WO2008152640A2

WO2008152640A2 - Three dimensional biocompatible scaffolds for ex-vivo expansion and transplantation of stem cells

Info

Publication number: WO2008152640A2
Application number: PCT/IL2008/000803
Authority: WO
Inventors: Shai Meretzki; Uriel Barkai
Original assignee: Pluristem Ltd
Current assignee: Pluri Biotech Ltd
Priority date: 2007-06-13
Filing date: 2008-06-12
Publication date: 2008-12-18
Anticipated expiration: 2009-12-13
Also published as: WO2008152640A3

Abstract

A method of transplanting undifferentiated hematopoietic stem cells into a recipient is disclosed. The method comprising (a) expanding the undifferentiated hematopoietic stem cells to increase the number of the hematopoietic stem cells by (i) culturing in a stationary phase plug-flow bioreactor stromal cells under continuous flow of a culture medium on a three dimensional biocompatible substrate to thereby generate a three dimensional stromal cell culture; and (ii) seeding undifferentiated hematopoietic stem cells into the stationary phase plug-flow bioreactor including the three dimensional stromal cell culture and under a continuous flow of a culture medium, thereby expanding the undifferentiated hematopoietic stem cells and obtaining a three dimensional stromal cell culture comprising increased number of hematopoietic stem cells; and (b) transplanting the three dimensional stromal cell culture comprising hematopoietic stem cells resulting from (a) into the recipient.

Description

THREE DIMENSIONAL BIOCOMPATIBLE SCAFFOLDS FOR EX-VIVO EXPANSION AND TRANSPLANTATION OF STEM CELLS

FIELD AND BACKGROUND OF THE INVENTION

The present invention, in some embodiments thereof, relates to biocompatible scaffolds and, more particularly, but not exclusively, to the use of same for expansion and transplantation of hematopoietic stem cells.

The hematopoietic system in mammals is composed of heterogeneous population of cells that range in function from differentiation-committed and mature cells with limited proliferative potential to pluripotent stem cells with extensive proliferative, differentiative and self renewal capacities. Hematopoietic stem cells (HSCs) are the most primitive (undifferentiated) cells of this system. HSCs possess self-renewal, expansion and differentiation capabilities that are exclusively required for the recovery of a hematopoietic system following transplantation. While differentiation committed and differentiated cells make the vast majority of the bone marrow cell population, HSCs are present therein at very low levels. During hematopoietic reconstitution of the bone marrow of a myeloablated patient, differentiation-committed progenitor cells direct short-term hematopoietic recovery while long-term hematopoiesis relies solely upon HSCs. The gold standard methodology for bone marrow (BM) reconstitution following natural or induced myeloablation relies upon allogeneic hematopoietic stem cell (HSC) transplantation. Different sources of HSCs have been implicated in hematopoietic regeneration. These include embryonic stem (ES) cells as well as HSCs from BM, peripheral blood (PB) or umbilical cord blood (CB). The least differentiated of these are embryonic stem (ES) cells. These pluripotent cells possess the capacity to differentiate into many cell types including blood cells [Keller et al., MoI Cell Biol. (1993) 13:473-86; Wiles and Keller, Development (1991) 111 :259-67]. However, ethical and religious constrains are limiting the use of ES cells. Another source of HSCs is the BM. Transplantation of BM-derived cells for bone marrow regeneration is used as a standard medical procedure. Stem cells from PB and from CB have recently been developed as alternative sources of HSCs. The major advantages for using PB and umbilical CB include their availability and ease of collection. However, the abundance of HSCs in PB is the lowest of all accessible sources. Likewise, there is a low absolute count of CB-HSCs for marrow recovery in any given CB unit. Nevertheless, umbilical CB transplantation is associated with durable engraftment and low incidence of severe graft- versus-host disease (GVHD), even when 1-2 human leukocyte antigen (HLA) mismatch cells are being employed [Rocha et al., N Engl J Med. (2004) 351:2276-85]. Due to the low available levels of HSCs, a major goal has been to expand these cells ex vivo. Expansion of HSCs has been previously described as for example in US Pat. App. 11/438,847 and US Pat. App. 10/727,580. However, the major challenge of HSC expansion lies in their predisposition to differentiate into more committed cells.

It is widely accepted that HSCs are intimately associated in vivo with discrete niches within the marrow [Wolf, Clin Haematol. (1979) 8:469-500; Askenasy et al., Stem Cells. (2002) 20:301-10], also referred to as hematons, which provide molecular signals that collectively mediate their differentiation and self renewal, via cell to cell contacts or short-range interactions [Dorshkind, Annu Rev Immunol. (1990) 8:111-37]. These niches are part of the hemopoietic inductive microenvironment (HIM), composed of marrow stromal cells, e.g., macrophages, fibroblasts, adipocytes and endothelial cells [Allen and Dexter, Exp. Hematol. (1984) 12; 517]. These cells maintain the functional integrity of the HIM by providing extra cellular matrix (ECM) macromolecules and basement membrane - components that facilitate cell to cell contact [Gupta et al. Blood (1996) 87:3229; Liesveld et al., Exp. Hematol. (1991) 19:63; Long et al., J. Clin. Invest. (1992) 90:251]. Furthermore, these cells secrete growth factors, chemokines and cytokines needed for controlled hemopoietic cell differentiation and proliferation [Eaves et al. Blood (1991) 78:110; Moore et al. Proc. Nat. Acad. Sci. (1997) 94:401 1; Li et al., Immunity (1998) 8:43].

In view of the above, it is not surprising that efforts have been made to establish HSC culture systems utilizing stromal cells to promote long-term maintenance and expansion of human HSC, some are summarized infra. U.S. Pat. No. 6,911,201 discloses three-dimensional stromal cell cultures for expanding/maintaining undifferentiated hematopoietic stem cells or progenitor cells using a stationary phase plug-flow bioreactor. The cultures are established on a non-woven fibrous matrix (e.g. polystyrene) forming a physiologically acceptable three-dimensional network of fibers. U.S. Pat. No. 5,541,107 discloses a three-dimensional bone marrow and tissue culture system utilizing a pre-established stromal support matrix. The stromal support matrix comprises stromal cells (e.g. fibroblasts) which provide the support, growth factors, and regulatory factors necessary to sustain long-term active proliferation of HSCs in culture. The cultures are maintained in tissue culture flasks. Furthermore, according to U.S. Pat. No. 5,541,107, the framework is composed of a biocompatible material, including biodegradable or non-biodegradable materials, and the three-dimensional cultures may be transplanted or implanted into a living organism.

U.S. Pat. No. 5,635,386 discloses methods for culturing human stem cells, including human hematopoietic progenitor cells and human stromal cells, in a liquid culture medium which is rapidly replaced. The stromal cells and hematopoietic progenitor cells may be co- cultured. According to the teachings of U.S. Pat. No. 5,635,386, the medium is perfused either continuously or periodically while removing metabolic products and replenishing depleted nutrients (e.g. growth factors) and at the same time maintaining the culture under physiologically acceptable conditions. The culture conditions results in cells which may be transplanted into a recipient.

SUMMARY OF THE INVENTION According to an aspect of some embodiments of the present invention there is provided a method of transplanting undifferentiated hematopoietic stem cells into a recipient, the method comprising the steps of (a) expanding the undifferentiated hematopoietic stem cells to increase the number of the hematopoietic stem cells by (i) culturing in a stationary phase plug-flow bioreactor stromal cells under continuous flow of a culture medium on a three dimensional biocompatible substrate to thereby generate a three dimensional stromal cell culture; and (ii) seeding undifferentiated hematopoietic stem cells into the stationary phase plug-flow bioreactor including the three dimensional stromal cell culture and under a continuous flow of a culture medium, thereby expanding the undifferentiated hematopoietic stem cells and obtaining a three dimensional stromal cell culture comprising increased number of hematopoietic stem cells; and (b) transplanting the three dimensional stromal cell culture comprising hematopoietic stem cells resulting from step (a) into the recipient.

According to an aspect of some embodiments of the present invention there is provided an article of manufacture comprising a three dimensional biodegradable substrate comprising at least 1 x 10⁷ stromal cells per ml and undifferentiated hematopoietic stem cells.

