METHODS FOR OBTAINING STEM CELLS

Abstract

The present invention relates to methods for obtaining stem cells from mammalian cadavers, methods for obtaining or purifying stem cells from a sample likely to contain non-stem cells, methods of regeneration of injured tissues and methods of treatment.

Claims

1-30. (canceled)

31. A method for culturing mammalian stem cells comprising using an anaerobic cell culture system for culturing isolated mammalian stem cells at an oxygen concentration equal to or less than 0.1% and at a temperature of 1-6 C. during a period of time of at least 2 days.

32. The method according to claim 31, wherein mammalian stem cells are cultured at 1-6 C. for 2 days to 30 days.

33. The method according to claim 31, wherein the mammalian stem cells are hematopoietic stem cells, neural stem cells or muscle stem cells.

34. The method according to claim 31, wherein isolated mammalian stem cells are cultured at an oxygen concentration equal to or less than 0.1% and at a temperature of 1-6 C. during a period of time of at least 4 days.

35. A method for regenerating an injured tissue in a subject in need thereof, said method comprising: a) maintaining a biological material which usually comprises stem cells at an oxygen concentration equal to or less than 0.1% and at a temperature of 1-6 C.; b) obtaining stem cells by selecting viable cells, wherein viable cells are stem cells; and c) administering the stem cells obtained at step b) to said subject.

36. The method according to claim 35, wherein the stem cells are mammalian stem cells and wherein, at step c), the stem cells are transformed or transfected with a vector, especially a vector of expression, or transduced with a virus vector, said vector comprising at least one polynucleotide sequence of interest, before being administered to said subject.

37. A method for regenerating an injured tissue in a subject in need thereof, said method comprising: a) maintaining a biological material which usually comprises stem cells at an oxygen concentration equal to or less than 0.1%, wherein the biological material is muscle sample, especially skeletal or smooth muscle sample; b) obtaining stem cells by selecting viable cells, wherein viable cells are muscle stem cells; and c) administering the stem cells obtained at step b) to said subject.

38. A method for regenerating an injured tissue in a subject in need thereof, said method comprising: a) maintaining a biological material which usually comprises stem cells at an oxygen concentration equal to or less than 0.1%, wherein the biological material is selected from the group consisting of brain, spinal cord and meninges sample; b) obtaining stem cells by selecting viable cells, wherein viable cells are neural stem cells; and c) administering the stem cells obtained at step b) to said subject.

39. A method according to claim 37, wherein the stem cells are mammalian stem cells, and wherein, at step c), the stem cells are transformed or transfected with a vector, especially a vector of expression, or transduced with a virus vector, said vector comprising at least one polynucleotide sequence of interest, before being administered to said subject.

40. A method according to claim 38, wherein the stem cells are mammalian stem cells, and wherein, at step c), the stem cells are transformed or transfected with a vector, especially a vector of expression, or transduced with a virus vector, said vector comprising at least one polynucleotide sequence of interest, before being administered to said subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0161] The present invention will be further illustrated by the additional description and drawings which follow, which refer to examples illustrating the obtaining of stem cells from cadavers, the enrichment of stem cells by maintaining them at 4 C. in the absence of oxygen and the use of hematopoietic stem cells and muscle stem cells obtained by this method of enrichment for regenerating bone marrow of irradiated mice or skeletal muscle of mice. It should be understood however that these examples are given only by way of illustration of the invention and do not constitute in anyway a limitation thereof.

[0162] FIG. 1 illustrates the percentage of animals giving rise to myogenic cultures (after 14 days in culture) every 4 days after death. N=10 at each time point.

[0163] FIG. 2.A. illustrates the number of viable stem cells extracted from one mg of muscle of mouse cadaver up to 8 days after death. The number of viable cells was evaluated by Flow Cytometry on calcein incorporation and propidium iodide exclusion (n>5 at each time point). The number of day after death is shown on the x-axis and the number of cells labelled with calcein is shown on the y-axis. This figure shows a linear decreasing of cells alive in muscle tissue after 4 and 8 days after death.

[0164] FIG. 2.B. illustrates the number of viable satellite cells (muscle stem cells) in a sample extracted from the tibialis anterior muscle of Tg:Pax7-nGFP transgenic mouse cadaver (n>5 at each time point). The number of viable satellite cells was evaluated by Flow Cytometry on GFP expression. The number of day after death is shown on the x-axis and the number of GFP positive cells is shown on the y-axis.

