WO2018051340A1

WO2018051340A1 - A process for retrieval of matrix dependent mesenchymal stromal cells from tissue

Info

Publication number: WO2018051340A1
Application number: PCT/IL2017/051034
Authority: WO
Inventors: Raphael Gorodetsky; Tamar PERETZ-YABLONSKI; Boaz ADANI; Astar LAZMI-HAILU; Maamoun BASHEER
Original assignee: Hadasit Medical Research Services & Development Ltd
Priority date: 2016-09-13
Filing date: 2017-09-13
Publication date: 2018-03-22

Abstract

A method for preparing a population of matrix dependent placental mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin, and preferably of fetal origin, is provided, as well as the placental mesenchymal stromal cells and their use in promoting a regenerative process in damaged tissue or reducing an inflammatory response associated with disease or injury and for use in mitigation of acute radiation syndrome (ARS) following exposure to high dose ionizing radiation and/or treating failing bone marrow. A composition for cryo-preservation of biological tissue is also provided.

Description

A PROCESS FOR RETRIEVAL OF MATRIX DEPENDENT

MESENCHYMAL STROMAL CELLS FROM TISSUE

FIELD OF THE INVENTION

The present invention relates in general to methods for efficient isolation of matrix - dependent cells from the placenta and possibly from other sources and their use in cell therapy applications.

BACKGROUND OF THE INVENTION

Isolated matrix-dependent cells are used for research or cell therapy applications such as enhanced regeneration of the hematopoietic system, mitigation of radiation effects and improvement of healing in models of neurodegenerative diseases.

Current methods for isolation of matrix-dependent mesenchymal stromal cells are based on full enzymatic digestion of the relevant tissue and expansion of the cells in bioreactors in 3D conditions provide relatively low yield. Mesenchymal stromal cells have also been isolated by various other researchers from terminal chorionic villi of human term placenta

Mesenchymal stem cells and stromal cells have been used in regenerative medicine, for example in mitigation of lethal radiation syndrome, ischemia induced limb injury, graft- versus-host disease (GVHD), transplanted organ rejection and autoimmune diseases⁽²⁾, ischemic brain injury⁽³⁾, critical limb ischemia⁴' ⁵⁾, myocardial infarction⁽⁶' ⁷⁾, IBD⁽⁸⁾ and diabetes mellitus type I or II ⁽⁹' ¹⁰' ⁵⁾. However, the low yield of current methods for isolation of matrix-dependent cells, and the low fraction of matrix-dependent cells of fetal origin among these cells, prevents development of cell-based treatment of these diseases WO 2012/140519; US 9,393,273; US 8,524,496; US20160271184; US20150216907; US 9,517,248).

Thus, there remains a considerable need for efficient methods for retrieval of fetal- origin matrix-dependent cells that provide higher yields and, therefore, enables to select the specific desired cells from a small volume of an adequate tissue.

SUMMARY OF INVENTION

In one aspect, the present invention is directed to a method for preparing a population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin, the method comprising (i) obtaining a tissue comprising matrix dependent mesenchymal stromal cells; (ii) optionally briefly contacting fragments of said tissue with a solution comprising a protease; (iii) optionally keeping the fragments for 48 hrs or more in motion, e.g. rotation, while suspended in growth medium at 37°C and an atmosphere with about 5% C0₂; (iv) contacting the fragments of the tissue with a solution comprising fibrinogen, evacuating the excess fibrinogen solution; (v) transferring the fragments to an unmodified/uncoated plastic surface; (vi) dispersing, distributing or spraying a solution comprising thrombin on said plastic surface and fragments thereby forming a thin fibrin film around the fragments that immobilizes the fragments to the plastic surface; (vii) covering the fragments with growth medium; (viii) harvesting matrix dependent mesenchymal stromal cells that have migrated out from the fragments, thereby preparing a population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin.

In another aspect, the present invention provides a population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin obtained by the method of the invention.

In another aspect the present invention provides a population of matrix dependent mesenchymal stromal derived solely from the fetal or the maternal tissue origin of the placenta as dictated by the tissue layers dissected from the whole donated placenta.

In an additional aspect, the present invention provides a pharmaceutical composition comprising the population of matrix dependent placental mesenchymal stromal cells of the present invention as defined above and a pharmaceutically acceptable carrier.

In a further aspect, the present invention provides a method for promoting a regenerative process in damaged tissue or organ or reducing an inflammatory response associated with disease or injury, the method comprising administering to a subject in need thereof a therapeutically effective amount of the population of matrix dependent placental mesenchymal stromal cells of the present invention or the pharmaceutical composition of the present invention.

In still another aspect, the present invention provides a population of matrix dependent placental mesenchymal stromal cells or pharmaceutical composition of the present invention for use in promoting a regenerative process in damaged tissue or organ or reducing an inflammatory response associated with disease or injury. In yet another aspect, the present invention provides a method for mitigation of acute radiation syndrome (ARS) following exposure to high dose ionizing radiation and/or treating failing bone marrow comprising administering to a subject in need thereof a therapeutically effective amount of the population of matrix dependent placental mesenchymal stromal cells.

In a further aspect, the present invention provides a population of matrix dependent placental mesenchymal stromal cells or pharmaceutical composition of the present invention for use in mitigation of acute radiation syndrome (ARS) following exposure to high dose ionizing radiation and/or treating failing bone marrow.

In yet another aspect, the present invention provides a population of matrix dependent mesenchymal stromal cells for the use in regeneration of damaged pancreas or damaged hair follicles.

In still another aspect, the present invention provides a method for regeneration of damaged pancreas or damaged hair follicles, the method comprising administering to a subject in need thereof a therapeutically effective amount of a population of matrix dependent mesenchymal stromal cells.

In still an additional aspect, the present invention is directed to a composition for cryo-preservation of biological tissue, said composition comprising fetal calf serum (FCS; e.g. 50-90%) or human plasma albumin (HAS; e.g. 5-50%) in addition to dimethyl sulfoxide (DMSO) and optionally polyethylene glycol or cell culture medium. In particular the composition for cryo-preservation comprises (i) 90% fetal calf serum (FCS) + 5% dimethyl sulfoxide (DMSO) + 5% polyethylene glycol (PEG); (ii) 90% FCS+10% DMSO; (iii) 10- 20% human plasma albumin (HSA) + 10% DMSO + 70-80% cell culture medium; or (iv) 10-20% HSA + 5% DMSO + 5% PEG + 70-80% cell culture medium.

In yet an additional aspect, the present invention provides a method for preserving biological tissue such as normal tissue or tumor tissue, comprising immersing said biological tissue in a composition for cryo-preservation of biological tissue of the present invention, and slowly reducing the temperature to -75°C to -80°C, thereby providing frozen biological tissue and storing the frozen biological tissue in liquid nitrogen.

BRIEF DESCRIPTION OF DRAWINGS

Fig. 1 illustrates normal full term placenta anatomy. A cross-section through a full term, normal placenta, exposing its different layers. The white dashed lines represent the area of interest, spread around the umbilical cord, for placental matrix dependent stromal cells (PSC) extraction.

Fig. 2 shows a flowchart of the cell migration isolation method leading to PSC expansion.

Fig. 3 shows micrographs depicting initial attachment of PSC on plastics after proteolytic digestion of the placental tissue using a conventional tissue digestion protocol. The yield is poor and the expansion is slow.

Fig. 4 shows micrographs depicting migration at different time points using the method of the invention. The population looks homogenous and the yield of migrated hPSC is high. Arrows indicate the migrating PSC from the plated placental tissue

Fig. 5 shows a bar-graph comparing the accumulative preliminary yield at passage 2 (before further expansion) for 1cm of tissue by the two different isolation procedures used (the known conventional method (enzymatic digestion) and the method of the invention (spontaneous migration), respectively).

Fig. 6 depicts an X & Y Centromere assay by fluorescence in-situ hybridization

(FISH). A sample of 200-300 cells from each batch stained for Y and X chromosomes by FISH was assayed to verify their fetal origin. Each cell contained a Y in one color (shown by arrow) and X chromosome (in a different color) validating their newborn placental tissue origin.

Fig. 7 shows a Fluorescence Activated Cells Sorter (FACS) histograms identifying the cells as mesenchymal. The PSC were found to be negatively stained for hematopoietic cell surface markers, (CD45, CD19, CDl lb, HLA-DR) and endothelial cells markers (HLA- G and CD34) and positive for typical mesenchymal cell surface markers (C105, CD90 and CD29 as well as CD146 and CD166).

Fig. 8 shows a flowchart of an experiment testing efficacy of treatment of lethal acute radiation syndrome (ARS) in a C3H/HENHSD/female mouse model. Mice were irradiated on day 0 and received PSC delivery on days 1 and 4 post irradiation. The delivery of the PSC was different (IM; SC). On day 23 post irradiation, when no further mortality is expected, the surviving mice were sacrificed and various samples were taken for further analysis.

Fig. 9 is a survival chart showing that both intramuscular (IM; diamonds) and subcutaneous (SC; squares) cell delivery were found to significantly improve the survival rate of irradiated mice as compared to the untreated group (triangles; from -20% to -85%). A delay in the initiation of the treatment by 48hrs yielded a similar effect (data not shown).

Fig. 10 shows a line graph of mice' weight over time. The mice were weighted on a daily basis following irradiation. The weight drop is visible after day 8. Only the PSC treated groups (IM, diamonds; SC, squares) recovered to gain their initial weight by the end of the experiment.

Figs. 11A-D show bar graphs of complete blood count taken on peripheral blood comparing the different arms of the experiment. A, red blood cells (RBC); B, white blood cells (WBC); C, hemoglobin (HBG); D, plateles (PLT). IM, intramuscular injection of the cells; SC, subcutaneous injection of the cells. * P<0.05; ** P<0.001; *** P<0.0001

Fig. 12 is a survival chart showing the short term (20 days) survival rate of the preliminary follow up of a group that underwent splenectomy (SPL-) prior to irradiation and IM PSC injection (crosses) was not significantly different from treated non-splenectomized group (diamonds).

Fig. 13 shows a line graph of weight follow-up of the mice on a daily basis after irradiation. A weight drop is evident after day 8. The splenectomized arm (circles) showed weight recovery tendency which was similar to the IM treated non splenectomized mice (diamonds). The mice in this group were sacrificed earlier after their initial recovery due to technical schedule constraints.

Figs. 14A-D show bar graphs of complete peripheral blood counts, comparing splenectomized and IM hPSC treated mice (IM (Spl-)) with non splenectomized mice and IM administration of placebo. The lower value of RDW in the splenectomized arm of IM PSC treated mice may indicate a lower rate of erythropoiesis relative to non splenectomized PSC treated mice. A, red blood cells (RBC); B, white blood cells (WBC); C, hemoglobin (HBG); D, plateles (PLT). * P<0.05; ** P<0.001; *** P<0.0001

Figs. 15A-C show micrographs of liver samples processed for cryo-section and stained for Terl 19(Cy-3) and DAPI.

Figs. 16A-B show a Fluorescence Activated Cells Sorter (FACS) complete erythropoietic population estimation in BM (4 bones times 4 for whole body BM) and spleen in the radiated and naive mice. Sorting order of erythropoiesis: Lin(-) — » TER119+— » CD71+→Size; Small=fully differentiated (ERY C). Med= partially differentiated (ERY B), Large= undifferentiated (ERY A). A, Dot blot showing the ERY A, ERY B and ERY C cell populations; B, Bar-graph quantifying the data displayed in A. In each arm, ERY A, left-most bar; ERY B, middle bar; ERY C, right-most bar. IM, non splenectomized and IM hPSC treated mice; SC, non splenectomized and SC hPSC treated mice; IM(Spl-), splenectomized and IM hPSC treated mice.