According to some embodiments of the invention, the undifferentiated hematopoietic stem cells comprise cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone-marrow. According to some embodiments of the invention, the stromal cells being derived from a tissue selected from the group consisting of a bone marrow, a placenta and an adipose tissue

According to some embodiments of the invention, the stromal cells and the undifferentiated hematopoietic stem cells share common HLA antigens.

According to some embodiments of the invention, the stromal cells and the undifferentiated hematopoietic stem cells are from a single individual.

According to some embodiments of the invention, the stromal cells and the undifferentiated hematopoietic stem cells are from different individuals. According to some embodiments of the invention, the stromal cells and the undifferentiated hematopoietic stem cells are from the same species.

According to some embodiments of the invention, the stromal cells and the undifferentiated hematopoietic stem cells are from different species.

According to some embodiments of the invention, the biocompatible substrate comprises a porrosive carrier.

According to some embodiments of the invention, the porrosive carrier comprises a porrosive sheet.

According to some embodiments of the invention, the porrosive sheet comprises a width of about 200-600 μm. According to some embodiments of the invention, the biocompatible substrate comprises a polymeric substrate.

According to some embodiments of the invention, the polymeric substrate comprises poly(caprolactone) (PCL).

According to some embodiments of the invention, the polymeric substrate is selected from the group consisting of polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co- glycolide (PLGA), polydioxanone, polygluconate, polylactic acid-polyethylene oxide copolymers, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes, aliphatic polyester polyacrylates, polymethacrylate, acyl substituted cellulose acetates, non- degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinyl imidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, teflon RTM, nylon silicon, poly(styrene-block-butadiene), polynorbornene, hydrogels, metallic alloys and oligo(ε-caprolactone)diol.

According to some embodiments of the invention, the biocompatible substrate comprises a synthetic polymer. According to some embodiments of the invention, the biocompatible substrate comprises a natural polymer.

According to some embodiments of the invention, the biodegradable substrate is selected from the group consisting of poly(caprolactone) (PCL), polyglycolic acid, poly

(DL-lactic-coglycolic acid), cat gut sutures, cotton, cellulose, gelatin, dextran, alginate, fibronectin, laminin, collagen, hyaluronic acid, polyhydroxyalkanoate, poly 4 hydroxybutirate (P4HB) and polygluconic acid (PGA).

According to some embodiments of the invention, the step of the seeding the undifferentiated hematopoietic stem cells into the stationary phase plug-flow bioreactor is effected while flow in the bioreactor is shut off for at least 10 hours following the seeding. According to some embodiments of the invention, the stromal cells are grown to a density of at least 1 x 10⁷ stromal cells per ml.

According to some embodiments of the invention, the culture medium is devoid of supplementary cytokines.

According to some embodiments of the invention, expanding the undifferentiated hematopoietic stem cells results in CD45+CD34+CD38- expressing cells.

According to some embodiments of the invention, expanding the undifferentiated hematopoietic stem cells results in CD45+CD34+CD38-CXCR4+ expressing cells.

Unless otherwise defined, all technical and/or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and/or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

BRIEF DESCRIPTION OF THE DRAWINGS

Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. [IF IMAGES, REPHRASE] With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced. In the drawings: FIG. 1 is a schematic illustration of the plug-flow bioreactor employed in the present invention (previously described in WOOO/46349). 1- medium reservoir; 2 - gas mixture container; 3 - gas filters; 4 - injection points; 5 - plug or container of plug flow bioreactor 20; 6 - flow monitors; 6a - flow valves; 7 - conditioned medium collecting/separating container; 8 - container for medium exchange; 9 - peristaltic pump; 10 - sampling point; 11- container for medium exchange; 12 - O2 monitor; 14 - steering device;

PH - pH probe.

FIGs. 2A-C are bar graphs depicting cell expansion following co-culturing. Monocultures of AFT024 murine fetal liver cell lines were cultivated on plastic culture plate surfaces (2D) or on non-biodegradable 3-D scaffolds (3D and bioreactor). Spatial cultures were cultivated either in 24- well static dishes (3D) or in flow-through bioreactor (bioreactor). When confluent, CD34+ cells were seeded onto the stromal surfaces and co- cultures were cultured for 14 days. Results are mean ± SEM of six independent measurements.

FIG. 3 is a line graph depicting the growth curve of AFT024 murine fetal liver cells on biodegradable scaffolds. PCL [poly(caprolactone)] porrosive sheets (4 mm x 4 mm) were loaded with 1 x 10⁵ murine fetal liver stromal cells and were cultured for 21 days in a plug flow bioreactor (FIG. 1). PCLl, PCL2 and PCL3 represent porrosive sheets of 0.25 mm, 0.35 mm and 0.55 mm width, respectively. Polystyrene is a non-biodegradable carrier. Results represent a mean of 5 independent measurements. FIG. 4 is a line graph depicting expansion of HSCs and stromal cells co-cultured on

3D polystyrene non-biodegradable disk shaped carriers (FibraCel, Bibby-Sterilin UK; 1200 cm effective surface growth area per gram). The stromal cells were grown using culture methodology without cytokine supplementation. 3 x 10⁴ cells/carrier BM-derived adherent human stromal cells were seeded on the disk carriers at day 0 and cultured in a plug flow bioreactor system. On growth day 50, 1 x 10⁴ human CB derived HSCs (CD34+) were seeded on the stromal cells and the co-culture was allowed to proliferate for additional 15 days within the bioreactor. DESCRIPTION OF SPECIFIC EMBODIMENTS QF THE INVENTION

The present invention, in some embodiments thereof, relates to biocompatible scaffolds and, more particularly, but not exclusively, to the use of same for expansion and transplantation of hematopoietic stem cells. The principles and operation of the present invention may be better understood with reference to the drawings and accompanying descriptions.

Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways. Also, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and should not be regarded as limiting.

While reducing the present invention to practice, the present inventors have uncovered that three dimensional (3D) biocompatible scaffolds comprising stromal cells can be effectively utilized to ex vivo expand undifferentiated hematopoietic stem cells.

Further, it was uncovered for the first time that these biocompatible scaffolds can be transplanted into a subject to thereby mimic the 3D environment of the bone marrow in vivo and enable on-going bone marrow (BM) regeneration.

As is shown hereinbelow and in the Examples section which follows, the present inventors have constructed a 3D niche/hematon-like microstructure that functions in a bioreactor (see Figure 1) and comprises stromal cells supporting the ex-vivo expansion of hematopoietic stem cells (HSCs, see Figures 2A-C). The stromal cells support the expansion of HSCs in an environment devoid of supplementary cytokines, thereby avoiding the risk of unrestrained growth. Moreover, the present inventors have shown that biodegradable scaffolds (e.g. PCL), which can be transplanted into a subject, support the growth of stromal cells in a similar manner to non-biodegradable scaffolds (see Figure 3).

Thus, according to one aspect of the present invention there is provided a method of transplanting undifferentiated hematopoietic stem cells into a recipient. The method according to this aspect of the present invention comprises: (a) expanding the undifferentiated hematopoietic stem cells to increase the number of the hematopoietic stem cells by:

(i) culturing in a bioreactor stromal cells under continuous flow of a culture medium on a three dimensional biocompatible substrate to thereby generate a three dimensional stromal cell culture; and (ii) seeding undifferentiated hematopoietic stem cells into the bioreactor including the three dimensional stromal cell culture and under a continuous flow of a culture medium, thereby expanding the undifferentiated hematopoietic stem cells and obtaining a three dimensional stromal cell culture comprising increased number of hematopoietic stem cells; and (b) transplanting the three dimensional stromal cell culture comprising the hematopoietic stem cells resulting from step (a) into the recipient.

As used herein, the phrase "undifferentiated hematopoietic stem cells" refers to uncommitted hemopoietic cells. Undifferentiated hematopoietic stem cells (HSCs) are self renewing cells that given the right growth conditions can give rise to all the mature blood cell types (e.g. myeloid and lymphoid lineages) present in the organism from which they were derived. Undifferentiated hematopoietic stem cells refer herein to both the earliest renewable hematopoietic cell population and the very early progenitor cells, which are somewhat more differentiated, yet are not committed. Typically undifferentiated hematopoietic stem cells are positively stained for the cellular markers CD34 and CXCR4. - Some of the cells are negative or dimly positive for CD38.