[0165] FIG. 2.C. illustrates the enrichment of Pax7 expressing satellite cells extracted from the cadaver of knock-in mouse Pax7.sup.LacZ/+. The enrichment was evaluated by measuring the percentage of X-Gal positive cells. The number of day after death is shown on the x-axis and the number of X-Gal+ cells is shown on the y-axis.

[0166] FIG. 2.D. illustrates the proportion of satellite cells that survive after death in a sample extracted from the cadaver of juvenile Tg:Pax7-nGFP transgenic mouse. The juvenile mice in which all SCs are activated have been used. This graph show the absence of decreasing of SCs between day 0 and day 4 post mortem showing that activated SCs can also resist 4 days post mortem. Eight days post mortem very few cells remain alive. Results are expressed as number of cells per mg of tissue. The number of day after death is shown on the x-axis and the number of GFP positive cells is shown on the y-axis.

[0167] FIG. 2.E. illustrates the viability of post mortem stem cells in a quiescent cell state Satellite cells were enumerated by FACS from resting or injured TA muscle of Tg:Pax7-nGFP mice at day 0 or 4 days post mortem (pm).

[0168] FIG. 3.A. is a graph which illustrates the expression level of two satellite cell key genes, i.e. Pax7 and MyoD, in skeletal muscle sample from mouse cadaver (n=5 at each time point). Gene expression was assessed by real time PCR (Taqman). The number of day after death is shown on the x-axis and the expression of Pax7 and MyoD is shown on the y-axis. 2-deltaCT indicates the threshold after which the signal is considered as significant and normalize with ubiquitously expressed gene (GAPDH: Glyceraldehyde 3-phosphate dehydrogenase).

[0169] FIG. 3.B. is a graph which illustrates the expression level of markers of myogenic cell commitment, i.e. Troponin, MyoD, Myogenin and, of markers of stem-like state, i.e. Pax7 and CD34 in skeletal muscle sample from mouse cadaver (n=5 mice for each gene). Gene expression was assessed by real time PCR (Taqman). The marker which is quantified is shown on the x-axis and the expression is shown on the y-axis. 2-deltaCT indicates the threshold after which the signal is considered as significant and normalize with ubiquitously expressed gene (TBP:tata binding protein)

[0170] FIG. 3.C. illustrates the percentage of clonogenicity of satellite cells (i.e. number of cells forming colonies) after FACS cell sorting and plating in 96 well dishes at day0, 4 and 8 post mortem. The number of day after death is shown on the x-axis and the expression of the percentage clonogenicity is shown on the y-axis.

[0171] FIG. 3.D. illustrates the time culture which is necessary to achieve the first division as a function of the number of day following death. The time culture is shown on the y-axis and the number of day after death is shown on the x-axis. This graph shows that cells that remain alive 4 and 8 days post mortem make their first division later than cells collected immediately after death.

[0172] FIG. 3.E. illustrates the measurement of the energizing state of satellite cells obtained from Tg:Pax7-nGFP transgenic mouse cadaver as a function of the number of day following death. Comparison was performed between day0, day4 and day8 post mortem. The number of active mitochondria in the different conditions was assessed by Mitotracker. The relative intensity of fluorescence which is the result of the staining of active mitochondria with Mitotracker is shown on the y-axis, and the ratio Day 0/Day X after death is shown on the x-axis.

[0173] FIG. 3.F. illustrates the quantity of ATP produced in satellite cells obtained from Tg:Pax7-nGFP transgenic mouse cadaver as a function of the number of day following death. The quantity of ATP was determined by fluorescence using luciferase activity as a readbout. Bioluminescence is shown on the y-axis and the number of day after death is shown on the x-axis.

[0174] FIG. 4.A. illustrates the percentage of survival in lethally irradiated mice which have been transplanted with bone marrow extracted from one single femur collected 0, 2, 3 and 4 days post mortem. The number of day after death is shown on the x-axis and percentage of mouse survival is shown on the y-axis.

[0175] FIG. 4.B. illustrates the percentage of blood chimerism (% of GFP+ leukocytes in circulating blood) after direct transplantation (black bars) and after a serial transplantation (gray bars) of bone marrow. The number of days after death when bone marrow used for transplantation was harvested is indicated on the x-axis, and percentage of mouse survival is shown on the y-axis. Blood chimerism after bone marrow transplantation using cadaver BM is strictly identical to what observe at day 0 post mortem and decrease lightly when using BM from day 4 post mortem.