Figs. 17A-B shows bar-graphs of complete erythropoietic population estimation in

BM (4 bones times 5 for whole body BM) in the irradiated and naive mice. A, ERY A population in BM; B, ERY B population in BM; IM, non splenectomized and IM hPSC treated mice; SC, non splenectomized and SC hPSC treated mice; IM(Spl-), splenectomized and IM hPSC treated mice. * P<0.05; ** P<0.001; *** P<0.0001

Fig. 18 shows a bar-graph of complete erythropoietic population identification in spleen in the irradiated and naive mice. In each arm, ERY A, left-most bar; ERY B, middle bar; ERY C, right-most bar. * P<0.05; ** P<0.001; *** P<0.0001

Fig. 19 shows a bar-graph of erythropoietic cell populations both in spleen (SPL) and bone marrow (BM) in naive (left bar) and IM (right bar) treated mice (*P<0.05; ** P<0.001; *** P<0.0001)

Figs. 20A-B show bar- graphs of counting of nucleated population in both BM (A) and spleen (B) in different arms of the experiment. BM cellularity in splenectomized mice was significantly higher compared to non splenectomized mice, possibly due to partial compensation of the absence of the spleen. IM, non splenectomized and IM hPSC treated mice; SC, non splenectomized and SC hPSC treated mice; IM(Spl-), splenectomized and IM hPSC treated mice. (*P<0.05; ** P<0.001; *** P<0.0001).

Fig. 21 shows a FACS dot plot of gating of hematopoietic stem cells (HSCs) population through lineage exclusion and double positive staining for Ckit and Sca-1 markers.

Fig. 22 shows a bar-graph of HSCs frequency both in BM (left bar) and spleen (right bar) in mice that underwent different treatments. The splenectomized arm has a significantly higher frequency of HSC compared to other arms which didn't undergo the surgery, indicating again the possibility to an attempt to compensate the absence of the spleen by the BM. (* P<0.05; ** P<0.001; *** P<0.0001).

Fig. 23 depicts a bar-graph of absolute HSCs counts in BM (left bar) vs spleen (right bar) in different arms of experiment. IM, non splenectomized and IM hPSC treated mice; SC, non splenectomized and SC hPSC treated mice; IM(Spl-), splenectomized and IM hPSC treated mice. * P<0.05; ** P<0.001;*** P<0.0001

Fig. 24 depicts a bar-graph of erythropoietin (EPO) secretion on day 8 post irradiation. Secretion Mouse EPO measured in blood plasma on day 8 following irradiation. Due to measurement saturation several sample dilution (1:2, 1:5) were tested to allow monitoring the full range of proteins concentrations. (* P<0.05; ** P<0.001; *** P<0.0001).

Fig. 25 shows a bar-graph of mouse VEGF levels measured in blood plasma on day 8 following irradiation. * P<0.05; *** P<0.0001

Fig. 26 shows photographs of bone histology comparing different arms of the experiment. Top panel, Naive; second from top, irradiated with IM PSC delivery; third from top, irradiated with IM PSC and SPL(-); bottom, irradiated with placebo; Right panels, white dashed squares represent the magnified area for detailed view of the BM.

Figs. 27A-D show micrographs of spleen histology comparing different arms of the experiment. Black arrows indicate megakaryocytes as indication for the presence of extra- medullary hematopoiesis (EMH) foci. A, irradiated with IM PSC delivery; B irradiated with IM PSC delivery; C, Naive; D, irradiated with placebo.

Fig. 28 shows the follow-up of mice' weight following 16Gy of head only irradiation by a linear accelerator (LINAC). Diamonds, 16 Gy irradiation + administration of PSCs; squares, 16 Gy irradiation; triangles, administration of PSCs only. Arrow, time wet food was provided.

Fig. 29 shows photographs of visually evident effects of subcutaneous PSC treatment on the regeneration of hair of mice at 2 time points. Upper row, 16 Gy irradiation + administration of PSCs; middle row, 16 Gy irradiation; lower row, administration of PSCs only.

Fig. 30 shows skin hair regeneration following 16Gy high dose irradiation of the head only on day 74 post irradiation. A, typical change in hair follicle density as recorded in histological sections on day 74; B, quantification of density of follicles as observed in A.

Fig. 31. Isolated effect of the PSC treatment on the skin only. Mice were irradiated by 18Gy with low penetrating irradiation by electrons, exposing only the back skin area. A, irradiation set-up; B,_skin surface follow-up, with selected representative mice, showing the difference of skin regeneration between the PSC treated (upper row) and non-treated (lower row) following 18Gy irradiation.

Figs. 32 A-F show migration of cells out of tissue pieces that have been frozen and thawed. (A) Day 18 post thawing and direct plating. (B) Day 18 post thawing and held in suspension (48 hrs) and then plating. (C) Tissue frozen in cryo-RG and analyzed on Day 11 post thawing and direct plating. (D) Tissue frozen in cryo-RG and analyzed on Day 18 post thawing and direct plating. (E) Tissue frozen in cryo-RG and analyzed on Day 11 post thawing, held in suspension 48hrs and direct plating. (F) Tissue frozen in cryo-RG and analyzed on Day 18 post thawing, held in suspension 48hrs and direct plating.

Figs. 33A-H show the effect of intracranial implantation of hPSC to EAE mice. hPSC were implanted in the periventricular white matter of mice on day 7 after EAE induction, prior to the development of clinical signs (n=12 mice). Significant (p<0.001) disease attenuation was observed in the clinical course of hPSC -implanted mice as compared to a control EAE group (n=ll, A). hPSC were detected 18 days after their implantation by HLA-ABC immunostaining of the mouse brain sections (B). EAE mice exhibited a typical inflammatory response in their brains, on day 25 post-EAE induction (C). A strong inflammatory response was noted in the hPSC transplanted mice in proximity to implantation site and in the brain meninges (D, E). Both meningeal and parenchymal spinal cord infiltrates were detected in the EAE mice (F, G) while hPSC implanted animals exhibited mostly meningeal infiltrates (H).

Fig. 34 shows the effect of collagenase treatment of fresh and thawed tissue. It can clearly be seen that collagenase treatment (0.75mg/ml) dramatically increases the number of cells migrating out of the source tissue, but not from fresh tissue. DETAILED DESCRIPTION OF THE INVENTION

Introduction

1. Embryonic stem cells and normal tissue development.

1.1 Cells sternness and commitment

When the ovum is fertilized and becomes a zygote it has the potential to differentiate into any kind of cell in the future organism to form, this potential of differentiation is called totipotent potency and has no commitment for any kind of tissue yet. Following the embryogenesis and the formation of blastocyst the cells become already committed to differentiate to the different germ lines. The inner cell mass (ECM) is the origin of the three germinal layers - endoderm, ectoderm and mesoderm in embryonic development. The ECM is also known as the origin on embryonic stem cells (ESC). Cells that can differentiate to different phenotypes and proliferate in high rate are called stem cells (SC). The degree of their sternness is associated with the spectrum of phenotypes into which the cells can differentiate. In normal process of embryonic development cells are specialized to form the different tissues types and organs in non-reversible manner. Most cells in fully formed adult organisms do not proliferate, with an exception for germ cells and a very small limited population of self-renewing tissue specific cells, such as hematopoietic stem cells, muscle progenitors and progenitors of intestinal crypts. The role of these cells is to sustain the tissue function and regenerate in case of injury. Tissues with high turnover of cells tend to contain a larger reservoir of SC or progenitors.

1.2 Tissue damage and normal regeneration.

Once tissues are damaged due to a physical injury, inflammation, or infection, they may release numerous signals and factors which encourage and mediate their regeneration. The fraction of stem cells in tissues which sustain tissue healing is variable in different types of tissues. A balanced potency of controlled cell renewal in tissues allows the organism a normal and healthy function. The well balanced homeostasis is very crucial for normal tissue function and maintenance. Disturbed homeostasis results in pathogenesis. When the tissue doesn't regenerate, the injury becomes chronic and/or inflammatory, as seen in diabetic patients. On the other hand, excessive cell proliferation in tissues results in hyperplasia in transformed cells by various neoplastic mutations or by viral induced proliferation.

2. Cell based tissue regeneration.

2.1 Major tissue damage.

Many damages and insults such as those resulting from high dose of ionizing radiation which affect many systems are too massive and complex to be handled by normal tissue regeneration processes. Such is a massive damage to the hematopoietic stem cell niche, which is one of the primary most radio- sensitive targets. The massive damage is affecting the renewal process of the blood cells from various germ lines. Other examples of tissue regeneration failures include the correction of damages due to autoimmune diseases, such as multiple sclerosis, or type I diabetes where the relevant cells are depleted with no ability to be regenerated. The damages of progressive degeneration are sustained with the relevant pathogenesis and clinical outcomes.

2.2 Cell based therapy in tissue regeneration.

Modern medicine and research seek for cell based procedures to increase the potential of tissue regenerations. Such process may use transplantation stem cells that serve as potential building blocks to replace cells in the damaged tissues.

For example in case of bone marrow (BM) failure in different kinds of anemia or hematopoietic tumors, bone marrow transplantation (BMT) can be used as a remedy. Unfortunately, such transplantations are often associated with different severe complications, such as graft versus host disease (GvHD) due to implanted T-lymphocytes recognize the host cells as foreign in skin, intestinal mucosa, bile ducts and lymph nodes and induce auto- rejection. In Host-versus-Graft Disease (HvGD) the transplantation fails since the host T- lymphocytes destroy the administered allogeneic BM cells.

Another ground breaking scientific achievement was the development of the induced pluripotent stem cells (iPSC), where fully differentiated mature cells were exposed to certain factors to undergo dedifferentiation with a potential to differentiate to multiple tissues. This set basis to new possible approaches in the regenerative medicine. Though iPSC technology raises high expectation in regenerative medicine, it is not without limitations, suggesting various, possible risks and obstacles in their in vivo application.

Various stem cell sources are being explored, so far with very little success, to treat diabetes since the proof-of-concept for cell therapy was laid down by transplanting cadaveric islets. Embryonic stem (hES) cells and iPSC could be potential sources for pancreatic progenitors. hES have got US-FDA approval to be used in clinical trials to treat type 1 diabetes mellitus by differentiating to insulin secreting islands. However, these progenitors more closely resemble their fetal counterparts and thus whether they will provide long-term regeneration of adult human pancreas remains to be demonstrated^-'-'¹. But so far the successes in using stem cells as building blocks for replacing the damaged tissue cells have been very limited.

Despite all the innovative technics and possible sources of stem cells, a major part of this field of research is focused on mesenchymal stem cells and its application for regenerative medicine. 3. Mesenchymal stromal and/or stem cells (MSC) and their role in cell based therapies.

Mesenchymal stromal cells (MSCs) are heterogeneous cell populations that derive from different tissue and have limited span of differentiation potential. They were initially described in the BM as BM-MSC They have putative roles in maintaining tissue homeostasis and are increasingly recognized as components of multipotent stem cell niches, especially when they derive from the BM niche. BM-MSC are able to differentiate into cells of multiple lineages to take part in regeneration of failing tissues. Nevertheless, the sternness and pluripotency of MSC from different other sources is controversial. MSC from different sources are heterogeneous with respect to phenotype and function in current isolation and cultivation regimes, which often lead to non-reproducible experimental results.

Due to their relatively easy isolation and expansion ^(li> and low immunogenicity, BM- MSC have been suggested as an extremely promising therapy in regenerative medicine for diverse diseases. Recent studies have identified several subsets of MSC which exhibit distinct features and biological activities, and high therapeutic potential for certain diseases^'" '¹, yet in most studies the activity of such cells seem to be indirect due to paracrine effects

3.1 Different sources of isolation of MSC.

MSCs have been retrieved from various tissues and used in a multitude of settings whereby numerous experimental protocols are available for expansion of MSCs in vitro/ A wide variety of methods has been established in the past years for their isolation, expansion cultivation and characterization as a prerequisite for clinical application with high numbers of cells carrying reproducible properties.

The BM stroma consists of a heterogeneous population of mesenchymal stem cells that provide the structural and physiological support for hematopoietic progenitors. Several experimental approaches have been used to characterize the development and functional nature of these cells in vivo and their differentiating potential in vitro. In vivo, presumptive osteogenic precursors have been identified by morphologic and immunohistochemical methods. In culture, the stromal cells can be separated from hematopoietic cells by their differential adhesion to tissue culture plastic and their prolonged proliferative potential. In cultures generated from single-cell suspensions of the BM the stromal cells can also be grown in colonies derived from a single precursor cell as colony-forming units. Many culture methods have been developed to expand marrow stromal cells derived from human, mouse, and other species. Various methods of cultivation and transplantation under appropriate conditions, have been studied and found to have substantial influence on the transplantation outcome/^™'¹

Due to the limited yield of MSC derived from the BM, similar stromal cells with sternness abilities were looked for in a wide range of adult and fetal tissues. Main cell sources included adipose tissue, bone, lung, peripheral blood and many other sources ^'. Despite the extensive research efforts, there are limited sources of such stromal cells with a wide trans-differential potential ^J .