The undifferentiated hematopoietic stem cells which are used for implementing the method of this aspect of the present invention can be from a tissue, such as, but not limited to, cord blood, cytokine-mobilized peripheral blood (collected by, for example, leukapheresis), bone-marrow (e.g. from femurs, hip, ribs or sternum), all of which are known to include undifferentiated hemopoietic stem cells. It will be appreciated that depending on the expansion method used, cells need not be further purified and unselected populations such as of peripheral blood mononuclear cells (PBMCs) may be used in accordance with the present teachings. PBMCs may comprise the entire complement of white blood cells present in a sample including a majority fraction of the cells having committed hematopoietic precursor cells, and an uncommitted minority fraction having pluripotent hematopoietic cells, which population has not undergone selection for hematopoietic stem cells. Suitable unselected mononuclear cells can be from any source relatively rich in hematopoietic cells, such as described above. Methods for identification and isolation of mononuclear cells are well known in the art, such as density centrifugation of whole blood followed by isolation of PBMCs from the "buffy coat". When needed, further steps of selection are employed. Methods of such separation are well known in the art, the most frequently used being fluorescence activated cell sorting in which cells are first tagged by affinity labeling with a fluorophore and are than collected. For example, anti-CD34 antibodies may be employed for selection of undifferentiated hematopoietic stem cells from a pool of cells (e.g. cord blood cells) and may further be isolated by a FACS sorter or by MACS (e.g. Miltenyl Biotech, see the Materials and Methods section which follows).

As used herein the terms "expanding" and "expansion" refer to substantially differentiation-less maintenance of the cells and ultimately cell growth, i.e., increase of a cell population (e.g., at least 2 fold) without differentiation accompanying such increase.

As used herein, the terms "stromal cells" or "mesenchymal cells" are used interchangeably and refer to adherent cells typically of a multipotent stem cell phenotype that can differentiate into more than one specific type of mesenchymal or connective tissue. Thus, stromal cells give rise to one or more mesenchymal tissues (e.g., adipose, osseous, cartilaginous, elastic and fibrous connective tissues, myoblasts) as well as to tissues other than those originating in the embryonic mesoderm (e.g., neural cells) depending upon various influences from bioactive factors such as cytokines.

As used herein the phrase "adherent cells" refers to a homogeneous or heterogeneous population of cells which are anchorage dependent, i.e., require attachment to a surface in order to grow in vitro.

Stromal cells may be isolated from various tissues including but not limited to bone marrow, peripheral blood, blood, placenta and adipose tissue. Stromal cells may also be derived from fetal tissues (e.g. fetal liver). A method of isolating stromal cells from peripheral blood is described by Kassis et al [Bone Marrow Transplant. 2006 May; 37(10):967-76]. A method of isolating stromal cells from placental tissue is described by Zhang et al [Chinese Medical Journal, 2004, 1 17 (6):882-887]. Methods of isolating and culturing adipose tissue, placental and cord blood stromal cells are described by Kern et al [Stem Cells, 2006; 24: 1294-1301]. Methods of isolating, purifying and expanding stromal cells are known in the arts and include, for example, those disclosed by Caplan and Haynesworth in U.S. Pat. No. 5,486,359 and Jones E.A. et al., 2002, Isolation and characterization of bone marrow multipotential mesenchymal progenitor cells, Arthritis Rheum. 46(12): 3349-60.

Thus, for example, BM aspirates (usually 20 ml) are diluted 3-fold in Hank's Balanced Salts Solution (HBSS; GIBCO BRL) and are subject to Ficoll-Hypaque (Robbins Scientific Corp. Sunnyvale, Calif.) density gradient centrifugation. Marrow mononuclear cells (<1.077 gm/cm³) are collected, washed 3 times in HBSS and resuspended in long- term culture (LTC) medium, consisting of DMEM supplemented with 12.5 % FCS, 12.5 % horse serum (Beit Ha'Emek), \0^A M β-mercaptoethanol (Merck) and 10^"6 mol/L hydrocortwasone sodium succinate (Sigma). Resuspended cells are plated in about 25 ml of medium in a tissue culture flasks (Coming) and incubated for 3 days at 37 °C (5 % CO2). Following 3 days in culture, non-adherent cells are discarded, and the adherent cells are thoroughly washed twice with phosphate buffered saline (PBS). The cells are further cultured with replacement of the culture medium weekly for about 5 weeks. Adherent cells are then trypsinized (using Trypsin/EDTA), dissociated by passage through a narrowed Pasteur pipette, washed 3 times in HBSS, resuspended in LTC medium, counted and seeded at 10⁶ cells/ml in 10 ml volumes into carriers in a bioreactor (explained in further detail hereinbelow). Other methods of expansion of BM-derived stromal cells are described by Colter

DC, et al. Rapid expansion of recycling stem cells in cultures of plastic-adherent cells from human bone marrow. Proc Natl Acad Sci USA. 97: 3213-3218, 2000].

When stromal cells are cultured under the culturing conditions of the present invention they exhibit negative staining for the hematopoietic stem cell markers CD34, CDl IB, CD43 and CD45. A small fraction of cells (less than 10 %) are dimly positive for CD31 and/or CD38 markers. In addition, mature stromal cells are dimly positive for the hematopoietic stem cell marker, CDl 17 (c-Kit), moderately positive for the osteogenic stromal cells marker, Stro-1 [Simmons, P. J. & Torok-Storb, B. (1991). Blood 78, 5562] and positive for the thymocytes and peripheral T lymphocytes marker, CD90 (Thy-1). On the other hand, the RS-I cells are negative for the CDl 17 and Strol markers and are dimly positive for the CD90 marker, and the RS-2 cells are negative for all of these markers.

As mentioned the placenta can be are readily available source for the stromal cells of the present invention. Placental cells may be obtained from a full-term or pre-term placenta. Placenta is preferably collected once it has been ex blooded. The placenta is preferably perfused for a period of time sufficient to remove residual cells. The term "perfuse" or "perfusion" used herein refers to the act of pouring or passaging a fluid over or through an organ or tissue. The placental tissue may be from any mammal; for example, the placental tissue is human. A convenient source of placental tissue is from a post partum placenta (e.g., 1-6 hours), however, the source of placental tissue or cells or the method of isolation of placental tissue is not critical to the invention.

Placenta derived adherent cells may be obtained from both fetal (i.e., amnion or inner parts of the placenta) and maternal (i.e., decidua basalis, and decidua parietalis) parts of the placenta. Tissue specimens are washed in a physiological buffer [e.g., phosphate- buffered saline (PBS) or Hank's buffer). Single-cell suspensions are made by treating the U tissue with a digestive enzyme (see below) or/and mincing and flushing the tissue parts through a nylon filter or by gentle pipetting (Falcon, Becton, Dickinson, San Jose, CA) with washing medium.

Adipose tissue derived adherent cells may be isolated by a variety of methods known to those skilled in the art. For example, such methods are described in U.S. Pat. No. 6,153,432. The adipose tissue may be derived from omental/visceral, mammary, gonadal, or other adipose tissue sites. One source of adipose tissue is omental adipose. In humans, the adipose is typically isolated by liposuction.

Isolated adherent cells from adipose tissue may be derived by treating the tissue with a digestive enzyme such as collagenase, trypsin and/or dispase; and/or effective concentrations of hyaluronidase or DNAse; and ethylenediaminetetra-acetic acid (EDTA); at temperatures between 25 - 50 °C, for periods of between 10 minutes to 3 hours. The cells may then be passed through a nylon or cheesecloth mesh filter of between 20 microns to 800 microns. The cells are then subjected to differential centrifugation directly in media or over a FicoU or Percoll or other particulate gradient. Cells are centrifuged at speeds of between 100 to 3000 x g for periods of between 1 minutes to 1 hour at temperatures of between 4- 50 °C (see U.S. Pat. No. 7,078,230).

In addition to placenta or adipose tissue derived adherent cells, the invention also envisages the use of adherent cells from other cell sources which are characterized by stromal cell phenotype (as is further described hereinabove). Tissue sources from which adherent cells can be retrieved include, but are not limited to, cord blood, hair follicles [e.g. as described in Us Pat. App. 20060172304], testicles [e.g., as described in Guan K., et al., Nature. 2006 Apr 27;440(7088):l 199-203], human olfactory mucosa [e.g., as described in Marshall, CT., et al., Histol Histopathol. 2006 Jun;21(6):633-43], embryonic yolk sac [e.g., as described in Geijsen N, Nature. 2004 Jan 8;427(6970): 148-54] and amniotic fluid [Pieternella et al. (2004) Stem Cells 22: 1338-1345], all of which are known to include stromal cells. Adherent cells from these tissue sources can be isolated by culturing the cells on an adherent surface, thus isolating adherent cells from other cells in the initial population. Regardless of the origin (e.g., placenta or adipose tissue), cell retrieval is preferably effected under sterile conditions. Once isolated the stromal cells are seeded on a three dimensional biocompatible adherent substrate.