[0176] FIGS. 4.C-F. illustrate immunophenotyping of circulating GFP+ cells after serial bone marrow transplantation assessed by flow cytometry.

[0177] FIG. 4.C shows the percentage of GFP+ B cells (cells expressing B22O cell surface marker).

[0178] FIG. 4.D shows the percentage GFP+ T cells (cells expressing CD5 cell surface marker).

[0179] FIG. 4.E shows the percentage GFP+ granulocytes (cells expressing Gr1 cell surface marker).

[0180] FIG. 4.F shows the percentage and GFP+ myeloid cells (cells expressing CD11b cell surface marker).

[0181] These experiments demonstrate the ability of cadaver bone marrow to fully reconstitute blood compartment.

[0182] FIG. 5. illustrates the number of cells that remain alive at 4 C. in normoxia (black bars) compared to complete anoxia (gray bars) as a function of days.

[0183] FIG. 6.A. illustrates the percentage of whole bone marrow cell loss many days after anoxia at 4 C. (black bars) Four days after storage in anoxia, 95.6% of cells are lost whereas bone marrow is still transplantable at this time point see FIG. 6B.

[0184] FIG. 6.B. illustrates the percentage of blood chimerism (% of GFP+ leukocytes in circulating blood) after direct transplantation (black bars) and after a serial transplantation (gray bars) of bone marrow. Transplanted was performed with bone marrow maintain 0 hours, 1 day, 2 days, 3 days or 4 days in anoxia before being transplanted. The number of day in which bone marrow was maintained in anoxia before being transplanted is indicated on the x-axis, and percentage of blood chimerism is shown on the y-axis. Note that n>5 and that 100% of mice survive at each time point.

[0185] FIGS. 6.C-F. illustrate immunophenotyping of circulating GFP+ cells after serial bone marrow transplantation assessed by flow cytometry.

[0186] FIG. 6.C shows the percentage of GFP+ B cells (cells expressing B22O cell surface marker).

[0187] FIG. 6.D shows the percentage GFP+ T cells (cells expressing CD5 cell surface marker).

[0188] FIG. 6.E shows the percentage GFP+ granulocytes (cells expressing Gr1 cell surface marker).

[0189] FIG. 6.F shows the percentage and GFP+ myeloid cells (cells expressing CD11b cell surface marker).

DETAILED DESCRIPTION OF THE INVENTION

Example I. Material and Methods

Ethical

[0190] Human samples were collected according to guidelines recommended by the national ethical committee.

[0191] All mice were housed in a level 2 biosafety animal facility, and received food and water ad libitum. Prior to manipulations, animals were anaesthetized using intraperitoneal injection of Ketamine and Xylazine (respectively 25% and 12.5% in PBS). This study was conducted in accordance with the local and EC guidelines for animal care (Journal Officiel des Communauts Europennes, L358, Dec. 18, 1986).

[0192] Mouse Strain

[0193] C57BL/6 mice (Ma-Credo, L'arbresle, France), Tg:Pax7-nGFP mice in which satellite cells can be easily visualize by their GFP expression (Sambasivan R, Dev Cell. 2009 June; 16(6):810-21), Pax7.sup.nlacZ/+ mice in which satellite cells can be easily visualize by their LacZ expression (Ramkumar Sambasivan and Shahragim Tajbakhsh unpublished), Tg:Pax7-nGFP::Tg:CAG:PLAP (Sambasivan 2009) in which all cells constitutively express placental alkaline phosphatise and satellite cell GFP, Tg: CAG-GFP mice (C57BL/6 TgN[actEGFP]Osb YO1) in which the GFP transgene is ubiquitinously expressed under the control of a non-tissue specific promoter, chicken beta-actin with cytomegalovirus enhancer, as a cytoplasmic protein (Okabe, et al, FEBS Lett. 1997 May 5; 407(3):313-9).

[0194] Tissue Preparation

[0195] Depending of the condition used, after animal sacrifice, tissues were snap-frozen immediately in liquid nitrogen-cooled isopentane for immunohistochemistry and histological analysis, or fixed using buffered 4% paraformaldehyde prior to cryopreservation in 30% sucrose overnight at 4 C. (a procedure that keep the spontaneous fluorescence of GFP in a tissue section). Serial 7 m thick cryosections were performed for analysis.