3.2 The controversies of differentiation potential and sternness of MSC from different sources

Many types of MSC were classified as stem cells with broad range of differentiation abilities. But various MSC which derived from different stromal sources do not possess the same differentiation potential (mostly cells isolated from adult organs which have limited proliferation capacity' " ' ' .

BM-MSC are able to differentiate in vitro and in vivo into several cell phenotypes such as bone, osteocytes, chondrocytes, adipocytes, and skeletal myocytes. Other phenotypes may transdifferentiate with less success. But during the last two decades, an increasing number of studies have suggested that MSC from different sources may treat indirectly neurodegenerative diseases, spinal cord damages, brain injuries, cardiovascular diseases, diabetes mellitus, and repair of the skeleton without differentiation into cells of relevant organs. Their relative immuno-privileged profile of such cell types allows both autologous and allogeneic use, which hints on their indirect paracrine effect, as they do no not participate as a building block of the repaired organs. The identification of MSC is currently based on three main criteria: plastic-adherence capacity, defined epitope profile, and capacity to differentiate in vitro into osteocytes, chondrocytes, and adipocytes. Due to that ability BM- MSC are called multipotent MSC. Later on some researchers managed to differentiate between the different MSC based on their activity. The two type of MSC (mesenchymal stromal cell and mesenchymal stem cell) are likely to be the same in many cases, though, the stromal cells are usually committed to certain lineage and have limited differentiation. 3.3 MSC and cell based therapy.

The expectations from the field of cell based tissue repair and wound healing has blossomed in the past 30 years. It has evolved from the use of recombinant growth factors, to living tissue engineering constructs based on differentiated stem cells. MSC consist of a heterogeneous population whose main role is to provide and maintain a healthy and rich microenvironment for supporting stem cells. This may be a basis for their use in cell based therapy and regenerative medicine ^(/'"^{' 2/>}. Due to the expectations that MSC contribute to neovascularization of the freshly formed tissues, as it occurs in the natural processes of healing in the organism, is very appealing to use them as a component of treatment for tissue regeneration ⁽—--—

Recent studies found that MSC can be found in small population in the umbilical cord and in peripheral blood It was suggested that these cells can be isolated and expanded to

? ⁷ )

high yields from fetal tissues and the placenta' . The evidence that placental MSC are multipotent and similar to BM-MSC is not sound. The phenotype of these stromal cells seems to be similar in terms surface markers, but their proliferation, size and secretome may be very different. Therefore, we termed these cells placental stromal cells (PSC) and not MSC. Major differences in their potency were even demonstrated between maternal and mostly fetal derived PSC ^{ .

4. Human Placental Mesenchymal Stromal Cells (hPSC)

4.1 Human placenta anatomy and development.

A mammalian placenta is an extraembryonic organ that is formed on the eight week into gestation in humans, the time of formation varies between species. The human placenta is composed from a number of layers of different tissues from different source. The subtypes can be roughly divided into two types of cells based on their origin - maternal source and fetal source^—'¹. The different layers that form the placenta through all the period of gestation provide the connection between the developing fetus and the uterine wall. Each layer has its own personalized role for normal fetus development. The chorion is formed from the mesoderm tissue and trophoblast cells derived from the forming fetus. The chorionic villi emerge from this chorion and enter the endometrium. The villi are microscopic, finger- like projections that contain capillaries for blood to flow through. The chorionic plate is covered by the amniotic tissue which contains the amniotic fluid and the fetus through all the gestation period. 4.2 PSC production.

Many researchers seeking for an easier access and a high yield of expanded focused on the human placenta as an easily available source of mesenchymal stromal cells. The placenta after a full term birth is often discarded and not needed, thus, allowing an easy and accessible source of such cells. PSC have been already produced from the placenta by different cell therapy industries. Our earlier studies were based on such cells ⁽³²⁾, but we established our own procedure for isolation of the desired population of PSC that may best suit our application.

4.3 PSC characterization and evaluation.

Stromal cells in general can be characterized by the presence of specific surface markers. These cells are positive to mesenchymal stromal markers such as CD90; CD29 and CD105, and negative to endothelial and hematopoietic markers such as CD34; CD45; CDl lb; CD19 and HLA-DR, respectively. They can be cultured in a standard tissue culture conditions on plastic surfaces and could be induced to differentiate in vitro to cells of different mesenchymal phenotypes, such as osteoblasts, adipocytes and chondroblasts. The placenta derived stromal cells correspond these requirements, furthermore they are positive to additional markers CD146 and CD166 and negative to the endothelial cells marker CD34.

4.4 PSC role in regenerative medicine and clinical applications.

PSC have been very attractive in clinical application and regenerative treatment. Many of which were applied in neurodegenerative diseases such as Parkinson disease or multiple sclerosis or in regenerative medicine and immunomodulatory practice. They cannot be termed stem cells due to their limited differentiation potential. Nevertheless, it is suggested that PSC are better immunomodulators and are less immunogenic, due to the absence of HLA-DR, than other types of MSC. This allows an efficient allogeneic treatment following their implantation, to receive a more potent indirect effect. We have shown that PSC are active even when injected in a distant site of the compromised tissues. This derives from the fact that their effect is indirect and is based on their highly potent secretome, as a response to a stress signal associated with the developing disease ' . 5. The attempt to use PSC therapy for mitigation of acute radiation syndrome (ARS).

5.1 Characteristics and pathogenesis of ARS.

Acute radiation syndrome, also known as radiation poisoning or radiation sickness, is potentially fatal disease caused by whole body acute exposure to high doses of ionizing radiation, beyond a few Gy. The early effects of very high doses may occur in some tissues almost immediately after exposure, while a wide range of symptoms may be evident up to several months and years later even in lower doses. Each organ and tissue may react differently, depending on their sensitivity and the time of manifestation of the effects. For example, organs and tissues such as the skin, the hematopoietic system, the gut the vascular system and the spermatogenic cells with high rate proliferation are known to be sensitive and express the acute radiation effects earlier. Though ionizing radiation may affect also proteins and lipids in the cell, the main lethal effect on the cells is caused due to DNA damage. Once exposed to ionizing radiation free radicals are formed and cause either single or double strand DNA breaks. But the double strand breaks seem to be more lethal causing cell cycle arrest and incomplete or faulty cell division. The pathogenesis of ARS is expressed earlier in the form of failure of hematopoietic systems in the BM, severe gut damages, as well as skin soars. The acute effect on the hematopoiesis is used when high dose total body radiation is given prior BMT in the treatment of lymphoma and leukemia patients to neutralize and eradicate the host's BM hematopoietic population to be replaced by new engrafted hematopoietic stem cells populations.

5.2 Practical treatments for ARS

Despite the fact that ARS is well documented in clinical level and its mechanism is well understood the treatment is still very limited. Most of the medical approaches are focused on relieving the symptoms and enhancing the regeneration of bone marrow after exposure to high dose of radiation.

The most practiced solution is BMT treatments, based on hematopoietic stem cells (HSCs) transplantation. If the exact dose of exposure is not known it can be very risky or fatal, not to mention that when speaking of a scenario of multiple individuals affected this solution is not practical due to transplantation limitation based on major histocompatibility complex and the risk of developing graft versus host disease. It was suggested to prescribe treatment with various growth factors such as G-CSF and GM-CSF or erythropoietin. However the higher the radiation dose the poorer the therapeutic effect of growth factors alone, due to the need of adequate doses or the complex growth factors, the instability of these factors once injected and the uncontrolled interaction of the poor remaining population of HSCs in the BM^. These treatments are accompanied by standard measures such as wide spectrum antibiotics, antifungals, blood transfusions and other supporting adjuvant treatments'^^-'¹.

5.3 MSCs as a treatment for ARS.

HSCs have been the main focus for treatment of ARS with the effect of BM failure. But due to limitation mentioned above HSCs availability is very poor when it comes to multiple casualties. HSCs can be available not only from the BM but also in small population in the umbilical cord and in a smaller population in the circulating blood, but those cannot be used as a solution for severe ARS. Recent reports suggest BM derived allogeneic MSC implantation could be used as a treatment for ARS resulting with a significant elevation of survival and BM regeneration Other reports suggested also the application of MSC in regenerative medicine unrelated to ARS in a combination with other treatments, where the MSC have an indirect proregenerative effect without serving as building blocks of the repaired tissues ⁽³⁸' ^39>. But all these proposed treatments still remain limited due to poor availability of BM derived MSC.

5.4 PSC as a proposed treatment for ARS.

An emergency treatment for life threatening radiation exposure is therefore of great interest. Such treatment should be easily available, not patient specific and should be effective even after a delayed administration after radiation exposure. Ideally it should have minimal adverse effects. Moreover, it a major advantage of such treatment is it availability "off the shelf.

Our initial work was performed using Placenta eXpanded (PLX) cells which were developed and manufactured by Pluristem Therapeutics. The PLX are human derived placental stromal cells, corresponding to all the requirement of mesenchymal characterization. PLX were expanded in bioreactors in 3D conditions. Pluristem debuted by presenting their placenta derived PLX-PAD as allogeneic transplant to treat chronic conditions such as limb ischemia in peripheral artery disease and suggested their potential for hematopoietic regeneration. The initial PLX were based on the cells from maternal origin in the placenta which yielded some protective effect. We identified batches that contained a mixture of maternal and fetal originated cells, which we termed PLX-RAD. We found this preparation to much better mitigate the high dose radiation effect, as reflected in survival and other clinical parameters^""'¹. Based on these findings a clinically applied cell therapy was developed by Pluristem. Based on these findings, our laboratory developed a unique technic to isolate and expand high yields of pure fetal PSC for their optimal effect in mitigation of radiation effect and other application in regenerative anti-inflammatory models. Our isolated PSC are now used for mitigation of ARS as well as in studies of other models including inflammatory bowel disease (IBD) and in neurodegenerative diseases.

6. BM and extramedullary hematopoiesis

Normally, in early development, in the third week of gestation specifically, HSCs are produced in the yolk sac and independently, from the wall of the embryonic aorta and vitelline arteries one week later. Then HSCs migrate and colonize the embryonic liver and later migrate to the BM where they remain permanently as the main producers of the hematopoietic cellular compartment. In normal conditions BM remains the only site of HSCs production and hematopoiesis. Like any other stem cells, HSCs have the ability for self- renewal and to give rise to all lineages of matured cells. HSCs are very dependent on their niche and their local microenvironment whose main role is to maintain and regulate their homeostasis. In some pathologic conditions extra-medullary hematopoiesis (EMH) occurs where multipotent hematopoietic progenitors scatter around the affected tissue, most likely in the spleen, liver, and lymph node. It is a secondary process which supports BM insufficiency in the adult organism. It is suggested that alteration of relationships between hematopoietic stem and progenitor cells and their original or new microenvironments occurs. EMH is very common among patients with severe hemolytic anemias such as thalassemia or sickle cell anemia, which results in a compensation mechanism leading to EMH in other tissues. EMH will be in most cases non-symptomatic and will require no clinical attention. However, other sites can be involved in EMH such as thymus, kidneys, retroperitoneum, paravertebral areas of the thorax, lungs, bowel and others organs. In these cases various adverse effects are observed and it can be fatal and is often correlated with poor prognosis of the corresponding disease.

The present invention is based on the finding that efficient isolation of matrix- dependent placental stromal cells can be achieved by allowing the cells to migrate out of relatively large fragments of the placental tissue onto an adhesive surface while immobilizing the tissue fragments to the surface with a thin layer of fibrin.

Thus, in one aspect, the present invention is directed to the present invention is directed to a method for preparing a population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin, the method comprising (i) obtaining a tissue comprising matrix dependent mesenchymal stromal cells; (ii) optionally briefly contacting fragments of said tissue with a solution comprising a protease; (iii) optionally keeping the fragments for 48 hrs or more in motion, e.g. rotation, while suspended in growth medium at 37°C and an atmosphere with about 5% C0₂; (iv) contacting the fragments of the tissue with a solution comprising fibrinogen, e.g. low concentration of fibrinogen, evacuating the excess fibrinogen solution; (v) transferring the fragments to an unmodified/uncoated plastic surface; (vi) dispersing, distributing or spraying a solution comprising thrombin on said plastic surface and fragments thereby forming a thin fibrin film around the fragments that immobilizes the fragments to the plastic surface; (vii) covering the fragments with growth medium; (viii) harvesting the mesenchymal stromal cells that have migrated out from the fragments, e.g. after a period of -7-20 days, and optionally culturing the cells for at least 2-3 passages, thereby preparing a population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin. The cells can be further expanded in tissue culture conditions and then potentially cryopreserved for further use.