As used herein "an adherent substrate" refers to a synthetic, naturally occurring or a combination of same of a non-cytotoxic (i.e., biologically compatible) material having a chemical structure (e.g., charged surface exposed groups) which may retain the cells on a surface.

As used herein, the term "substrate" refers to a 3D scaffold or matrix upon which cells may be cultured (i.e., survive and preferably proliferate for a predetermined time period).

Much of the success of scaffolds in cell culture depends on identifying an appropriate material to address the critical physical, mass transport, and biological design variables inherent to each. Any scaffold material which is structurally similar to the extracellular matrix of tissues (e.g. hydrogels), is biocompatible and may be delivered in a minimally invasive manner (i.e. by transplant) may be utilized according to the present teachings. The scaffold of the present invention may be made uniformly of a single polymer, a co-polymer or blend thereof. However, it is also possible to form a scaffold according to the invention of a plurality of different polymers. There are no particular limitations to the number or arrangement of polymeric substrates used in forming the scaffold. Any combination which is biocompatible (biodegradable and/or nonbiodegradable) and may act as adhesive substrate for cells, may be formed into fibers, and degrades at a suitable rate, may be used.

The term "biodegradable" refers to materials that are bioresorbable and/or degrade and/or break down by mechanical degradation upon interaction with a physiological environment into components that are metabolizable or excretable, over a period of time from minutes to years, while maintaining the requisite structural integrity. As used in reference to polymers, the term "degrade" refers to cleavage of the polymer chain, such that the molecular weight stays approximately constant at the oligomer level and particles of polymer remain following degradation. Biocompatible polymers and materials suitable for polymer scaffold fabrication include polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polygluconate, polylactic acid-polyethylene oxide copolymers, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester, poly(alpha- hydroxy acid), polycaprolactone, polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes, aliphatic polyester polyacrylates, polymethacrylate, acyl substituted cellulose acetates, non- degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinyl imidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, teflon RTM, nylon silicon, a porous silicone, ceramic, and shape memory materials, such as poly(styrene-block-butadiene), polynorbornene, hydrogels, metallic alloys, and oligo(.epsilon.-caprolactone)diol as switching segment/oligo(p-dioxyanone)diol as physical crosslink. Other suitable polymers can be obtained by reference to The Polymer Handbook, 3rd edition (Wiley, N. Y., 1989).

In certain embodiments, the scaffold materials may be a biodegradable polymer or material. Biodegradable scaffold materials may be biocompatible. Examples of biocompatible biodegradable polymers which are useful as scaffold materials include, but are not limited to, polylactic acid (PLA), poly (DL-lactic-co-glycolic acid), polyglycolic acid (PGA), polycaprolactone (PCL), and copolymers thereof, polyesters such as polyglycolides, polyanhydrides, polyacrylates, polyalkyl cyanoacrylates such as n-butyl cyanoacrylate and isopropyl cyanoacrylate, polyacrylamides, polyorthoesters, polyphosphazenes, polypeptides, polyurethanes, polystyrenes, polystyrene sulfonic acid, polystyrene carboxylic acid, polyalkylene oxides, hyaluronic acid, polyhydroxyalkanoate, poly 4 hydroxybutirate (P4HB) and polygluconic acid (PGA), alginates, agaroses, dextrins, dextrans, cat gut sutures, cotton, cellulose, gelatin, polyanhydrides, biopolymers such as collagens and elastin, fibronectin, laminin, alginates, chitosans, glycosaminoglycans, and mixtures of such polymers.

In other embodiments, the scaffold materials may be non-biodegradable polymers or materials. Such non-biodegradable scaffold materials may be used to fabricate materials which are designed for long term or permanent implantation into a host organism. Nonbiodegradable structural scaffold materials may be biocompatible. Examples of biocompatible non-biodegradable polymers which are useful as scaffold materials include, but are not limited to, polyethylenes, polyvinyl chlorides, polyamides such as nylons, polyesters, rayons, polypropylenes, polyacrylonitriles, acrylics, polyisoprenes, polybutadienes and polybutadiene-polyisoprene copolymers, neoprenes and nitrile rubbers, polyisobutylenes, olefinic rubbers such as ethylene-propylene rubbers, ethylene-propylene- diene monomer rubbers, and polyurethane elastomers, silicone rubbers, fluoroelastomers and fluorosilicone rubbers, homopolymers and copolymers of vinyl acetates such as ethylene vinyl acetate copolymer, homopolymers and copolymers of acrylates such as polymethylmethacrylate, polyethylmethacrylate, polymethacrylate, ethylene glycol dimethacrylate, ethylene dimethacrylate and hydroxymethyl methacrylate, polyvinylpyrrolidones, polyacrylonitrile butadienes, polycarbonates, polyamides, fluoropolymers such as polytetrafluoroethylene and polyvinyl fluoride, polystyrenes, homopolymers and copolymers of styrene acrylonitrile, cellulose acetates, homopolymers and copolymers of acrylonitrile butadiene styrene, polymethylpentenes, polysulfones, polyesters, polyimides, polyisobutylenes, polymethylstyrenes, and other similar compounds known to those skilled in the art. Other biocompatible non-degradable polymers that are useful in accordance with the present disclosure include polymers comprising biocompatible metal ions or ionic coatings which can interact with DNA. Such metal ions include, but are not limited to gold and silver ions, Al, Fe, Mg, and Mn.

It will be appreciated that a mixture of non-biodegradable and/or biodegradable scaffold materials may be used to form a biomimetic structure of which part is permanent and part is temporary.

According to the teachings of the present invention and as illustrated in the Examples section which follows, scaffolds which are particularly suitable for expansion of stromal cells include, but are not limited to, PCL scaffolds.

Preparation of scaffold material varies with the desired character of the scaffold. Scaffold material may comprise natural or synthetic organic polymers that can be gelled, or polymerized or solidified (e.g., by aggregation, coagulation, hydrophobic interactions, or cross-linking) into a 3-D open-lattice structure that entraps water or other molecules, e.g., to form a hydrogel. As mentioned above, the scaffold materials may comprise a single polymer or a mixture of two or more polymers in a single composition. Additionally, two or more scaffold materials may be co-deposited so as to form a polymeric mixture at the site of deposition. In exemplary embodiments, structural scaffold materials are easy to process into complex shapes (e.g. sheet) and have a rigidity and mechanical strength suitable to maintain the desired shape under in vivo conditions.

It will be appreciated that the polymeric scaffolds of the present invention can comprise, for example, a porous, non-woven array of fibers (i.e. porrosive carrier). Fabrication and use of porous scaffolds for support of cells in ex vivo cultures is well known in the art [for a review, see Drury et al, Biomaterials 2003; 24: 4337-51; Levene (US Patent No. 6,337,198, which is incorporated by reference as if fully set forth by reference herein) discloses the use of such biodegradable porous polymer scaffolds for cell and tissue growth]. Hence, the polymeric scaffold can be shaped to maximize surface area (e.g. shape of squares, rings, discs, cruciforms), to allow adequate diffusion of nutrients and growth factors to the cells. Taking these parameters into consideration, one of skill in the art could configure a polymer scaffold having sufficient surface area for the cells to expand using methods known in the art. Furthermore, the polymeric scaffolds can comprise a fibrillar structure. The fibers can be round, scalloped, flattened, star-shaped, solitary or entwined with other fibers. Branching fibers can be used, increasing surface area proportionately to volume.

According to an exemplary embodiment, the scaffold of the present invention is in the form of a sheet.