[0196] Images were captured on a Zeiss Axiophot microscope with an Apotome (Carl Zeiss Inc., Germany) and Orca ER digital camera (Hamamatsu Photonics, Japan) using Simple PCI (C-Imaging, Compix Inc) software.

[0197] Immunohistochemistry

[0198] For human cases, 5 m cryosections of muscles from cadavers were immunostained with mouse anti-human CD56 (1:20 dilution; NHK-1-RD1; Beckman Coulter) and revealed using peroxidase Vectastain ABC kit (Vector Laboratories).

[0199] For mouse tissues, immunostainings were done without antigen unmasking. The following protocol was always used: after rehydratation of sections with PBS, non-specific protein binding was blocked with 20% goat serum and cells were permeabilised with 0.5% triton-X100 (Sigma-Aldrich, St-Louis, Mo.) 20 minutes. Incubation with primary antibody was done overnight at 4 C., and signal was revealed with secondary antibody incubated 1 hour at 37 C.

[0200] Dako Diluent buffer (Dako, Glostrup, Denmark) was used for diluting antibodies.

[0201] The following antibodies were used as primary: mouse monoclonal antibodies against human Placental Alkaline Phosphatase [8B6] (1:300, GenTex, Irvine, Calif.), M-cadherin (1:50, Alexis Biochemicals, Lausen, Switzerland), myogenin (1:50, BD Pharmingen, San Jose, Calif.), rabbit polyclonal anti-human (or mouse) desmin (1:50, Abcam, Cambridge, UK), rabbit polyclonal antibodies against mouse myogenin (1:50, Santa Cruz biotechnologies, Santa Cruz, Calif.) and Laminin-1 (1:50, Sigma-Aldrich, Saint-Louis, Mo.).

[0202] The secondary antibodies used was Cy3 conjugated donkey anti-mouse (1:400, Jackson lmmunoresearch lab., Baltimore, Pa.), FITC conjugated goat anti-mouse (1:200, Jackson), Cy3 conjugated donkey anti-rabbit (1:200, Jackson), biotinilated horse anti-mouse (1:200, Vector laboratories, Burlingame, Calif.), Cy5 conjugated donkey anti-rabbit (1:200, Jackson), FITC conjugated donkey anti-goat (1:200, Jackson) and DTAF conjugated-streptavidin (1:400, Immunotech Beckman, Brea, Calif.).

[0203] X-Gal Staining

[0204] Cytocentrifuged cells were rehydrated with PBS before fixation with PFA 4% followed by overnight incubation with X-Gal 40 mg/ml (reconstitute in Dimthylsulfoxide, Invitrogen, Paisley, UK) in a solution containing 4 mM each of potassium ferrocyanide, potassium ferricyanide, 2 mM MgCl, and 0.02% NP40 in PBS at 37 C.

[0205] Bone Marrow Transplantation

[0206] Briefly, donor BM cells were obtained by flushing 2 femurs of donor mice (various times post mortem) with RPMI medium (Invitrogen, Paisley, UK) and 0.1% heparin (Choay 5000 Ul/ml). In the case of late post-mortem cadavers the BM cell suspension was incubated with 50 g/ml DNAse I (Roche, Mannheim, Germany)I to preclude clogging of cells. After washing, retro-orbital injection of cells was done in 0.1 ml fresh mouse serum and Hanks Buffer (PAA Laboratories GmbH, Pasching, Austria) (1:1), in 9.5 Gy-irradiated, 4 week-old B6 mice (.sup.60Co rays within 1 day before BM transplantation). After transplantation, mice received 10 mg/kg/day ciprofloxacin for 10 days to prevent infection during the aplastic phase.

[0207] Flow Cytometry Analysis

[0208] To quantify the amount of engraftment, the peripheral blood mononuclear cells of transplanted mice were analyzed by flow cytometry using a Cyan cytometer (DakoCytomation, Glostrup, Denmark) 1 month post-transplantation. Red blood cells were lysed using ACK buffer (NH.sub.4Cl 0.15M, KHCO.sub.3 1 mM, Na.sub.2EDTA 0.1 mM) and immunostainings were done at +4 C. for 30 mn using rat anti mouse CD16/CD32 (Mouse BD Fc Block) (BD Biosciences) to preclude cell activation and adherence to plastic. In all the FC experiment cells were also labelled with Propidium Iodide 1 mg/ml (Sigma-Aldrich, St-Louis, Mo.) to exclude dead cells from analysis.