Step (iv) may be done by immersing the fragments in a low concentration of fibrinogen, such as up to 1%, and removing the extra liquid before transferring the fragments to the plastic dish. After distribution of the fragments on the dish the extra fibrinogen is aspired leaving only fibrinogen on the surface of the tissue fragments. The stable fibrin formation may be allowed to occur in a cell culture incubator at 37°C for 5-30 min. The immobilized tissue fragments are then covered slowly and carefully with growth medium.

For example, the method may be performed in the following non-limiting way:

The placenta is opened and rinsed extensively in phosphate buffer saline with 10% antibiotics (Pen-Strep, Beit-Haemek) to clean residual blood. The isolation is aimed at harvesting fetal cells only. Therefore, these cells of interest are isolated from specific tissues taken from the outer area of the placenta chorion on the border of the alantois, adjacent to the connection with the umbilical cord which is derived from embryonic cells. 2

Tissue pieces of ~lcm , 2-3 mm deep are cut off from the internal layers of the placenta using scalpel/scissors and fine forceps. The large tissue pieces are rinsed with 10% antibiotics in PBS. Then the epithelium and soft sponge tissue are carefully scrapped off. The pieces are trypsinized for 30 mins with 10% trypsin EDTA in PBS to reduce remaining epithelial tissue while getting rid of most residual trapped circulating blood cells. After trypsinization the tissue fragments are chopped into small pieces of 1-2 mm and rinsed extensively in 10% antibiotics. Then the chopped tissue fragments are trypsinized again for 15 min.

Followed by further washing, the smaller tissue fragments are immersed with fibrinogen (1.5-2.5% in normal saline) and about 10-20 fragment are spread on the surface of large petri dishes (15cm diameter). All the fibrinogen solution is aspired out to leave only residual fibrinogen on the surface of the tissue fragments. Thrombin (lOOu/ml) is then briefly sprayed on the dish' surface to activate the residual fibrinogen on the fragments surface to form fibrin clot, which allowes the tight attachment to the plastic dish surface. Followed by 20-30 min incubation in 37°C, the plates with the small fragments attached to the plastic surface are carefully covered with pluripotent stem cells (PSC) medium, with care that the fragments on the plastic surface will stay attached without floating. First signs of PSC migrating from the tissues fragments through the fibrin clot are visible from day 5 post plating. Within the following ~2 weeks a wave of excessive number of large PSCs can be seen migrating out from the attached tissue fragments to populate large areas of the plate surface. The fibroblast-like stromal cells are harvested when a significant area of the plate is covered with high cell density, about 15-17 days post-plating. Then the PSC are subjected for further expansion in common tissue culture procedures flasks. Upon verification of the fetal source of the cells by Fluorescence in-situ Hybridization (FISH) with staining of the X-Y centromeres and analysis of the cell surface markers by Fluorescence Activated Cells Sorter (FACS), the cells are further expanded for up to 10 passages, usually human PCS (hPSC) of passages 5-8 were used.

Variations of the above protocol have also been developed by the inventors:

1. Once washed from blood, the umbilical cord is cut off and disposed and the placenta is soaked in phosphate buffered saline (PBS) or normal saline +10% antibiotics (such as neomycin, streptomycin and penicillin) for at least 10 minutes. 2. Using a sterile surgical tool the placenta is spread open with the fetal membrane side up.

3. If the target cells are neonate stromal cells, the tissue samples are to be collected on the fetal side mainly in the decidua, within -0.5-2 cm in size and no thicker than 2-3 mm.

4. Maternal or mix PSC samples are collected in deeper layers

5. For neonate samples 2-4 mm deep from the fetal membrane (decidua) are collected, internal spongy tissues and surface epithelium are scrubbed away.

6. The extracted pieces are washed in PBS or saline with 10% antibiotics to remove excess of blood and undesired surface loose cells.

7. The tissue fragments pieces are processed with 0.02% trypsin/EDTA for half an hour (possibly in rotation in a C0₂ incubator).

8. The large source tissue samples are then chopped into very small fragments to a final size of a -1-3 mm using scissors and scalpel and processed again in the above conditions in trypsin for another 15 minutes.

9. The pieces are either (a) Immediately processed as follows; or (b) Incubated in rotation in a C0₂ incubator for 24-72 hrs and then processed as follows:

10. The liquid is discarded and the pieces washed with medium to stop trypsinization.

Immediate direct cell migration protocols

1. The rinsed small tissue fragments, preferably but not limited to 1-2 mm are mixed with small amount of dilute sterile fibrinogen (~lmg/ml) and spread on a tissue culture plastic plate/flask for a few minutes.

2. The flasks/cell culture dishes are positioned sidewise to allow the drip off of excess fibrinogen solution leaving only a minute film on the samples.

3. A solution of 20-100 units of thrombin is prepared and loaded in a small sterile spray device.

4. The surface of the plate with the dispersed tissue fragments is sprayed briefly with the thrombin solution in a sterile environment and the flasks are put in the C0₂ incubator at 37°C for 10-30 min till the activated fibrin clot is stabilized, immobilizing the tissue fragments to the surface. 5. The plate is then covered with medium and the desired matrix dependent mesenchymal cells start migrating from the attached tissues within 7-10 days of incubation.

Cell migration protocols after tissue pieces suspension

1. Extracted tissue pieces after trypsinization processing are held in 50 ml tubes with medium and put into C0₂ incubator in 37°C in rotation for more than 48 hours.

2. The tissue pieces are then washed and plated as above (Immediate direct cell migration protocols).

3. A similar yield, but with possibly higher rate is expected to be achieved in the migration from tissue pieces held in suspension for 1-7 days.

It has been found in accordance with the present invention that living tissue immersed in certain solutions developed by the inventors can be frozen, kept frozen and used to isolate viable matrix-dependent cells. It has further been found that the components and their relative concentration in the cryo-preservation solution used determine the efficiency of recovery of viable cells from the frozen tissue, and that treating the thawed tissue with proteases results in mild softening of the tissue from which the cells could be more efficiently isolated by migration.

Thus, in certain embodiments, said fragments of tissue are made from fragments or bulk tissue previously immersed in a cryo-preservation solution, frozen by slowly reducing the temperature to -75°C to -80°C, and then stored in liquid nitrogen, thawed and treated with a protease of step (ii) selected from trypsin and/or collagenase, wherein said cryo- preservation solution comprises: fetal calf serum (FCS; e.g. 50-90%) or human plasma albumin (HAS; e.g. 5-50%) in addition to dimethyl sulfoxide (DMSO) and optionally polyethylene glycol or cell culture medium. In particular the composition for cryo- preservation comprises (i) 90% fetal calf serum (FCS) + 5% dimethyl sulfoxide (DMSO) + 5% polyethylene glycol (PEG); (ii) 90% FCS+10% DMSO; (iii) 10-20% human plasma albumin (HSA) + 10% DMSO + 70-80% cell culture medium; or (iv) 10-20% HSA + 5% DMSO + 5% PEG + 70-80% cell culture medium.

In certain embodiments, the collagenase concentration used to treat the thawed tissue is between about 0.2- 3mg/ml, for example 0.75 mg/ml. The tissue may be treated with trypsin and rinsed prior to the collagenase treatment.

In certain embodiments, the source tissue is selected from placenta, breast, liver, kidney, tumors, uterus, bone, cartilage, spleen and skin. In certain embodiments, the tissue is placenta and the fragments are obtained from the fetal side of the surface decidua of the chorion. In certain embodiments, the tissue is essentially lacking terminal chorionic villi.

In certain embodiments, the matrix dependent mesenchymal stromal cells are stromal cells, such as placental stromal cells. The cells may be identified by detection of specific surface markers. For example, the isolated matrix matrix dependent mesenchymal stromal mesenchymal stromal stromal cells being isolated are human cells expressing CD 105, CD44, CD29 and CD90. In certain embodiments, more than 95% of the cells are of fetal origin and the population is essentially free of cells of maternal origin.

The population of matrix dependent mesenchymal stromal cells isolated by the method of the invention is essentially free of cells of hematopoietic and endothelial origin, which may be identified by the expression of CD45, CD19, CDl lb, HLA-DR and CD34. These cells may make up less than 1% of the population of matrix dependent mesenchymal stromal cells, thus making the population of matrix dependent mesenchymal stromal cells isolated by the method of the invention essentially free of cells of hematopoietic and endothelial origin.

In certain embodiments, the solution comprising fibrinogen used in the method described above comprises about 0.5-3% fibrinogen, e.g. about 1% fibrinogen; and the solution comprising thrombin may independently comprise at least about 10 U/ml thrombin, e.g. about 100 U/ml thrombin.

In certain embodiments, the protease used in the method of the present invention is trypsin, e.g. at a concentration of 1%.

It has been found in accordance with the present invention that frozen tissue will provide more viable cells after thawing when used in accordance with the method of the invention if the thawed tissue is treated with trypsin and collagenase prior to contacting with the dilute fibrinogen solution. Thus the thawed tissue may be treated with about 1% fibrinogen, rinsed and then treated with 0.2-3mg/ml collagenase.

In certain embodiments, the method of the present invention for isolating cells from placenta, comprises: (i) obtaining a tissue from the fetal side of the surface decidua of the chorion of placenta; (ii) briefly contacting fragments of said tissue with a solution comprising trypsin, e.g. 1 % trypsin; and optionally rinsing the fragments, cutting the fragments into finer fragments and contacting said finer fragments with a solution comprising trypsin following a rinsing and washing; (iii) optionally keeping the fragments for 48 hrs or more in suspension in motion/rotation while suspended in growth medium at 37°C and an atmosphere with about 5% C0₂ before treating with trypsin followed by exposure to fibrinogen; (iv) briefly contacting fragments of said tissue of (ii) or (iii) with a solution comprising about 0.5-3% fibrinogen, e.g. about 1% , fibrinogen and removing excess fibrinogen leaving only a film on the fragments; (v) transferring the fragments to an unmodified or uncoated plastic surface; remove excess liquid with fibrinogen; (vi) distributing, e.g. spraying, a solution comprising at least about 10 U/ml thrombin, e.g. about 100 U/ml thrombin, on said plastic surface and fragments thereby forming a thin fibrin film around the fragments that immobilizes the fragments to the plastic surface; (vii) covering the fragments with growth medium; (viii) harvesting matrix dependent mesenchymal stromal cells that have migrated out from the fragments; thereby preparing a population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin. In certain embodiments, said fragments of tissue used as the source for the placental stromal cells are fragments having previously been immersed in a cryo-preservation solution, frozen by slowly reducing the temperature to -75°C to -80°C, and then stored in liquid nitrogen, thawed and treated with a protease of step (ii) selected from trypsin and/or collagenase, wherein said cryo-preservation solution comprises: fetal calf serum (FCS; e.g. 50-90%) or human plasma albumin (HAS; e.g. 5-20%) in addition to dimethyl sulfoxide (DMSO) and optionally polyethylene glycol or cell culture medium. In particular the composition for cryo-preservation comprises (i) 90% fetal calf serum (FCS) + 5% dimethyl sulfoxide (DMSO) + 5% polyethylene glycol (PEG); (ii) 90% FCS+10% DMSO; (iii) 10-20% human plasma albumin (HSA) + 10% DMSO + 70-80% cell culture medium; or (iv) 10-20% HSA + 5% DMSO + 5% PEG + 70-80% cell culture medium.

In another aspect, the present invention provides a population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin obtained by any one of the methods of the invention as defined above. In another aspect the present invention provides a population of matrix dependent mesenchymal stromal derived from only the fetal or the maternal tissue origin of the placenta as dictated by the tissue layers dissected from the whole donated placenta.

In certain embodiments, more than 95% of said cells obtained with the method of the present invention are of fetal origin and the population is essentially free of cells of maternal origin.