When the scaffold is used in the form of a sheet, such as a non-woven fiber sheets or sheets of open-pore foamed polymers (i.e. porrosive sheet), the width of the sheet is about 50 to 1000 μm or more, there being provided adequate porosity for cell entrance, entrance of nutrients and for removal of waste products from the sheet. According to an exemplary embodiment the surface area of the sheet is about 16 mm². The diameter of the pores comprising the scaffold is typically about 10 μm to 100 μm. Such sheets can be prepared from fibers of various thicknesses, the preferred fiber thickness or fiber diameter range being from about 0.5 μm to 20 μm, still more preferred fibers are in the range of 10 μm to 15 μm in diameter. Furthermore, the scaffolds of the present invention may be supported by, or bonded to, a porous support sheet or screen providing for dimensional stability and physical strength. Such matrix sheets may also be cut, punched, or shredded to provide particles with projected area of the order of about 0.2 mm² to about 10 mm², with the same order of width (about 50 to 1000 μm). Therapeutic compounds can also be incorporated into the scaffolds material.

Campbell et al (US Patent Application No. 20030125410) discloses methods for fabrication of 3D scaffolds for stem cell growth, the scaffolds having preformed gradients of therapeutic compounds such as analgesics, growth factors, cytokines, immune modulators, etc. The scaffold materials, according to Campbell et al, fall within the category of "bio-inks". Such "bio-inks" are suitable for use with the bioreactors and methods of the present invention. Frondoza et al (US Patent No. 6,662,805, and US Patent Application No. 20010014475) have disclosed methods for the in-vitro preparation of implantable tissue replacements grown from stem and other cells, on microcarriers.

Thus, factors, including but not limited to nutrients, growth factors, inducers of differentiation or de-differentiation, products of secretion, immunomodulators, inhibitors of inflammation, regression factors, hormones, or other biologically active compounds can be incorporated into or can be provided in conjunction with the polymer scaffold. Following generation of the biocompatible scaffolds of the present invention, the scaffolds are typically sterilized, for example by oxygen plasma, following which they are seeded with stromal cells.

As used herein, the term "seeding" refers to plating, placing and/or dropping the cells of the present invention into the biocompatible scaffolds of the present invention. It will be appreciated that the concentration of cells which are seeded on or within the biocompatible scaffolds depends on the type of cells used (as described above) and the composition of the scaffolds.

To ensure that stromal cells are seeded on the scaffolds, immediately following seeding, circulation may be stopped for at least 10 hours to allow the cells to settle on the scaffolds.

According to a preferred embodiment, the stromal cells are cultured in a bioreactor.

As used herein, the term "bioreactor" refers to any device in which biological and/or biochemical processes develop under monitored and controlled environmental and operating conditions, for example, pH, temperature, pressure, nutrient supply and waste removal. According to one embodiment of the invention, the basic classes of bioreactors suitable for use with the present invention include stationary phase plug-flow bioreactors, roller bottle bioreactors and stirred flask bioreactors.

The stationary phase plug-flow bioreactors described herein have been previously disclosed in WO00/46349 and are unique in that they combine both 3D stromal cell cultures with a continuous flow system. As described in detail in the Examples section which follows, stationary phase plug-flow bioreactors are easy to monitor and regulate and enable propagation of large cell numbers in a relatively small volume. The stationary phase plug-flow bioreactors contain a sampling and injection point allowing the sequential seeding of stromal cells and hematopoietic stem cells onto carriers (e.g. scaffolds). Furthermore, these bioreactors supply the cells with a constant flow of air (gas mixture containing air/CO2 02) and with a continuous medium exchange at a rate of about 10-50 ml/day.

Stirred flask or spinner flask bioreactors are also suitable for use in accordance with the present teachings. Spinner flasks are either plastic or glass bottles with a central magnetic stirrer shaft and side arms for the addition and removal of cells and medium, and gassing with CO₂ enriched air. Stirred bioreactors provide a homogeneous environment and are easy to operate, allowing sampling, monitoring and control of culture conditions. Typical operating modes include batch, fed-batch and perfusion mode (medium exchange with retention of cells by means of an external filtration module or of internal devices such as spin filters). Stirred suspension culture systems are relatively simple and readily scalable. In addition, their relatively homogeneous nature makes them suited for the investigation of different culture parameters. Spinner and stirrer flask systems designed to handle culture volumes of 1-12 liters are commercially available, such as the Corning ProCulture System (Corning, Inc., Acton, MN), Techne Stirrer System (Techne Incorporated, Burlington, NJ), cell culture (Bell-Flo) and bioreactor systems from Bellco Inc. (Vineland, NJ), for example, Bellco Prod. No's. Z380482-3L capacity and Z380474- IL capacity. Roller bottle bioreactors may also be employed according to the present teachings.

A roller bottle bioreactors is a bottle- shaped vessel supplied with a culture medium and cells, which is continuously rotated to cause the cells to be attached to the inside thereof for growing. The rotation of the roller bottle causes the cell surface to make rotational movements between the inside and outside of the culture medium, so that the cells repeat the process of absorbing nutrients in the inside and oxygen in the outside.

It will be appreciated that in order to support cell growth, the stromal cells are cultured in the presence of a culture medium.

The culture medium used by the present invention can be any liquid medium which allows at least cell survival. Such a culture medium can include, for example, salts, sugars, amino acids and minerals in the appropriate concentrations and with various additives and those of skills in the art are capable of determining a suitable culture medium to specific cell types. Non-limiting examples of such culture medium include, phosphate buffered saline, DMEM, MEM, RPMI 1640, McCoy's 5A medium, medium 199 and IMDM (available e.g., from Biological Industries, Beth Ha'emek, Israel; Gibco-Invitrogen Corporation products, Grand Island, NY, USA).

The culture medium may be supplemented with various antibiotics (e.g., Penicillin and Streptomycin), growth factors or hormones, specific amino acids (e.g., L-glutamin) and the like. It will be appreciated that according to the present teachings, the culture medium is devoid of supplementary cytokines. For example, as is shown in the Examples section which follows, a culture medium which includes Dulbecco's high-glucose medium (DMEM), fetal calf serum (FCS), horse serum, β-mercaptoethanol and hydrocortwasone sodium succinate is capable of maintaining long term culture of stromal cells. The medium can be designed devoid of animal (xeno) contaminants for future use of the cells in the clinic. Thus, the bioreactor (e.g. 3D stationary phase plug-flow bioreactor) described by the present invention is capable of supporting the long-term growth of stromal cell lines as well as primary marrow stromal cells. The use of stromal cells in the bioreactor is not only essential for the establishment of stromal cell contact (via unique "niches" and cell-cell, cell-ECM interactions), but also for stromal cell production of known and novel soluble and membrane-bound cytokines. Stromal cells can further facilitate the supplementation of such bioreactors with appropriate cytokines by using genetically engineered cytokine- producing variants. Thus, bioreactor stromal cells can also be molecular engineered.

As described in the Examples section below, the stromal cells are typically cultured in the stationary phase plug- flow bioreactor for a period of about 2-10 weeks for establishment of spatially organized three dimensional stromal cell culture. The three dimensional stromal cell culture described herein comprises at least 1 x 10⁷ stromal cells per ml.

Following generation of the three dimensional stromal cell cultures of the present invention, the undifferentiated hematopoietic stem cells are seeded thereon. Seeding the undifferentiated hematopoietic stem cells is effected directly in the bioreactor such as under a continuous flow of a culture medium.

According to the present teachings, expansion of the undifferentiated hematopoietic stem cells on the three dimensional stromal cell cultures results in an increase in the number of undifferentiated hematopoietic stem cells. The increase in cell number is, at least by about 2 fold, at least by about 5 fold at least by about 10 fold, at least by about 20 fold, at least by about 40 fold, at least by about 60 fold, at least by about 80 fold, or at least by about 100 fold.

Furthermore, according to the present teachings, the expansion of undifferentiated hematopoietic stem cells results in a cell population comprising CD45+CD34+CD38- expressing cells and CD45+CD34+CD38-CXCR4+ expressing cells (see Figures 2B-C).

As mentioned, the present inventors have uncovered that the three dimensional biocompatible stromal cell cultures comprising the expanded undifferentiated hematopoietic stem cells are highly suitable for in vivo bone marrow (BM) reconstitution. Thus, the three dimensional stromal cell cultures comprising the undifferentiated hematopoietic stem cells are transplanted into a recipient.