[0209] Leukocytes were gated on, and GFP fluorescence was measured under the fluorescein isothiocyanate channel. Specific fluorescence stainings were done using PE-Cy5-conjugated anti-Ly-6C (Gr1) (eBioscience San diego, USA), PE-conjugated anti-CD11 b (eBioscience San diego, USA), PE-conjugated anti-CD5 (BD Biosciences), PE-conjugated anti-B220 (BD Biosciences), Abs and their respective isotypes. All analyses and quantitation were performed using Summit v4.3 software from DakoCytomation.

[0210] For the assessment of active mitochondria immediately after isolation of cells by FACS Mitotracker (invitrogen, M22246) deep red at 500 nM was used for 30 minutes at 37 C. Then the intensity of far red staining was analysed.

[0211] ATP Level Measurement

[0212] For measuring the levels of ATP, cells were isolated by FACS directly in lysis buffer and maintained at 4 C. An ApoSENSOR kit from biovision (Catalog #K254-200, -1000) was used to measure ATP levels where luciferin reacts with ATP and emits signal in proportion to ATP content; emitted light was measured using luminometer (GLOMAX 20/20 luminometer promega).

[0213] FACS Cell Sorting and Analysis

[0214] MoFlo Legacy (Beckman Coulter, Brea, Calif.) was used for cell sorting and CyAn ADP for cell analysis (Beckman Coulter).

[0215] Cell Suspension Preparation from Muscle Tissue

[0216] After sacrifice, muscles from mice were carefully dissected, minced in small pieces and washed in PBS before digestion with Pronase (protease from streptomyces griseus (Sigma-Aldrich, St-Louis, Mo.) reconstituted in DMEM with penicillin Streptomycin 0.4%). All supernatants were collected and enzyme activity immediately blocked by adding 20% foetal calf serum. This procedure was performed serially until complete digestion of the tissue (4 to 5 rounds of 20 minutes digestion at 37 C.). Cells were then washed and filtered with a 40 m cell strainer before 10 minutes treatment with an antibiotic/antifungus cocktail.

[0217] Cell Cultures

[0218] Unless otherwise indicated, culture media components were obtained from GIBCO (Invitrogen, Paisley, UK) and culture plastics were obtained from TPP (Trasadingen, Switzerland). Human or mouse muscle cells were cultured from muscle samples as described previously (Chazaud et al., 2000). In standard conditions (spontaneous in vitro myogenesis), cells were grown in Ham's F12 medium containing 20% FCS (growing medium) 1% UltroserG (PALL Life Sciences, Saint Germain en Laye, France), 0.2% Vitamins, 1% non essentials amino acids 100, 0.4% Penicillin Streptomycin 10000 U/ml without serum withdrawal. In differentiating conditions, growing medium was replaced by Ham's F12 medium containing 5% FCS (differentiating medium) at time of subconfluence.

[0219] For culture without oxygen, GenBag (Biomerieux, Craponne, France) devices were used.

[0220] RNA Extraction, RT and qPCR

[0221] Total RNA was extracted from cells isolated by FACS on GFP positivity directly in lysis buffer using the Quiagen RNAeasy Micro purification Kit. 400-600 ng of DNAse-treated (Roche). RNA was processed for random-primed reverse transcripion using the SuperScript II reverse transcriptase protocol of Invitrogen. The cDNAs were then analyzed by real-time PCR using Taqman universal Master Mix and an ABI Prism 7700 (Perkin-Elmer Applied Biosystems) and a StepOnePlus (Applied Biosystems). TBP reference transcript levels were used for the normalisation of each target within each sample (=CT). Custom primers were designed using the Primer3Plus online software.

[0222] Statistical Analysis

[0223] In all experiments the n value was at least 5. The t test was used for statistical analyses (GraphPad-InStat software). P<0.05 was considered significant.

Example II. Stem Cells Survive for Extended Periods Post Mortem

[0224] To determine how long muscle cells would survive in dead tissue, human cadavers were obtained from the centre du don des corpsFacult de Mdecine Paris Descartes. After death, cadavers were store at 4 after an initial and variable period lasting from several hours to 24 hours at room temperature. In all cases (n=16) patients were from 57-95 y.o. in age (mean 84 y.o.). A deltoid muscle biopsy (2 grams) was performed from 6-17 days post mortem. None of the patients were suffering from neoplasia. Histological analysis of the muscle showed a necrotic appearance and chromatin from myonuclei usually appeared leaky. CD56 immunostaining which labelled satellite cells (SCs) (i.e. muscle tem cells) in human showed a few positive cells were not necrotic, but they exhibited a compact appearance.