In certain embodiments, the cells are formulated for subdermal, intraperitoneal, intra- cerebroventricular, intrathecal, intravenous or intramuscular injection.

In an additional aspect, the present invention provides a pharmaceutical composition comprising the population of matrix dependent mesenchymal stromal cells of the present invention as defined above and a pharmaceutically acceptable carrier.

In certain embodiments, the pharmaceutical composition may be formulated for subdermal, intraperitoneal, intra-cerebroventricular, intrathecal, intravenous or intramuscular injection.

Mesenchymal stem cells and stromal cells have been used in regenerative medicine, for example in mitigation of lethal radiation syndrome, ischemia induced limb injury, graft- versus-host disease (GVHD), transplanted organ rejection and autoimmune diseases⁽²⁾, ischemic brain injury⁽³⁾, critical limb ischemia*⁴' ⁵⁾, myocardial infarction⁽⁶' ⁷⁾, IBD ⁽⁸⁾ and diabetes mellitus type I or II ⁽⁹' ¹⁰' ⁿ⁾ (all references cited are hereby expressly incorporated herein in their entireties by reference).

It has further been found in accordance with the present invention that matrix- dependent cells isolated according to the methods of the present invention can be used to ameliorate symptoms of a mice model of human multiple sclerosis, or models of acute radiation syndrome (ARS) or failing bone marrow.

Thus, in a further aspect, the present invention provides a method for promoting a regenerative process in damaged tissue or organ or reducing an inflammatory response associated with disease or injury, comprising administering to a subject in need thereof a therapeutically effective amount of the population of matrix dependent mesenchymal stromal cells of the present invention or the pharmaceutical composition of the present invention.

In still another aspect, the present invention provides a population of matrix dependent mesenchymal stromal cells or pharmaceutical composition of the present invention for use in promoting a regenerative process in damaged tissue or organ or reducing an inflammatory response associated with disease or injury.

In certain embodiments, the promotion of a regenerative process of damaged tissue comprises treatment or mitigation of acute radiation syndrome (ARS) following exposure to high dose ionizing radiation, or failing bone marrow.

In certain embodiments, the damaged tissue is bone marrow.

In certain embodiments, the damaged organ is hair follicles, or the cells are for use in treating alopecia, i.e. partial or complete hair loss. In certain embodiments, the alopecia is a result of the ARS, i.e. it is caused by exposure to high dose ionizing radiation.

In certain embodiments, in the case of treatment of ARS and failing bone marrow, the population of matrix dependent mesenchymal stromal cells is injected intramuscularly, subdermally or intraperitoneally, or is formulated for intramuscularl, subdermal or intraperitoneal injection,.

In certain embodiments, the disease or injury is selected from an autoimmune disease, an inflammatory disease, allergy, ischemic brain injury, critical limb ischemia, myocardial infarction, an immune-mediated neurodegenerative disorder, a metabolic disorder, g graft- versus-host disease (GVHD) or transplanted organ rejection.

In certain embodiments, the autoimmune disease is selected from multiple sclerosis, Crohn's disease, ulcerative colitis and type 1 diabetes mellitus, in particular multiple sclerosis.

In certain embodiments, the damaged organ is damaged pancreas or the metabolic disorder is diabetes mellitus type 2. In certain embodiments, in the case of treatment of multiple sclerosis, the population of matrix dependent mesenchymal stromal cells is injected intra-cerebroventricularly or is formulated for intra-cerebroventricular injection,.

In a further aspect, the present invention is directed to a method for mitigation of acute radiation syndrome (ARS) following exposure to high dose ionizing radiation and/or treating failing bone marrow comprising administering to a subject in need thereof a therapeutically effective amount of the population of matrix dependent mesenchymal stromal cells described herein above, or the pharmaceutical composition described herein above.

In a further aspect, the present invention provides a population of matrix dependent mesenchymal stromal cells or pharmaceutical composition of the present invention for use in mitigation of acute radiation syndrome (ARS) following exposure to high dose ionizing radiation and/or treating failing bone marrow.

In certain embodiments, the damaged hair follicles causes hair loss and thus the method is for alleviating or treating partial or complete hair loss.

In certain embodiments, the damaged hair follicles are damaged following or by exposure to high dose radiation.

In certain embodiments, more than 95% of the population of matrix dependent mesenchymal stromal cells used in the methods for promoting a regenerative process in damaged tissue or organ or reducing an inflammatory response associated with disease or injury, are cells of fetal origin and the population is essentially free of cells of maternal origin.

In still a further aspect, the present invention provides a composition for cryo- preservation of biological tissue, said composition comprising fetal calf serum (FCS; e.g. 50- 90%) or human plasma albumin (HAS; e.g. 5-50%) in addition to dimethyl sulfoxide (DMSO) and optionally polyethylene glycol or cell culture medium. In particular the composition for cryo-preservation comprises (i) 90% fetal calf serum (FCS) + 5% dimethyl sulfoxide (DMSO) + 5% polyethylene glycol (PEG); (ii) 90% FCS+10% DMSO; (iii) 10- 20% human plasma albumin (HSA) + 10% DMSO + 70-80% cell culture medium; or (iv) 10-20% HSA + 5% DMSO + 5% PEG + 70-80% cell culture medium.

In yet another aspect, the present invention provides a method for preserving biological tissue such as normal tissue or tumor tissue, comprising immersing said biological tissue in a composition for cryo-preservation of biological tissue described above, and slowly reducing the temperature to -75°C to -80°C, thereby providing frozen biological tissue and storing the frozen biological tissue in liquid nitrogen As used herein, the terms "subject" or "individual" or "animal" or "patient" or "mammal," refers to any subject, particularly a mammalian subject, for whom diagnosis, prognosis, or therapy is desired, for example, a human.

Pharmaceutical compositions for use in accordance with the present invention may be formulated in conventional manner using one or more physiologically acceptable carriers or excipients. The carrier(s) must be "acceptable" in the sense of being compatible with the other ingredients of the composition and not deleterious to the recipient thereof.

The following exemplification of carriers, modes of administration, dosage forms, etc., are listed as known possibilities from which the carriers, modes of administration, dosage forms, etc., may be selected for use with the present invention. Those of ordinary skill in the art will understand, however, that any given formulation and mode of administration selected should first be tested to determine that it achieves the desired results.

Methods of administration include, but are not limited to, parenteral, e.g., intravenous, intraperitoneal, intramuscular, subcutaneous, mucosal (e.g., oral, intranasal, buccal, vaginal, rectal, intraocular), intrathecal, topical and intradermal routes. Administration can be systemic or local. In certain embodiments, the pharmaceutical composition is adapted for oral administration.

The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the active agent is administered. The carriers in the pharmaceutical composition may comprise a binder, such as microcrystalline cellulose, polyvinylpyrrolidone (polyvidone or povidone), gum tragacanth, gelatin, starch, lactose or lactose monohydrate; a disintegrating agent, such as alginic acid, maize starch and the like; a lubricant or surfactant, such as magnesium stearate, or sodium lauryl sulphate; and a glidant, such as colloidal silicon dioxide.

The compositions may be formulated for parenteral administration by injection, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers, with an added preservative. The compositions may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents. Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile pyrogen free water, before use.

For purposes of clarity, and in no way limiting the scope of the teachings, unless otherwise indicated, all numbers expressing quantities, percentages or proportions, and other numerical values recited herein, should be interpreted as being preceded in all instances by the term "about." Accordingly, the numerical parameters recited in the present specification are approximations that may vary depending on the desired outcome. For example, each numerical parameter may be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

The term "about" as used herein means that values of 10% or less above or below the indicated values are also included.

The invention will now be illustrated by the following non-limiting examples.

EXAMPLES Section I: Isolation of human fetal placental stromal cells and their use in treatment of lethal acute radiation syndrome (ARS).

Materials and methods

Isolation of Human fetal placental stromal cells (f-hPSC)

f-hPSC were isolated from placentas of healthy mothers with healthy normal full term male delivered by Caesarean section. The use of the donated placentas was approved by Institutional Ethical Committee (Helsinki) with the full consent of the mothers.

We employed a unique new method of harvesting cells of interest which was developed in our laboratory, allowing to selectively isolate and expand high yields of pure fetal population of hPSC from the placenta.

The placenta was opened and rinsed extensively in phosphate buffer saline with 10% antibiotics (Pen-Strep, Beit-Haemek) to clean residual blood.

The isolation aimed at harvesting fetal cells only. Therefore, these cells of interest were isolated from specific tissues taken from the outer area of the placenta chorion on the border of the alantois, adjacent to the connection with the umbilical cord which is derived from embryonic cells (Fig. 1).

Tissue pieces of ~lcm , 2-3 mm deep were cut off from the internal layers of the placenta using scalpel/scissors and fine forceps. The large tissue pieces were rinsed with 10% antibiotics in PBS. Then the epithelium and soft sponge tissue were carefully scrapped off. The pieces were trypsinised for 30 mins with 10% trypsin EDTA in PBS to reduce remaining epithelial tissue while getting rid of most residual trapped circulating blood cells. After trypsinization the tissue fragments were chopped into small pieces of 1-2 mm and rinsed extensively in 10% antibiotics. Then the chopped tissue fragments were trypsinized again for 15 min.

Followed by further washing, the smaller tissue fragments were immersed with fibrinogen (1.5-2.5% in normal saline) and about 10-20 fragment were spread on the surface of large petri dishes (15cm diameter). All the fririnogen solution was aspired out to leave only residual fibrinogen on the surface of the tissue fragments. Then thrombin (lOOu/ml) was briefly sprayed on the dish' surface, to activate the residual fibrinogen on the fragments surface to form fibrin clot which allowed the tight attachment to the plastic dish surface. Followed by 20-30 min incubation in 37°C, the plates with the small fragments attached to the plastic surface were carefully covered with PSC medium, with care that the fragments on the plastic surface will stay attached without floating. First signs of PSC migrating from the tissues fragments through the fibrin clot were visible from day 5 post plating. Within the following ~2 weeks a wave of excessive number of large PSCs could be seen migrating out from all the attached tissue fragments to populate large areas of the plate surface. The fibroblast- like stromal cells were harvested when a significant area the plate was covered with high cell density, about 15-17 days post plating. Then the PSC were subjected for further expansion in normal tissue culture procedures flasks. Upon verification of the fetal source of the cells by Fluorescence in-situ Hybridization (FISH) with staining of the X-Y centromeres and analysis of the cell surface markers by Fluorescence Activated Cells Sorter (FACS), the cells were further expanded for up to 10 passages. In most experiments f-hPSC of passages 5-8 were used.

Materials for tissue culture and PSC isolation

- Fibrinogen 75mg/ml vial, VITEX, USA

- Human Thrombin (800-1200 IU/ml), Quixil, Omrix, Israel or VITEX, USA.

- The following materials for tissue culture were purchased from BI (Beit Ha Emek):

Dulbeco modified eagle medium (DMEM) low glucose (lg/ml)

Phosphate buffer saline XI (PBS)

Heat inactivated fetal calf/bovine serum (FCS/FBS)

L- Glutamine 200mM/Liter

Antibiotics - Penicillin (10,000U/ml); streptomycin (lOmg/ml)

Trypan blue 0.05%

Trypsin EDTA solution B - Trypsin 0.25% & EDTA 0.05% in Puck's saline A HeraCell temp, controlled C0₂ incubator for cell culturing, Heraeus Eppendorf centrifuge, 5810R

Main solutions used:

Placenta washing medium - 10% antibiotics; PBSX1

Trypsin washing medium - 10% Trypsin EDTA solution B; PBSX1

PSC medium - DMEM low glucose (lg/ml); 10%FCS/FBS; 1% antibiotics; 1% L- Glutamine 200mM/Liter (medium).

Freezing medium - DMSO 10%; DMEM low glucose (lg/ml) 50%; FCS/FBS 40%.

Injection medium - PlasmaLyte A multiple electrolytes injection type 1 USP, Baxter.

Disposable tissue culture materials purchased from Bar Naor:

75ml and 50 ml flasks

Well plates

Petri dishes

50ml and 15 ml tubes

1.5ml and 2 ml tubes

1.5 ml cryovials

f-hPSC characterization and identification

When the isolated f-hPSC from the different excised bulk tissue samples reached passage 2, their fetal origin was determined by FISH of X and Y chromosomal centromeres. The f-hPSC were also characterized as mesenchymal stromal cells by FACS with specific surface markers staining. They were negative to hematopoietic and endothelial markers: CD45; CDl lb; CD34; CD19; HLADR, and positive to mesenchymal stromal markers: CD29; CD90 and CD105.