Following expansion of undifferentiated hematopoietic stem cells on the three dimensional stromal cell cultures (typically for about 2 weeks), the scaffolds are implanted into a recipient (e.g., a subject suffering from a pathology requiring bone marrow regeneration as described hereinbelow). In such cases the cells seeded on the scaffold for ex vivo expansion can be derived from the treated individual (autologous source) or from allogeneic or syngeneic sources such as cord blood which are not expected to induce an immunogenic reaction. It will be appreciated that the stromal cells and hematopoietic stem cells can be obtained from the same species (e.g. human), from different species (e.g. human and porcine), from a single individual (i.e. autologous), from different individuals (e.g. autologous and allogeneic, autologous and syngeneic, autologous and xenogeneic, or any other combination of same). Furthermore, the stromal cells and hematopoietic stem cells may share common HLA antigens (at least one common HLA antigen) or on the contrary may comprise different HLA antigens.

Those skilled in the art are capable of determining when and how to implant the biocompatible scaffold comprising the stromal cells and HSCs to thereby induce bone marrow regeneration and treat the pathology. The three dimensional stromal cell cultures comprising the undifferentiated hematopoietic stem cells are preferably implanted into a tissue such as, but not limited to, the portal vein, the kidney capsule or the hepatic artery. The three dimensional stromal cell cultures comprising the undifferentiated hematopoietic stem cells are suitable for ex vivo bone marrow regeneration to be utilized in, for example, but not limited to, anemia, aplastic anemia, sickle cell anemia, myelodysplasia, leukemia, lymphoma, a bone marrow disorder or a hematopoietic disease or disorder, including but not limited to, Myeloproliferative disorders (MPD), Myelodysplastic Syndrome (MDS), Plasma cell disorders.

Since the biocompatible scaffolds comprising the stromal cells and HSCs of the present invention may be used to regenerate bone marrow, they may be used for treating diseases characterized by loss of regenerative capacity of the hematopoietic system. Methods of implanting scaffolds in a subject are known in the art (see for example,

Artzi Z, et al., 2005, J. Clin. Periodontol. 32: 193-9; Butler CE and Prieto VG, 2004, Plast. Reconstr. Surg. 114: 464-73).

As mentioned, the cells which can be used according to the teachings of the present invention may comprise non-autologous cells. Non-autologous cells (e.g. allogeneic cells or xenogeneic cells), such as human cadavers, human donors or xenogeneic donors (e.g. porcine), may induce an immune reaction when administered to the subject. Several approaches have been developed to reduce the likelihood of rejection of non-autologous cells. These include either suppressing the recipient immune system or encapsulating the non-autologous cells in immunoisolated, semipermeable membranes before transplantation.

Suppressing the immune system may be effected by administration of immunosuppressant drugs. Examples of immunosuppressive agents include, but are not limited to, methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, infliximab (REMICADE), etanercept, TNF.alpha. blockers, a biological agent that targets an inflammatory cytokine, and Non-Steroidal Anti- Inflammatory Drug (NSAIDs). Examples of NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors and tramadol. These agents may be administered individually or in combination. Immunosuppressant drugs may be administered prior to, concomitantly with, or following transplantation of the cells. Encapsulation techniques are generally classified as microencapsulation, involving small spherical vehicles and macroencapsulation, involving larger flat-sheet and hollow- fiber membranes (see for example, Uludag, H. et al. Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000; 42: 29-64). Pollok et al were able to successfully encapsulate a polymer scaffold seeded with islets using porcine chondrocytes [Dig Surg 2001;18:204-210].

Methods of preparing microcapsules are known in the arts and include, for example, those disclosed by Lu MZ, et al., Cell encapsulation with alginate and alpha- phenoxycinnamylidene-acetylated poly(allylamine). Biotechnol Bioeng. 2000, 70: 479-83, Chang TM and Prakash S. Procedures for microencapsulation of enzymes, cells and genetically engineered microorganisms. MoI Biotechnol. 2001, 17: 249-60, and Lu MZ, et al., A novel cell encapsulation method using photosensitive poly(allylamine alpha- cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules may be prepared by complexing modified collagen with a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA), methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in a capsule thickness of 2-5 μm. Such microcapsules can be further encapsulated with additional 2-5 μm ter-polymer shells in order to impart a negatively charged smooth surface and to minimize plasma protein absorption (Chia, S.M. et al. Multi-layered microcapsules for cell encapsulation

Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide (Sambanis, A. Encapsulated islets in diabetes treatment. Diabetes Thechnol. Ther. 2003, 5: 665-8) or its derivatives. For example, microcapsules can be prepared by the polyelectrolyte complexation between the polyanions sodium alginate and sodium cellulose sulphate with the polycation poly(methylene-co-guanidine) hydrochloride in the presence of calcium chloride.

It will be appreciated that cell encapsulation is improved when smaller capsules are used. Thus, the quality control, mechanical stability, diffusion properties, and in vitro activities of encapsulated cells improved when the capsule size was reduced from 1 mm to

400 μm (Canaple L. et al., Improving cell encapsulation through size control. J Biomater

Sci Polym Ed. 2002; 13:783-96). Moreover, nanoporous biocapsules with well-controlled pore size as small as 7 nm, tailored surface chemistries and precise microarchitectures were found to successfully immunoisolate microenvironments for cells (Williams D. Small is beautiful: microparticle and nanoparticle technology in medical devices. Med Device

Technol. 1999, 10: 6-9; Desai, T. A. Micro fabrication technology for pancreatic cell encapsulation. Expert Opin Biol Ther. 2002, 2: 633-46).

The three dimensional biocompatible scaffolds comprising the stromal cells and HSCs scaffold of the present invention may be implanted to a subject per se, or it may be mixed with suitable carriers or excipients.

Hereinafter, the term "carrier" refers to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the scaffold. Exemplary carriers include Hank's solution, Ringer's solution, or physiological salt buffer.

Typically a therapeutically effective amount of the biocompatible scaffolds comprising the stromal cells and HSCs are administered to the subject - i.e. an amount of cells effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., bone marrow disorder) or to regenerate bone marrow in the subject being treated. Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated from animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in experimental animals. The data obtained from these animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 p.l). Dosage amount and interval may be adjusted individually to provide cell numbers sufficient to induce bone marrow regeneration. The minimal effective concentration (MEC) will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations. The amount of biocompatible scaffolds comprising the stromal cells and HSCs to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

Efficiency of treatment, e.g. bone marrow regeneration, may be determined using any known animal model (e.g. murine model) in need of bone marrow reconstitution, such as SCID/NOD mice or Brown Norway rat acute myelocytic leukemia model (BNML). Such animal models are well known to one of ordinary skill in the art. Following transplantation under the kidney capsule of the biocompatible scaffolds comprising the stromal cells and HSCs of the present invention, the animals are monitored for generation of newly transplanted bone marrow using any method known in the art [e.g. short tandem repeat (STR) assay, HLA assays, FISH, etc.].

Compositions of the present invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agency of the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as if further detailed above.

It is expected that during the life of a patent maturing from this application many relevant biocompatible scaffolds will be developed and the scope of the term biocompatible scaffolds is intended to include all such new technologies a priori. As used herein the term "about" refers to ± 10 %. The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to".

The term "consisting of means "including and limited to".

The term "consisting essentially of means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof. Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from

1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween. As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

As used herein, the term "treating" includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition. It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

EXAMPLES

Reference is now made to the following examples, which together with the above descriptions, illustrate the invention in a non limiting fashion.