[0225] Mononuclear cells were extracted from muscle biopsies using standard protocols (Chazaud B, et al. Exp Cell Res. 2000; 258: 237-44.) and cultured for two weeks in gelatin coated dishes and in a classical medium composed with HamF12, 20% fetal calf serum, 0.4% penicillin/streptomycin, 1% ultroserG, 0.2% vitamin, 1% non essential amino-acids. In all cases, including latepost mortem time point (17 days), after a maximum of 4 days, a few cells were observed that were attached to the bottom of the dish. They grew slowly from small colonies and when the density reached a critical threshold, some cells align to fuse. Two weeks post plating, differentiating medium (HamF12, 5% normal horse serum, 0.4% penicillin/streptomycin, 0.2% vitamin, 1% non essential amino-acids) was added to the culture and cell fused forming numerous myotubes.

[0226] Immunostainings confirm that more than 90% of the attached cells forming small colonies were expressing the myogenic marker Desmin. This was also the case at later stages when these cells fused and differentiated into myotubes, expressing both Desmin and the differentiation transcription factor Myogenin. Due to the extensive decomposition of tissues, we were not able to obtain cadavers after 17 days post mortem.

[0227] To test the survival potential of SCs in muscle samples, we take advantage of organ donors with beating heart in who we perform a surgical muscle biopsy. These Donors were younger in age (n=15; from 41-77 y.o., mean 57 y.o.). We kept the muscle sample in a buffered medium (DMEM, 1 mM HEPES, 0.4% penicillin/streptomycin), at 4 C. in a sealed container. The time of tissue sampling (i.e. number of days between the sampling and the culture) was noted (see Table I below).

TABLE-US-00001 TABLE I D2 D4 D6 D10 D14 D20 D25 D30 D35 D40 D50 D55 D60 D77 F 56 y.o. + + + F 58 y.o. + + F 43 y.o. + + M 50 y.o. F 59 y.o. + + F 57 y.o. + + + + F 74 y.o. + M 69 y.o. + + + + + M 55 y.o. F 43 y.o. + + + M 65 y.o. + + M 54 y.o. M 55 y.o. + + F 41 y.o F 77 y.o + +

[0228] As observed in cadavers, and from day 4 post-biopsy, muscle exhibited a necrotic appearance with some remaining CD56 immunopositive and other compact small cells adjacent to myofibers. Depending of the size of the sample, culturing muscle cells was possible many days after sampling. The samples were assayed regularly from day 2-77. Prior to day 30 post-sampling, the cultured cells yielded large numbers of cells, the majority (>80%) being myogenic as assessed by the formation of myotubes that expressed Myogenin and Desmin. After 35 days post-sampling viable cells were no longer obtained (assayed for 15 days in culture).

[0229] Similar results were obtained with mouse cadavers. C57BL6 mice (n=10 per time point) were sacrificed using CO.sub.2 and kept for several days at 4 C. (FIG. 1). Skeletal muscles displayed a necrotic and oedematous appearance with some remaining M-cadherin expressing cells a marker for SCs. Muscle SCs were cultured up to 10 days after isolation. One hundred percent (n=10) of the cases gave rise to a large number of SCs. After 10 days post mortem, most of the cases did not yield viable cells in culture, in part because of the contamination of the medium by bacteria arising during tissue decomposition. Mouse cadavers were more sensitive to bacterial proliferation (even at 4 C.) than humans. Up to ten days post mortem, all cultured cells were myogenic as assessed by the formation of myotubes and the expression of myogenic markers.

Example III. Characterisation of Cell Types that Survive after Organismal Death

[0230] To determine if stem cells have a greater capacity to survive after organismal death, cell suspensions obtained from cadavers were stained with calcein that labels only live cells and evaluated the number of cells that remained alive by flow cytometry (FC). As shown in FIG. 2.A. the number of cells incorporating calcein per mg of tissue (n=7 animals at each time point) varies from 2678718 at day 0 to 82033 at day 4 and 17922 at day 8 post mortem.