8 million cells were taken from random isolated bulk tissue samples for surface staining (1 million cells per sample). Each sample was stained for a combination of surface markers as followed: CD31, CD34, CD45, CDl lb, HLA-DR for the lack of hematopoietic and endothelial cells and CD29, CD105, CD90, CD146, CD166 for identification of the specific mesenchymal stromal cells origin of the cells

FACS was performed on a Maxquant analyzer, Miltenyl using a standard FACS buffer (0.5% FCS/FBS in PBS pH=7). FISH X-Y centromeres analysis

FISH analyses of centromeres of X and Y chromosomes were performed at the Labs of the Department Genetics of Hadassah Medical Center, using the kit purchased from Abbott Molecular, Vysis.

Mice

Female C3H/HEN-HSD mice aged 8-9 weeks were used according to the Institutional Animal Welfare Ethical Committee permission (MD - 16 -14727-4). Throughout all the experiments, the mice were kept in the animal facility under the SPF conditions and monitored on a daily basis.

Anesthetics:

The solutions used for anesthesia were

Ketamine 10ml (lOOml/ml).

Xylazine 2% 25ml.

Rymadil 20ml (50mg/ml).

For post-surgery treatment mice were administered Rimadyl injections (4.4 mg/kg) once a day, for three days.

For the terminal procedure an anesthetic cocktail of 0.9ml of ketamine with 0.1ml of Xylazine are diluted in 1ml normal saline. The dosage is administrated per mouse' weight (0.1ml/20g mouse).

Splenectomy

Mice were anesthetized using a ketamine-xylazine cocktail. Once fully anesthetized a qualified surgeon removed their spleen and stitched the wound. Mice were given Rymadil and normal saline (0.7ml).

Mice were kept warm until waking up and put back to their cage.

The radiation exposure occurred 10 days after performing the surgery. Allowing the animals to heal and stabilize.

The surgery was performed at the animal's facility laboratory under SPF conditions.

Irradiation

After the required minimal acclimatization period of a few days the mice were exposed to high dose rate lethal whole body radiation by a total dose of 8Gy. The mice were transferred for irradiation in a special jig which restricted them in a certain height, covered with two plastic bags to ensure the SPF condition and to allow their return to the animal's facility after the procedure.

Irradiation was performed in the radiotherapy unit at the Sharett Institute of Oncology at Hadassah with a clinical linear accelerator 6-18 MeV LINAC (Varian, Medical Systems, CA, USA, calibrated by the Radiotherapy physicists.

f-hPSC delivery

On day 1 and 4 post radiation exposure (day 0), mice were injected with a total of 4 million cells each. The cells were harvested from confluent flasks, between passage 5-7, using Trypsin solution, counted, and centrifuged 1400 rpm for 5 min at 4C°, then re- suspended with plasmaLyte A to a final concentration of 20 million cells/ΙΟΟμΙ. Each mouse was injected 50μ1 to each leg in two time points. The control mice were injected with PlasmaLyte A only.

Protocol for terminating the animal experiments

The mice surviving the radiation treatment at the day of interest were sacrificed. They were anaesthetized and blood was collected from cardiac puncture. Then the mice were euthanized and various samples were harvested such as spleen, liver samples. Femurs and tybiae were excised from both legs for bone marrow extraction.

Complete blood count (CBC)

CBC was performed on the collected blood using a COULTER® LH-750A

Hematology Analyzer, Beckman Coulter, Inc (Nyon, Switzerland)

ELISA of selected human and mouse proteins

Plasma was separated from blood by centrifugation (2500 rpm, 10 min) and stored at - 20C° until the ELISA essay. Assays of 4 different murine and human proteins were performed on the collected plasma samples using antibodies as follows:

Antihuman VEGF; Antimouse VEGF; Antihuman PLGF and Antihuman EPO.

All the ELISA kits were purchased from R&D systems, Quantikine.

Harvesting cells from spleen and bone marrow (BM)

After extraction of the spleens, they were crushed and processed through a 40μιη mesh, centrifuged and the extracted cells were counted (whole spleen count). A volume of ΙΟΟμΙ was taken from each sample for further centrifugation. These samples were treated with 1ml of RBC lysis buffer for 3 min of incubation at room temperature. Then the samples were aggressively vortexed and centrifuged again to perform an additional counting (nucleated Spleen cells count).

BM was extracted from the four long bones (both tibias and femurs) using mortar and pestle set and filtered through a 40μιη mesh. The extracted BM cells was centrifuged and counted for whole BM cell count and nucleated BM count similarly to the procedure performed for spleen derived cells.

Flow cytometry

Both bone marrow and spleen cells samples were stained for Erythropoiesis and HSCs populations. (100μ from each sample for each staining procedure)

The FACS assays were run on FACS Maxquant analyzer, Miltenyl. The analysis and the statistics were performed on FCSexpress 4.0.

The population of erythropoietic cells were selected based on the negative to the linage surface markers (Lin- cocktail: B220; CD3; CD8; CD115; GR1).

The erythroid cells, positive to TER119 and CD71 were divided into three populations, where Ery-A were un-differentiated cells, Ery-B were partially differentiated cells and Ery-C were fully differentiated erythrocytes.

For HSCs population evaluation, cells are to be negative to the lineage markers (Lin- cocktail: B220; CD3; CD8; TER119; CD115; CD41; CD48) and positive to Ckit and Ska-1.

The antibodies for FACS were purchased from Applied Biolegend.

Histology of Bones, Spleen and Liver samples

Tissue samples for histology were kept 24 hours in formaldehyde 4% at room temperature and then transferred to 70% ethanol and then processed for histology and stained by H&E at the Hadassah Pathology Laboratories.

• Materials for histology

Slides super frost ++ were purchased from Bar Naor.

Formaldehyde (4%) was purchased from BioLab ltd.

Frozen sections preparation

Whole livers were extracted from the euthanized mice and held overnight in freshly prepared paraformaldehyde, then the paraformaldehyde was removed by suction and replaced by sucrose once again for overnight incubation. Samples then were frozen in OCT and cut using a cryostat. Once the slide were prepared they were stained for TER-119 (CY-3) and DAPI and kept in 8°C.

• Materials for histology

Paraformaldehyde (Fluka, Chemica)

Slides super frost ++, purchased from Bar Naor.

Sucrose purchased from Deajung

O.C.T. Tissue plus (Scigen)

Terl l9 primary antibody purchased from Applied Biolegend.

Secondary Cy-3 conjugated antibody (donkey anti mouse) purchased from Jackson ImmunoResearch laboratories.

Mounting medium DAPI (Sigma Aldrich)

Example 1. Retrieval and characterization of stromal / matrix dependent mesenchymal stromal cells from tissues, as demonstrated by isolation of human fetal placental stromal cells (f-hPSC)

A commonly used method for cell retrieval from tissues is by the digestion of the tissue and collection of the relevant cells These methods consist basically of the tissue proteolysis with collagenase and DNAse, leading to a very limited yield of mixed cell populations. A new migration-isolation method developed in our Laboratory enabled the high yield isolation of the newborn cells, based on the migratory potential of the PSC from the selected tissue fragments of interest (migration-isolation) as shown in the scheme in Fig. 2.

The placental cells from the newborn origin were isolated from a desired location at the outer area of the placenta chorion, on the border of the allantois, adjacent to the connection with the umbilical cord which is derived from embryonic cells as shown in Fig. 1.

The migration-isolation technique allowed a selective isolation and expansion of high yields of pure fetal population of hPSC from the selected placental tissues. We compared this procedure with the regular tissue digestion protocol and the migration-isolation (Figs. 3 & 4) The migration based cell retrieval method had a lag time of 5 -10 days before the initiation of cells migration from the tissue fragments to the plastic (Fig. 2). In spite of the lag period this procedure was proven to be the most effective yield the highest number of target cells with faster expansion (Fig. 5). The downloaded cells were of more uniform stromal phenotype, with minimal contamination with endothelial cells, while the regularly used digestion procedure yielded in early stages a very few mixed populations of attached cells. These cells progressed very slowly and needed a longer expansion period to reach the required yields.

Characterization f-hPSC as fetal and stromal mesenchymal cells

FISH X/Y centromere staining for verification of the fetal male origin of the cells. The X & Y centromeres were stained to show the presence of the Y chromosome, thus showing that the cells are from a male fetal origin (Fig 6).

Flow cytometry of cell markers profile of the isolated and expanded hPSC.

The hPSC were expanded in conventional monolayer culture and were used for experiments following expansion by 5-7 passages. Our f-hPSC differed from maternal derived cells by size (-20% larger), but had similar expression of typical stromal mesenchymal antigens (not shown).

Isolated PSC positively stained for mesenchymal markers (PE) and negatively stained for endothelial and hematopoietic markers (FITC).

The pure fetal hPSC were isolated and expanded to high cell numbers. The cells seemed stable over the long culture and expansion period without any spontaneous trans- differentiation or change in the cell phenotype expressing stromal-mesenchymal markers phenotype and no expression of both hematopoietic and endothelial markers (Fig 7).

Mitigation of acute lethal radiation syndrome by f-hPSC treatments

The lethal acute radiation syndrome (ARS) was studied with a C3H/HENHSD/female mouse model. The mice were given a lethal dose of ~8Gy whole body ionizing irradiation.

In a previous study systemic IV administration of commercially produced PSC was associated with severe adverse effects. Therefore, we adopted remote cell delivery by either IM or SC administration, which were found to be associated with no apparent complications. A higher number of cells were delivered (4xl0⁶ per mouse) at two time points following irradiation (Fig. 8). A dramatically enhanced survival of the treated mice (both IM and SC administered) was noticeable (Fig. 9) with enhancement of a fast regeneration of the hematopoietic system in the peripheral blood count (Figs. 11 A-E).

Mice model for mitigation of radiation effects and follow up

Following irradiation the mice showed a weight loss from day 9 onwards. The PSC treated arms (IM and SC) recovered to their initial weight unlike the placebo treated arm (Fig 10), where most mice died before the end of the experiment and the survivors failed to recover to their initial weight.

Example 2. Effect of irradiation alone or with PSC treatment on complete blood count profile

Complete blood count (CBC) was performed for all groups of irradiated mice (IM; SC and Vehicle controls) and non-irradiated (Naive, PSC only). On day 23, at the end of the experiments, the peripheral blood cells in irradiated mice following both IM and SC PSC delivery recovered better as compared to the placebo treated mice. No significant difference was found between the Naive mice and those that had IM injection of PSC without being irradiated (Fig. 11).

The role of extramedulary hematoiesis in the recovery of the mice from high dose irradiation

In order to examine the possible induction of hematopoietic regeneration following high dose irradiation, a group of mice were splenectomized a week before irradiation, following treatment with IM PSC delivery. Their survival and weight were followed up and compared to non-splenectomized irradiated mice treated with IM injection of PSC or placebo The splenectomized mice showed no significant difference in survival and weight follow up (Figs 12 & 13).

Effect of irradiation on complete blood count profile and the effect PSC treatment in splenectomized mice

Complete blood count (CBC) was performed for all groups Irradiated mice (IM; SC; SPL- and Vehicle) and non-irradiated (Naive, PSC only). Peripheral blood cells in irradiated mice following IM PSC delivery recovered better, as compared to the splenectomized mice. The CBC results in IM (Spl-) were closer to the Vehicle group than to the IM and SC cell treated groups (Fig. 14).

Evaluation of possible extra-medullary hematopoiesis (EMH) in the Liver in Cryo-section as compensation for splenectomy.

In order to evaluate liver's potential to compensate the spleen absence in the irradiated mice, liver samples were taken from naive non irradiated mice and irradiated IM hPSC treated mice, with or without splenectomy. No clear evidence of clear Erythropoietic foci was noticed in these sections (Fig. 15). FACS profile of HSCs and Erythropoiesis, bone marrow and spleen.

The observation regarding the mitigation of ARS were further supported by FACS analysis, showing a significant difference in erythropoiesis and HSC counts between bone marrow (BM) and spleen (SPL) samples in hPSC treated mice

A highly significant increase of the proportion of spleen relative to bone marrow hematopoietic cells is demonstrated in the PSC treated irradiated mice.