Generally, the nomenclature used herein and the laboratory procedures utilized in the present invention include molecular, biochemical, microbiological and recombinant DNA techniques. Such techniques are thoroughly explained in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al., (1989); "Current Protocols in Molecular Biology" Volumes I-III Ausubel, R. M., ed. (1994); Ausubel et al., "Current Protocols in Molecular Biology", John Wiley and Sons, Baltimore, Maryland (1989); Perbal, "A Practical Guide to Molecular Cloning", John Wiley & Sons, New York (1988); Watson et al., "Recombinant DNA", Scientific American Books, New York; Birren et al. (eds) "Genome Analysis: A Laboratory Manual Series", VoIs. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); methodologies as set forth in U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", Volumes I-III Cellis, J. E., ed. (1994); "Current Protocols in Immunology" Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), "Basic and Clinical Immunology" (8th Edition), Appleton & Lange, Norwalk, CT (1994); Mishell and Shiigi (eds), "Selected Methods in Cellular Immunology", W. H. Freeman and Co., New York (1980); available immunoassays are extensively described in the patent and scientific literature, see, for example, U.S. Pat. Nos. 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521; "Oligonucleotide Synthesis" Gait, M. J., ed. (1984); "Nucleic Acid Hybridization" Hames, B. D., and Higgins S. J., eds. (1985); "Transcription and Translation" Hames, B. D., and Higgins S. J., Eds. (1984); "Animal Cell Culture" Freshney, R. L, ed. (1986); "Immobilized Cells and Enzymes" IRL Press, (1986); "A Practical Guide to Molecular Cloning" Perbal, B., (1984) and "Methods in Enzymology" Vol. 1-317, Academic Press; "PCR Protocols: A Guide To Methods And Applications", Academic Press, San Diego, CA (1990); Marshak et al., "Strategies for Protein Purification and Characterization - A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference as if fully set forth herein. Other general references are provided throughout this document. The procedures therein are believed to be well known in the art and are provided for the convenience of the reader. All the information contained therein is incorporated herein by reference. GENERAL MATERIALS AND METHODS

Bioreactor

The bioreactor used in accordance with the teachings of the present invention was constructed in accordance with the design described in Figure 1. The glassware was designed and manufactured at the Technion (Israel) and connected by silicone tubing (Degania, Israel). The carriers were rotated overnight in phosphate buffered saline (PBS; Beit Ha'Emek Industries, Israel) without Ca⁺² and Mg⁺², followed by removal of the PBS and released debris. Each column was loaded with 10 ml packed carrier. The bioreactor was filled with PBS-Ca-Mg, all outlets were sealed and the system was autoclaved (120 °C, 30 minutes). The PBS was removed via container [depicted by the no. 8] and the bioreactor was circulated in a 37 ⁰C incubator with 300 ml Dulbecco's high-glucose medium (DMEM; GIBCO BRL) containing 10 % heat-inactivated fetal calf serum (FCS; Beit Ha'Emek Industries, Israel) and a Pen-Strep-Nystatin mixture (100 U/ml:100 μg/ml:1.25 μn/ml; Beit Ha'Emek), for a period of 48 hours. Circulating medium was replaced with fresh DMEM containing the above +2 mM L-glutamine (Beit Ha'Emek). Stromal cells

Stromal cell lines, i.e. AFT024 murine fetal liver cell lines (ATCC #SCRC), were maintained in DMEM supplemented with 10 % FCS and were incubated at 37 °C in 5 % CO2. Cells were grown in tissue culture flasks (Coming) and were split by trypsinization upon reaching confluence.

Primary human marrow stromal cultures were established from aspirated sternal marrow of hematologically healthy donors undergoing open-heart surgery. Briefly, marrow aspirates were diluted 3-fold in Hank's Balanced Salts Solution (HBSS; GIBCO BRL) and were subject to Ficoll-Hypaque (Robbins Scientific Corp. Sunnyvale, Calif.) density gradient centrifugation. Marrow mononuclear cells (<1.077 gm/cm³) were collected, washed 3 times in HBSS and resuspended in long-term culture (LTC) medium, consisting of DMEM supplemented with 12.5 % FCS, 12.5 % horse serum (Beit Ha'Emek), 10^"4 M β-mercaptoethanol (Merck) and 10^"6 mol/L hydrocortwasone sodium succinate (Sigma). Cells were incubated in 25 ml tissue culture flasks (Coming) for 3 days at 37 °C (5 % CO2) and then at 33 °C (idem) with weekly culture refeeding. Stromal cells from individual donors were employed for each bioreactor. For 3D and monolayer studies, primary stromal cell cultures were split by trypsinization (0.25 % Trypsin and EDTA in Puck's Saline A; Beit Ha'Emek) every 10 days, to allow sufficient stromal cell expansion. Seeding of stromal cells

Confluent cultures of stromal cell lines or 5-week primary marrow stromal cells were trypsinized and the cells were washed 3 times in HBSS, resuspended in bioreactor medium (see above), counted and seeded at 10⁶ cells/ml in 10 ml volumes via an injection point (depicted by the no. 4, Figure 1) onto 10 ml carriers in the glass column of the bioreactor. Immediately following seeding, circulation was stopped for 16 hours to allow the cells to settle on the carriers. Stromal cell growth in the bioreactor was monitored by removal of carriers and cell enumeration by the MT method [Hansen et al., J Immunol

Methods (1989) 119:203]. When stromal cells were confluent, medium was replaced with LTC medium.

Preparation of stromal cell conditioned medium (SCM)

At equivalent cell densities, monolayer and bioreactor stromal cells were recharged with fresh LTC culture medium. Stromal conditioned media (SCM) was collected following overnight incubation of the cells. For this purpose, medium flow in the 3D cultures was stopped for 16 hours and removed directly from the column prior to reinitiation of circulation. Stromal cells were also grown in the bioreactor in serum-free medium.

Isolation ofCD34+ cells

Umbilical cord blood (CB) samples were taken under sterile conditions during delivery and were fractionated on Ficoll-Hypaque and buoyant (< 1.077 gr/cm³) mononuclear cells were collected. Cells from individual CB samples were pooled, incubated with anti-CD34 antibodies and isolated by midi MACS (Miltenyl Biotech). Stromal-stem cell co-cultures

Isolated, pooled CB CD34+ cells were seeded at equivalent numbers (about 5 x 10⁵) onto monolayer or bioreactor containing equivalent densities of confluent stromal cells. Upon addition to the bioreactor, medium flow was stopped for 16 hours to enable contact with stromal cells and was re-initiated at a rate of 0.1 -1.0 ml per minute. CD34+ cell seeded-stromal cell carriers were removed for control studies in the absence of medium exchange. Co-cultures were maintained in LTC medium without cytokines. At various times (up to 4 weeks), non-adherent cells were collected from monolayer supernatants or from circulating culture medium via a container (depicted by the no. 8, Figure 1). Adherent cells were collected via sequential trypsinization and exposure to EDTA-based dissociation buffer (GIBCO BRL), followed by gentle pipetting of the cells. To avoid the presence of stromal cells in the resulting suspension, the cells were re- suspended in HBSS + 10 % FCS and were subjected to a 60 minutes adhesion procedure in plastic tissue culture dishes (Corning), at 37 °C. Circulating and carrier-isolated hematopoietic cells were washed, counted and assayed separately for CD34+/38- /CXCR4+ by flow cytometry (FACS, see details below). Flow Cytometry

Cells were incubated at 4 ⁰C for 30 minutes with saturating concentrations of monoclonal anti-CD34+PerCP (Beckton-Dickinson), anti-CXCR4-fluorescein isothiocyanate (FITC, R&D systems) and phycoerythrin (PE, Beckton-Dickinson) antibodies. The cells were washed twice in ice-cold PBS containing 5 % heat-inactivated FCS and resuspended for three-color flow cytometry on a FACSscan (Beckton- Dickinson).

EXPERIMENTAL RESULTS

The bioreactor system employed by the present invention (illustrated in detail in Figure 1) was previously described in WO00/46349 (fully incorporated herein by reference). In short, the bioreactor system contained four parallel plug flow bioreactor units [depicted by the no. 5]. Each bioreactor unit contained 1 gram of porrosive carriers (4 mm in diameter) which enabled propagation of large cell numbers in a relatively small volume (described in further detail hereinbelow). The bioreactor was maintained in an incubator of 37 °C. The flow in each bioreactor was monitored [depicted by the no. 6] and regulated by a valve [depicted by the no. 6a]. Each bioreactor contained a sampling and injection point [depicted by the no. 4], allowing the sequential seeding of stromal and hematopoietic cells. Culture medium was supplied at pH 7.0 from a reservoir [depicted by the no. I]. The reservoir was supplied by a filtered [depicted by the no. 3] gas mixture containing air/CO2 02 [depicted by the no. 2] at differing proportions in order to maintain 5 - 40 % dissolved oxygen at exit from the column, depending on cell density in the bioreactor. The 02 proportion was suited to the level of dissolved 02 at the bioreactor exit, as was determined by a monitor [depicted by the no. 12]. The gas mixture was supplied to the reservoir via silicone tubes. The culture medium was passed through a separating container [depicted by the no. 7] which enabled collection of circulating, non-adherent cells. Circulation of the medium was obtained by means of a peristaltic pump [depicted by the no. 9] operating at a rate of 0.1-3 ml/minute. The bioreactor units were equipped with an additional sampling point [depicted by the no. 10] and two containers [depicted by the nos. 8, 11] for continuous medium exchange at a rate of 10-50 ml/day. The use of four parallel bioreactor units enabled periodic dismantling for purposes such as cell removal, scanning electron microscopy, histology, immunohistochemistry, RNA extraction, etc.

