niche

(31) Evaluation of the Factors Secreted by the 3D Niche

(32) The main molecules involved in the regulation of hematopoiesis and produced inside the thereby modeled niches were quantified by an ELISA test after 7 days of co-culture in the presence of HSC/HP CD34.sup.+. The results are expressed in pg/ml per 10.sup.5 stromal cells (MSCs, osteoblasts, osteoclasts and adipocytes) and 5.10.sup.4 initially seeded CD34.sup.+ cells. Table 1 below shows a significant production of various factors; the osteoblasts and the MSCs being the majority of producing cells. These results testify the functional capability of the cells forming the thereby modeled niches.

(33) TABLE-US-00001 TABLE 1 Evaluation of the factors secreted by a cellularized niche Factor Concentration at D7 (pg/ml) Osteopontin 17000 5000 Osteoprotegerin 9 7 Flt3-L 21000 8000 Interleukine 8 4500 3500 Interleukine 6 5000 100 Stem Cell Factor 5900 100 Angiopoietin 1 800 40 Hepatocyte Growth factor 1700 50 SDF1 380 80

(34) Evaluation of the Stromal Cell Power of the Modeled 2D and 3D Niches

(35) The 2D modeled niches (osteoblasts, adipocytes, MSCs and osteoclasts) promote expansion of the CD34.sup.+ cells from a population of Lin.sup. cells from peripheral blood of non-mobilized healthy subjects (richness in CD34.sup.+ cells equivalent to 30%). The results show that after 7 days of co-culture, the CD34.sup.+ population represents about 96.25%+/1.71% (n=4) of the produced viable cells (FIG. 2).

(36) The percentage of CD34.sup.+ cells is significantly higher than that observed in the so-called <<niche-free>> condition, i.e. in the presence of biomaterials not colonized by stromal cells (97%+/2% vs 85%+/8%; p<0.05). Although the proliferation rate of the total cells is slightly smaller under the niche conditions (7.75%+/3.5%) relatively to niche-free conditions (13.67%+/3.06%; not significant), the amplification rate of the number of CD34.sup.Strong cells within the niches is about 3.5 times higher than on day 0. On the contrary, when the CD34 cells are cultivated under <<niche-free>> conditions, the number of CD34.sup.Strong cells is less than the initial figure. The thereby modeled niches allow amplification of the CD34.sup.Strong cells to the detriment of the CD34.sup.Weak cells, which suggests that taking into account the niche may be of interest in the optimization of the culture procedures of CD34.sup.Strong cells, giving the possibility of promoting proliferation to the expense of differentiation.

(37) Moreover, when the CD34.sup.+ cells are separated from the 2D niche by a transwell system, the results in terms of the maintaining of the number of CD34.sup.+ cells are inferior to the condition reproducing a complete niche, which suggests the importance of cell contacts and of the factors secreted in majority by the stromal cells in this process.

(38) Evaluation of the Maintaining/Acquisition of the SP Functionality by Modeled Niches

(39) In order to validate the capability of the hematopoietic niches of maintaining a pool of primitive hematopoietic cells, we also evaluated their capability of maintaining the SP functionality of the Lin.sup. medullary cells after 3 to 7 days of culture. The SP functionality is defined by the capability of the stem cells and in particular of the HSCs of causing effluence of the fluorescent coloring agent Hoechst-33342 or of other coloring agents and drugs. This functionality may be evaluated by flow cytometry by quantification of the residual fluorescence of the cells in the form of a characteristic cytogram, the cells having the greatest effluence capability being considered as the most primitive. For this purpose, we cultivated Lin.sup. medullary cells (2.10.sup.5) in the presence or in the absence of 2D cellularized niches (n=3 to 4).

(40) FIG. 3 shows that in the presence of cellularized niches (osteoblasts, adipocytes, osteoclasts, MSCs), the Lin.sup. medullary cells preserve their SP functionality, as compared with co-cultures on non-cellularized biomaterials (niche-free condition) or in a transwell system (Boyden condition). Thus, in the presence of a cellularized niche, the percentage of SP cells (1.46+/0.33) is maintained relatively to the first day (before cultivation: 1.02+/0.03, NS), or even increased as compared with the niche-free condition (0.05+/0.01, p<0.01) and to the transwell condition (0.17+/0.05, p<0.05). Thus, in terms of the absolute number of SP cells, the culture on a 2D niche gives the possibility of maintaining the pool of SP cells in a co-culture for three days (about 2920, in the presence of a niche), which is not the case in the absence of a niche or in a transwell condition (about 100, in the absence of a niche). These results suggest that the cellular interaction between the stromal cells and hematopoietic cells are required for maintaining the SP functionality.

(41) Unlike the HSCs/HPs of bone marrow, the cells of the peripheral blood do not express the SP functionality. When the latter were seeded for 3-5 days on a model niche, a certain percentage (0.5 to 2%) of them acquired SP functionality. Experiments conducted by using the various cell constituents separately show that the MSCs seem to have a predominate role in this acquisition (FIG. 4).