FACS profile erythropoietic cell populations in bone marrow.

A significant difference was shown in both ERY A and ERY B population between different arms of the experiment, while no significant difference was found in the fully differentiated population ERY C as shown in Figs. 17 &18.

ERY A and ERY B population in BM in different arms of the experiment.

FACS profile Erythropoietic population in spleen

Erythropoietic population was also found in the spleen indicating possible EMH complementing the BM regeneration (Fig. 18).

Comparing the erythropoietic population in Naive and IM treated mice (BM vs spleen)

It is clearly demonstrated that erythropoiesis is significantly higher in the spleen compared to the BM in the IM treated mice as compared to normal erythropoiesis rate in BM in the naive mice (Fig. 19).

BM and spleen cellularity in different arms of the experiment.

To assess the hematopoietic stem cells population, firstly nucleated BM and spleen was counted to evaluate their cellularity, next FACS analysis was done to estimate HSC frequency to find the net number of HSC in the whole body BM and spleen (Fig. 20).

HSCs were characterized by FACS (Fig 21).

BM and spleen HSCs frequency were estimated in different arms of the experiment

(Fig. 22).

Absolute HSCs count in spleen was compared with that of BM in different arms of the experiment (Fig. 23).

Peripheral blood plasma proteins screening by ELISA assays

Our results clearly showed that PSC injected remotely (IM or SC) had still a significant effect on the BM of the irradiated animal. Therefore, it is expected that the hPSC are likely to secrete different agents capable of triggering and encouraging regenerative processes and hematopoiesis. ELISA assays were performed for selected relevant proteins in different time points following irradiation and PSC treatment. Since previous unpublished experiments found that the highest secretion of the cytokines from IM injected PSC is detectable on days 6-9 following irradiation, proteins of interest were examined on day 8 (Figs. 24 and 25) as well as at later time points. Therefore the mouse VEGF levels in plasma in the different arms of the experiments were measured also at days 11 and 23 following irradiation, showing no significant difference is the plasma VEGF levels (not shown).

Other ELISA performed.

Additional ELISA were performed to identify the presence of human and mouse PLGF (VEGEF analogue secreted from placenta) and Human VEGEF. No detectable levels of these factors were monitored (data not shown).

Histological bone sections for the evaluation of BM cell density

Bones were extracted from mice with different treatment and processed for histology and stained for H&E. The density of the BM was found to be significantly higher in the irradiated treated mice, as compared to the placebo treated mice (Fig. 26).

Evaluation of extra-medullary hematopoiesis (EMH) through spleen histology.

In some of the mice treated with PSC the spleens seemed to be enlarged. Histology with H&E staining showed numerous foci of EMH in the irradiated treated groups as compared to irradiated and placebo treated mice (Fig. 27).

Example 3. The effect of placental stromal cells (f-PSC) on hair protection after exposure to irradiated by high dose of radiation.

Fig. 28 shows the follow-up of mice' weight following 16Gy of head-only irradiation by a linear accelerator (LIN AC). As treatment, the mice were injected locally (subcutaneously) to the neck area with expanded isolated fetal placental stromal cells (f-PSC) on days 18 and 53 after irradiation. The irradiation retarded the growth rate of the mice, probably, among other causes due to the damages to the oral/ esopharinx tissues. In the non- PSC-treated mice a more severe weight loss was recorded at later stages from day 50. In mice injected with PSC no such drop in weight was recorded at this period. A recovery of the non- PSC-treated mice followed due to the introduction of wetted food, probably since the mice ate less due to radiation induced effects on the teeth. The high dose irradiation induced late effects to the skin, as recorded by dry desquamation, progressive hair loss and loss of hair pigmentation with time (Fig. 29). On day 46 onward a major effect of radiation was evident as hair loss in all irradiated mice. In the mice injected locally (subcutaneously) to the neck area with f-PSC (days 18 and 53 after irradiation) regenerating non-pigmented hair is seen in the center of the irradiation field. In non-PS C -treated mice a large area of baldness is seen with minimal regeneration of the hair. On day 74 following the 2^nd PSC injection a full regeneration of mostly pigmented hair is seen with no baldness. In the non-PS C -treated mice a poor recovery of the hair is seen with mostly non-pigmented low density thin hair Altogether, this demonstrates the high effectiveness of the PSC treatment for inducing regeneration and recovery of the skin and hair.

Mice injected subcutaneously to the neck area with PSC on day 18 and 53 after 18Gy irradiation had much higher density of more normal looking hair follicles. Typical changes in hair follicle density as recorded in histological sections on day 74 are presented in Fig. 30A. The irradiated only skin had lower density of enlarged inflamed follicles with signs of fibrosis. The density of the follicles was assayed in histological slides of all mice and the averaged data are presented in Fig. 30B. Clearly, while there was no significant difference between the density of the follicles in the control injected with PSC only, in the non-PSC treated there was very significant decrease in the number of the follicles at that time point after initial damage which was recorded in earlier time points (as demonstrated in Fig. 29).

In a further ongoing experiment the mice were irradiated by 18Gy with low penetrating irradiation by electrons, exposing only the back skin area (see irradiation set-up in Fig. 31A). This procedure assured no exposure of other tissues and organs to radiation. A clear process of recovery is seen only in the skin subcutaneously treated with PSC on days 18 and 35. The skin surface follow-up is seen in Fig. 31B, with selected representative mice, showing the different of skin regeneration between the PSC treated and non-treated following 18Gy irradiation.

The experiments will terminate on -day 60 and skin samples will be taken for histology to evaluate the skin follicles density and condition. SECTION II. Isolation of human fetal placental stromal cells and their use in treatment of multiple sclerosis.

Isolation of placental stromal cells.

A placenta was obtained in sterile conditions immediately following caesarian section of healthy male child by a healthy mother, with her written consent. The placenta was then rinsed extensively and small fractions from the surface decidua of the chorion in the fetal side were cut into small pieces which were then further minced to smaller tissue fragments. Those were briefly digested twice with trypsin immersed in medium with highly diluted (1%) fibrinogen (Omrix) then distributed on a plastic dish and sprayed with thrombin (lOOU/ml) to adhere them to the plastic surface of culture dish, the pieces were then cultured with low glucose DMEM medium with 10% FCS, 1% antibiotics (penicillin streptomycin, optionally with neomycin) and 1% glutamine (all from Biological Industries (BI) Beit Haemek, Israel). The PSC from these selected tissue fragments spontaneously migrated to the plastic surface to reach a uniform culture of pure stromal cells (CD91+, CD29+, CD 105+ partially positive and CDl lb, CD34 and CD45 negative within 2 passages within 1-2 weeks to (Figs. 32A-F). The cells had very low HLA-DR expression. Fish staining of the X/Y centromeres enables to identify the source of the cell from the placenta, mother's being X/X and new born X/Y. Cultures of isolated cells in which most where from fetal source (>80%) were used for the experiments. The cells were then expanded for 5-8 passages before their injection. They were harvested for implantation and counted, rinsed and re-suspended in PlasmaLyte A (Baxter Healthcare). Cell preparation with a viability of <90% was injected IM within 1-2 hours from their harvest.

The protocol may be performed as follows:

1. Once washed from blood, the umbilical cord is cut off and disposed and the placenta is soaked in phosphate buffered saline (PBS) or normal saline +10% antibiotics

(such as neomycin, streptomycin and penicillin) for at least 10 minutes.

2. Using a sterile surgical tool the placenta is spread open with the fetal membrane side up.

4. Maternal or mix PLC samples are collected in deeper layers 5. For neonate samples 2-4 mm deep from the fetal membrane (decidua) are collected, internal spongy tissues and surface epithelium are scrubbed away.

7. The tissue fragments pieces are processed with 0.02% trypsin/EDTA for half an hour (possibly in rotation in rotation in a C02 incubator).

9. The pieced are either (a) Immediately processed as follows; or (b) Incubated in rotation in a C0₂ incubator for 24-72 hrs and then processed as follows:

10. The liquid is discarded and the pieces washed with medium to stop trypsinisation.

Immediate direct cell migration protocol

1. The rinsed small tissue fragments, preferably but not limited to 1-2 mm are mixed with small amount of dilute sterile fibrinogen (~lmg/ml) and spread on a tissue culture plastic plate/flask for a few minutes .

4. The surface of the plate with the dispersed tissue fragments is sprayed briefly with the thrombin solution in a sterile environment and the flasks are put in the C0₂ incubator at 37°C for 10-30 min till the activated fibrin clot is stabilized, immobilizing the tissue fragments to the surface.

5. The plate is then covered with medium and the desired stromal/matrix dependent mesenchymal stromal cells start migrating from the attached tissues within 7-10 days of incubation.

Cell migration protocols after tissue pieces suspension

1. Extracted tissue pieces after trypsinisation processing can be held in 50 ml tubes with medium and put into C02 incubator in 37°C in rotation for more than 48 hours. 2. The tissue pieces can then be washed and plated as above (Immediate direct cell migration protocol).

The resulting cell population comprises essentially only placental stromal cells staining positive for CD105; CD44; CD29, CD90, CD146 and CD166 markers and is essentially free of cells of hematopoietic and endothelial origin (Fig. 7).

Example 4. Cell migration protocols after cryo-preserved tissue pieces

It is of prime interest to develop methods in which cells could be extracted from deep frozen samples at different time points after their storage. This could be implemented for both normal tissues and tumour samples. No current successful method is available.

We propose a new medium for such cryo-preservation of tissue fragments which will allow using the tissue/tumour fragments for isolation of their cells long after their harvest.

1. After processing, tissue pieces are preserved in the following medium:

Cryo - RG: (50% FCS +10% DMS + 40% Cryo-mix)

2. Cryo-mix (1L) includes: DMEM( 100ml), water(823ml), Sodium Bicarbonate(26.6ml), HEPES(25ml), Folic acid solution (2mg/ml), L-Glut(20ml), Sodium hydroxide solution 4.4ml), Chondroitin sulphate (25.5gr).

3. Cells were placed overnight in -80°C and then placed into liquid nitrogen storage.

After normal thawing procedure, part of the tissue pieces went through migration procedure as mentioned above. The other part left for two days in suspension and then plated through migration procedure.

As can be seen in Figs. 32A-F, no migrating was seen on day 18 post thawing and direct plating (A) or on day 18 post thawing and held in suspension (48 hrs; B) and then plating. However, cells could be seen migrating from tissue pieces frozen in cryo-RG, 11 days post thawing (direct plating; C) 18 days post thawing (direct plating; D), slightly 11 days post thawing (held in suspension 48 hrs and then plating; E) and 18 days post thawing (held in suspension 48 hrs and then plating; F). Example 5. Comparison between existing methods and the migration method of the present invention.

As can be seen in Figs. 2 to 4, the new method has several advantages of existing methods:

1. The migration method had a lag time of 5 -10 days to show the first migrating cells from the tissue fragments, though it was proven to be the most effective and bring the highest yield of the target cells and a quicker expansion of their population.

2. The cells are of more uniform stromal cells with minimal contamination of endothelial cells.

3. The regularly used digestion procedure showed immediately a very few mixed populations of attached cells forming very slowly foci with much lower yield and longer expansion period.

4. The digestion method did not allow delayed extraction of the cells from the tissue fragments

Example 6. Treatment of experimental autoimmune encephalomyelitis.

6.1 Experimental induction of autoimmune encephalomyelitis with intra cranial or IM hPSC delivery.

Mice were immunized by subcutaneous injection of 300μg of myelin oligodendrocyte glycoprotein (MOG) peptide corresponding to amino acids 35-55, diluted in normal saline (0.9% NaCl) and emulsified with complete Freund's adjuvant. Bordetella pertussis toxin (300 ng; List Biological Laboratories, Inc.) was diluted in 0.2 ml normal saline and injected intraperitoneally immediately after MOG immunization and 2 days later. Clinical signs of EAE typically appeared 10-12 days post-immunization, reaching the peak neurological disability within additional 6-10 days. Following the induction, 85% of the animals developed the disease. Disease progression and severity was scored daily as follows: 0 = normal; 1 = limp tail; 2 = ataxia; 3 = partial hind limb weakness and/or inability to flip over; 4 = hind limb scored independently by three researchers at turns, in a non-blinded manner. The data was analyzed by two of them at the end of the experiment. For relapse induction mice were immunized again with MOG peptide and pertussis toxin, starting on day 40, as previously described ^⁴¹⁾. Intra-muscular implantation to the quadriceps and hamstrings in total amount of 2xl0⁶ hPSC in ΙΟΟμΙ of PlasmaLyte A was performed on day 0 and 5 after EAE induction for studying preventive effect, and on days 11 and 15 after EAE induction for studying treatment protocol. Intra cranial stereotactic implantation (bregma 0mm, lateral 0.5mm) of 0.5X10⁶ hPSC in ΙΟμΙ of PlasmaLyte A was performed on day 7 post EAE-induction. Control mice were followed without intervention. The mice were anesthetized for the invasive procedures with ketamine/xylazine.