To start the cell cultivation process, stromal cells derived from AFT024 murine fetal liver cell lines were seeded onto a porrosive carrier comprising unvarnished, non- biodegradable polystyrene 3-D scaffolds (FibraCel, Bibby-Sterilin UK) and were placed in a flow-through bioreactor to form a cell-matrix unit. Following the establishment of a spatially organized culture, HSCs from umbilical cord blood (CB), were plated onto the stromal cells to form a complete unit that was cultivated for an additional two weeks in the bioreactor. The cultivation and expansion process was executed in a unique environment that was devoid of supplemented cytokines thereby avoiding unwanted effects such as the risk of teratoma formation and reduced rate of engraftment. As a control, the stromal cells and HSCs were cultured on plastic culture dishes (2D) or on non-biodegradable polystyrene 3-D scaffolds (FibraCel, Bibby-Sterilin UK) cultured in 24-well static dishes (3D). Using standard FACS analyses to evaluate the HSC output of the system (compared to the control systems), the competitive advantage of the present invention . over the conventional cultivation methods was fully recognized (Figures 2A-C). As is illustrated in Figures 2A- C, preferred expansion of HSC defined as CD45+CD34+CD38- expressing cells (Figure 2B, bioreactor) and CD45+CD34+CD38-CXCR4+ expressing cells (Figure 2C, bioreactor) over the more differentiated hematopoietic CD34+ expressing cells (Figure 2A, bioreactor) is apparent using the cultivation and expansion process of the present invention utilizing the bioreactor. Moreover, specific expansion of these cells couldn't be observed using the conventional cell cultivation conditions (2D and 3D, Figures 2 A-C).

The growth of stromal cells (AFT024 murine fetal liver cells) on biodegradable scaffolds compared to non-biodegradable scaffolds was carried out next. PCL [poly(capro lactone)] porrosive sheets (4 mm x 4 mm) were loaded with 1 x 10⁵ cells and cultured for 21 days in DMEM medium fortified with 10 % FCS, 2 mM glutamine and antibiotics. Three types of PCL porrosive sheets encompassing different width were used (PCLl, PCL2 and PCL3 of 0.25 mm, 0.35 mm and 0.55 mm width respectively). For comparison, the non-biodegradable carrier polystyrene was employed. As is illustrated in Figure 3, stromal cells exhibited similar growth rates on biodegradable PCL scaffolds and on non-biodegradable scaffolds.

The cultivation process initially required a period of 2-10 weeks for the establishment of spatially organized stromal cells on the 3D-scaffolds. As described in detail above, the HSCs were then plated onto the stromal cells for an additional two weeks. The ratio of stromal to HSCs was 8.5:1 at time HSCs were plated onto the scaffolds. As is illustrated in Figure 4, the expansion rate of hematopoietic cells (defined as CD45+CD34+CD38-) was very significant and the final ratio of HSC/stromal at the end of culture was nearly 1.

Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

All publications, patents and patent applications mentioned in this specification are herein incorporated in their entirety by reference into the specification, to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting.

Claims

WHAT IS CLAIMED IS:

1. A method of transplanting undifferentiated hematopoietic stem cells into a recipient, the method comprising the steps of:

(a) expanding the undifferentiated hematopoietic stem cells to increase the number of the hematopoietic stem cells by:

(i) culturing in a stationary phase plug-flow bioreactor stromal cells under continuous flow of a culture medium on a three dimensional biocompatible substrate to thereby generate a three dimensional stromal cell culture; and

(ii) seeding undifferentiated hematopoietic stem cells into said stationary phase plug-flow bioreactor including said three dimensional stromal cell culture and under a continuous flow of a culture medium, thereby expanding the undifferentiated hematopoietic stem cells and obtaining a three dimensional stromal cell culture comprising increased number of hematopoietic stem cells; and

(b) transplanting said three dimensional stromal cell culture comprising hematopoietic stem cells resulting from step (a) into the recipient.

2. An article of manufacture comprising a three dimensional biodegradable substrate comprising at least 1 x 10⁷ stromal cells per ml and undifferentiated hematopoietic stem cells.

3. The method or article of manufacture of claim 1 or 2, wherein said undifferentiated hematopoietic stem cells comprise cells isolated from a tissue selected from the group consisting of cord blood, mobilized peripheral blood and bone-marrow.

4. The method or article of manufacture of claim 1 or 2, wherein said stromal cells being derived from a tissue selected from the group consisting of a bone marrow, a placenta and an adipose tissue.

5. The method or article of manufacture of claim 1 or 2, wherein said stromal cells and said undifferentiated hematopoietic stem cells share common HLA antigens.

6. The method or article of manufacture of claim 1 or 2, wherein said stromal cells and said undifferentiated hematopoietic stem cells are from a single individual.

7. The method or article of manufacture of claim 1 or 2, wherein said stromal cells and said undifferentiated hematopoietic stem cells are from different individuals.

8. The method or article of manufacture of claim 1 or 2, wherein said stromal cells and said undifferentiated hematopoietic stem cells are from the same species.

9. The method or article of manufacture of claim 1 or 2, wherein said stromal cells and said undifferentiated hematopoietic stem cells are from different species.

10. The method of claim 1, wherein said biocompatible substrate comprises a porrosive carrier.

11. The method of claim 10, wherein said porrosive carrier comprises a porrosive sheet.

12. The method of claim 11, wherein said porrosive sheet comprises a width of about 200-600 μm.

13. The method of claim 1, wherein said biocompatible substrate comprises a polymeric substrate.

14. The method of claim 13, wherein said polymeric substrate comprises poly(caprolactone) (PCL).

15. The method of claim 13, wherein said polymeric substrate is selected from the group consisting of polylactic acid (PLA), poly-L-lactic acid (PLLA), poly-D-lactic acid (PDLA), polyglycolide, polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polydioxanone, polygluconate, polylactic acid-polyethylene oxide copolymers, modified cellulose, collagen, polyhydroxybutyrate, polyhydroxpriopionic acid, polyphosphoester, poly(alpha-hydroxy acid), polycarbonates, polyamides, polyanhydrides, polyamino acids, polyorthoesters, polyacetals, polycyanoacrylates, degradable urethanes, aliphatic polyester polyacrylates, polymethacrylate, acyl substituted cellulose acetates, non-degradable polyurethanes, polystyrenes, polyvinyl chloride, polyvinyl flouride, polyvinyl imidazole, chlorosulphonated polyolifins, polyethylene oxide, polyvinyl alcohol, teflon RTM, nylon silicon, poly(styrene-block-butadiene), polynorbornene, hydrogels, metallic alloys and oligo(ε-caprolactone)diol.

16. The method of claim 1, wherein said biocompatible substrate comprises a synthetic polymer.

17. The method of claim 1, wherein said biocompatible substrate comprises a natural polymer.

18. The article of manufacture of claim 2, wherein said biodegradable substrate is selected from the group consisting of poly(caprolactone) (PCL), polyglycolic acid, poly (DL-lactic-co-glycolic acid), cat gut sutures, cotton, cellulose, gelatin, dextran, alginate, fibronectin, laminin, collagen, hyaluronic acid, polyhydroxyalkanoate, poly 4 hydroxybutirate (P4HB) and polygluconic acid (PGA).

19. The method of claim 1, wherein said step of said seeding said undifferentiated hematopoietic stem cells into said stationary phase plug-flow bioreactor is effected while flow in said bioreactor is shut off for at least 10 hours following said seeding.

20. The method of claim 1, wherein said stromal cells are grown to a density of at least 1 x 10⁷ stromal cells per ml.

21. The method of claim 1, wherein said culture medium is devoid of supplementary cytokines.

22. The method of claim 1, wherein said expanding said undifferentiated hematopoietic stem cells results in CD45+CD34+CD38- expressing cells.

23. The method of claim 1, wherein said expanding said undifferentiated hematopoietic stem cells results in CD45+CD34+CD38-CXCR4+ expressing cells.