The animals were scored daily for neurological symptoms, and perfused on day 28 or 60 for histopathological analysis.

6,2 Intracranial implantation of hPSC attenuates EAE

To examine whether hPSC exhibit effective immuno-modulatory properties in the animal model of MS, we first performed an intracranial implantation of hPSC to periventricular white matter tracts of EAE mice (n=l l and 12 per group). hPSC were implanted on day 7 after EAE induction, prior to appearance of clinical signs. A significant disease attenuation was observed in the hPSC implanted EAE mice, as compared to non- implanted mice (p<0.0005, Fig. 33A). The mice were sacrificed for histopathological evaluation at day 25 post EAE induction (18 days after cell implantation). The hPSC were detected by immunostaining for HLA-ABC at the site of implantation in the mice brains (Fig. 33B) and in the ventricles. Typical inflammatory process was found in the spinal cords and brains of EAE mice (Fig. 33C). In the cell implanted mice, a strong local inflammatory response was noted around the site of implantation, as well as in the brain meninges (Fig. 33D & E). The number of inflammatory infiltrates was quantified throughout the spinal cord. There were 4.42+1.18 infiltrates per section in control mice, as compared to 4.02+0.64 infiltrates per section in the hPSC-treated mice (n.s.). In control mice the inflammatory process involved also the spinal cord parenchyma (Fig. 33F & G), whereas in hPSC-treated mice it was almost exclusively meningeal (Fig. 33H). These differences may reflect the meningeal immune response towards the graft itself.

Figs. 32A-F show a comparison between a known (B, C) and the new (D-F) method for isolating matrix-dependent cells. (A) Time line representation for migration separation method. Figures B-C show micrographs showing cells obtained with the known digestion process at 3 days (B) and 5 days (C) after digestion. Figures D-F show cells migrating out of the tissue fragment on day 5 (D), day 7 (E) and day 17 (F) after the attachment of the fragments to the surface. Compare with brief description of figs. 2-4.

Fig. 34 shows the effect of collagenase treatment of fresh and thawed tissue. It can clearly be seen that collagenase treatment (0.75mg/ml) dramatically increases the number of cells migrating out of the source tissue, but not from fresh tissue.

SECTION III.

Example 7. Treatment of diabetes mellitus type II by administration of mesenchymal stromal cells

In order to examine the possible regeneration of pancreas, the Cohen rats diabetes model was used. Diabetes was induced by a sugar reach diet. In the cell-treated group rats PSC were injected IP. The damaged pancreas tissues in the mice treated with IP PSC were significantly regenerated almost to their original size (not shown).

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Claims

1. A method for preparing a population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin, the method comprising:

(i) obtaining a tissue comprising matrix dependent mesenchymal stromal

cells;

(ii) optionally briefly contacting fragments of said tissue with a solution

comprising a protease;

(iii) optionally keeping the fragments for 48 hrs or more in motion while

suspended in growth medium at 37°C and an atmosphere with about 5% C0₂;

(iv) contacting the fragments of said tissue with a solution comprising

fibrinogen, and then evacuating excess fibrinogen solution;

(v) transferring the fragments to an unmodified plastic surface;

(vi) dispersing a solution comprising thrombin on said plastic surface and

fragments thereby forming a thin fibrin film around the fragments that immobilizes the fragments to the plastic surface;

(vii) covering the fragments with growth medium;

(viii) harvesting matrix dependent mesenchymal stromal cells that have

migrated out from the fragments,

thereby preparing a population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin.

2. The method of claim 1, wherein said fragments of tissue are made from fragments or bulk tissue previously immersed in a cryo-preservation solution, frozen by slowly reducing the temperature to -75°C to -80°C, stored in liquid nitrogen, thawed and treated with a protease of step (ii) selected from trypsin and/or collagenase, wherein said cryo-preservation solution comprises:

fetal calf serum (FCS; e.g. 50-90%) or human plasma albumin (HAS; e.g. 5-50%) in addition to dimethyl sulfoxide (DMSO) and optionally polyethylene glycol or cell culture medium.

3. The method of claim 2, wherein said cryo-preservation solution comprises:

(i) 90% fetal calf serum (FCS) + 5% dimethyl sulfoxide (DMSO) + 5% polyethylene glycol (PEG);

(ii) 90% FCS+10% DMSO;

(iii) 10-20% human plasma albumin (HSA) + 10% DMSO + 70-80% cell culture medium; or

(iv) 10-20% HAS + 5% DMSO + 5% PEG + 70-80% cell culture medium.

4. The method of claim 2 or 3, wherein said collagenase is used at a concentration of about 0.2-3 mg/ml.

5. The method of any one of claims 1 to 4, wherein said tissue is selected from placenta, breast, liver, kidney, tumors, uterus, bone, cartilage, spleen and skin.

6. The method of claim 5, wherein said tissue is placenta.

7. The method of claim 6, wherein said tissue is obtained from the fetal side of the surface decidua of the chorion.

8. The method of any one of claims 1 to 7, wherein said matrix dependent mesenchymal stromal cells are stromal cells, preferably placental stromal cells.

9. The method of claim 8, wherein more than 95% of said cells are of fetal origin and the population is essentially free of cells of maternal origin.

10. The method of claim 8 or 9, wherein said stromal cells are human cells expressing CD 105, CD44, CD29 and CD90.

11. The method of any one of claims 1 to 10, wherein said hematopoietic and endothelial origin are human cells expressing significant levels of CD45, CD19, CDl lb, HLA-DR and CD34.

12. The method of claim 8, wherein said cells of hematopoietic and endothelial origin make up less than 1% of the population of matrix dependent mesenchymal stromal cells.

13. The method of any one of claims 1 to 12, wherein said solution comprising fibrinogen comprises about 0.5-3% fibrinogen, e.g. about 1% fibrinogen.

14. The method of any one of claims 1 to 13, wherein said solution comprising thrombin comprises at least about 10 U/ml thrombin, e.g. about 100 U/ml thrombin.

15. The method of any one of claims 1 to 14, wherein said protease is trypsin, and said solution comprising trypsin comprises e.g. 1 % trypsin.

16. The method of any one of claims 1 to 15 for isolating placental stromal cells from placenta, comprising:

(i) obtaining a tissue from the fetal side of the surface decidua of the

chorion of placenta;

(ii) briefly contacting fragments of said tissue with a solution comprising

trypsin, e.g. 1 % trypsin; and optionally rinsing the fragments, cutting the fragments into finer fragments and contacting said finer fragments with a solution comprising trypsin following a rinsing and washing;

(iii) optionally keeping the fragments for 48 hrs or more in suspension in

rotation while suspended in growth medium at 37°C and an atmosphere with about 5% C0₂

(iv) briefly contacting fragments of said tissue of (ii) or (iii) with a

solution comprising about 0.5-3% fibrinogen, e.g. about 1% fibrinogen, and removing excess fibrinogen leaving only a film on the fragments;

(v) transferring the fragments to an uncoated plastic surface; remove

excess liquid with fibrinogen;

(vi) spraying a solution comprising at least about 10 U/ml thrombin, e.g.

about 100 U/ml thrombin, on said plastic surface and fragments thereby forming a thin fibrin film around the fragments that immobilizes the fragments to the plastic surface;

(vii) covering the fragments with growth medium; (viii) harvesting matrix dependent mesenchymal stromal cells that have migrated out from the fragments;

thereby preparing a population of placental matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin.

17. A population of matrix dependent mesenchymal stromal cells essentially free of cells of hematopoietic and endothelial origin obtained by the method of any one of claims 1 to 16.

18. The population of matrix dependent mesenchymal stromal cells of claim 17, wherein more than 95% of said cells are of fetal origin and the population is essentially free of cells of maternal origin.

19. The population of matrix dependent mesenchymal stromal cells of claim 17 or 18, formulated for subdermal, intraperitoneal, intra-cerebroventricular, intrathecal, intravenous or intramuscular injection.

20. A pharmaceutical composition comprising the population of matrix dependent mesenchymal stromal cells of any one of claims 17 to 19 and a pharmaceutically acceptable carrier.

21. The pharmaceutical composition of claim 20 formulated for subdermal, intraperitoneal, intra-cerebroventricular, intrathecal, intravenous or intramuscular injection.

22. A population of matrix dependent mesenchymal stromal cells of any one of claims 17 to 19 or pharmaceutical composition of claim 20 or 21 for use in promoting a regenerative process in damaged tissue organ or reducing an inflammatory response associated with disease or injury.

23. The population of matrix dependent mesenchymal stromal cells or the pharmaceutical composition for the use of claim 22, wherein said promoting a regenerative process of damaged tissue comprises treatment or mitigation of acute radiation syndrome (ARS) following exposure to high dose ionizing radiation, or failing bone marrow.

24. The population of matrix dependent mesenchymal stromal cells or the pharmaceutical composition for the use of claim 23, wherein said damaged tissue is bone marrow.

25. The population of matrix dependent mesenchymal stromal cells or the pharmaceutical composition for the use of claim 23, wherein said damaged organ is hair follicles, or for the use in treating alopecia caused by exposure to high dose ionizing radiation.

26. The population of matrix dependent mesenchymal stromal cells or the pharmaceutical composition for the use of claim 22, wherein said promoting a regenerative process of damaged tissue comprises treatment or mitigation of alopecia.

27. The population of matrix dependent mesenchymal stromal cells or the pharmaceutical composition for the use of any one of claims 22 to 26, wherein said population of matrix dependent mesenchymal stromal cells is injected intramuscularly, subdermally or intraperitoneally .

28. The population of matrix dependent mesenchymal stromal cells or the pharmaceutical composition for the use of claim 22, wherein said disease or injury is selected from an autoimmune disease, an inflammatory disease, allergy, ischemic brain injury, critical limb ischemia, myocardial infarction, an immune-mediated neurodegenerative disorder, a metabolic disorder, graft- versus-host disease (GVHD) or transplanted organ rejection.

29. The population of matrix dependent mesenchymal stromal cells or the pharmaceutical composition for the use of claim 28, wherein said autoimmune disease is selected from multiple sclerosis, Crohn's disease, ulcerative colitis and type 1 diabetes mellitus.

30. The population of matrix dependent mesenchymal stromal cells or the pharmaceutical composition for the use of claim 28, wherein said damaged organ is damaged pancreas or said metabolic disorder is diabetes mellitus type 2.

31. A population of matrix dependent mesenchymal stromal cells for the use in regeneration of damaged pancreas or damaged hair follicles.

32. The population of claim 31, wherein said damaged hair follicles are damaged following exposure to high dose radiation.

33. The population of matrix dependent mesenchymal stromal cells or the pharmaceutical composition for the use of any one of claims 22 to 33, wherein more than 95% of said population of matrix dependent mesenchymal stromal cells are cells of fetal origin and essentially free of cells of maternal origin.

34. A composition for cryo-preservation of biological tissue, said composition comprising fetal calf serum (FCS; e.g. 50-90%) or human plasma albumin (HAS; e.g. 5-50%) in addition to dimethyl sulfoxide (DMSO) and optionally polyethylene glycol or cell culture medium.

35. The composition of claim 35, comprising

(ii) 90% FCS+10% DMSO;

(iv) 10-20% HAS + 5% DMSO + 5% PEG + 70-80% cell culture medium.

36. A method for preserving biological tissue such as normal tissue or tumor tissue, comprising immersing said biological tissue in a composition of claim 34 or 35, and slowly reducing the temperature to -75°C to -80°C, thereby providing frozen biological tissue and storing the frozen biological tissue in liquid nitrogen.