[0231] To determine the number of viable SCs in a tissue after organism death, the Tg:Pax7nGFP mice were used in order to take advantage that all their satellite cells are GFP-positive and that SCs could be prospectively isolated by FACS based on GFP epifluorescence (Sambasivan R, Dev Cell. 2009 June; 16(6):810-21). The number of SCs in one Tibialis anterior (TA) muscle was enumerated every four days after death from 8 week old mice kept at 4 C. As shown in FIG. 2.B. four days after death, 50% of SCs remained in the TA. Eight days after death, 30% of the SCs survived in the TA. By 12 or 16 days post mortem only a few viable SCs remained (2% and 1%, respectively). All GFP+ cells isolated by FACS were viable as assayed by the exclusion of propidium iodide, and the ability to give colonies when cultured.

[0232] To determine the proportion of SCs that survive after death, the knock-in mouse Pax7.sup.nlacZ/+ was used. In this mouse, bacterial lacZ reporter gene expression reflects the expression of the Pax7 gene in all satellite cells (R. Sambasian and S. Tajbakhsh, unpublished). Between day 0 and day 4 post mortem, the proportion of X-gal positive cells increased by 3.4 fold (see FIG. 2.C.) indicating that a higher proportion of muscle stem cells were present after organismal death.

[0233] To investigate the mechanism that permits muscle stem cells to survive after organismal death, it was examined whether cellular quiescence conferred a survival advantage compared to proliferating cells. To do this, we counted SCs from Tg:Pax7-nGFP in juvenile mice before SCs entered quiescence (growth paradigm). At P10 (10 days postnatal), satellite cells proliferate actively and myofibres continue to increase in size due to the addition of nascent myoblasts (Shinin V, et al. Nat Cell Biol. 2006 July; 8(7):677-687; White R B, et al. BMC Dev Biol. 2010 22; 10:21). In this scenario, no significant drop in SCs number between day 0 and day 4 post mortem (FIG. 2.D.) was observed after sacrifice of the pups. However, dramatic decrease was observer after 8 days post mortem.

[0234] To confirm that quiescent state might confer a survival advantage to the stem cells, three cohorts of mice were examined. In the first, satellite cells were enumerated from uninjured TA muscle of Tg:Pax7-nGFP mice at 4 days post mortem (1980212 GFP.sup.+/TA; n=5 mice; see FIG. 2.E). Satellite cells in the second cohort were enumerated 5 days after a severe muscle injury with a myotoxin, thereby promoting stem cell re-entry into the cell cycle to effect myofibre regeneration. At this time, myogenic cells proliferate actively (mean 103,0008494 GFP.sup.+/TA; n=5 mice; see FIG. 2.E). The third cohort was treated similarly, but 5 days post-injury, mice were sacrificed, then satellite cells were isolated by FACS at 4 days post mortem (mean 46080 GFP.sup.+/TA, n=5 mice). Therefore, the majority of proliferating myogenic cells do not survive in post mortem tissue suggesting that cellular quiescence, in part, protects stem cells from death in post mortem tissue.

Example IV. Characterisation of Viable Cells after Organismal Death

[0235] To characterise the surviving SCs sub-population after death in mouse skeletal muscle, RT-qPCR were performed on purified satellite cells isolated by FACS and lysed directly in buffer. A library of cDNA was synthesized by reverse transcription and real time PCR (Taqman) was performed to assess the gene expression level of key satellite cell genes. The level of Pax7 and MyoD were similar in surviving cells day 4 and day 8 post mortem vs. cells extracted immediately after death (n=5) (FIG. 3.A.). Complementary experiments were carried out to further assess the extent of lineage priming and hence, the commitment status of the muscle stem cells post mortem. To do that, we performed RT-qPCR on purified satellite cells isolated by FACS. Cell determination and differentiation markers Myod and Myogenin, and the myofibre structural protein Troponin T were used as readouts for myogenic cell commitment whereas Pax7 and the receptor stem cell marker CD34 were used as readouts of the more stem-like state. Interestingly, a progressive increase in the levels of CD34 was observed from day 0 to day 8 post mortem, whereas an inverse trend was noted for MyoD, Myogenin and Troponin T transcript levels (n=5 mice/condition; see FIG. 3.B). This suggests that the post mortem derived muscle stem cells are less transcriptionally primed for myogenic commitment compared to those isolated from freshly isolated tissue.

[0236] Further, these data clearly show that the post mortem derived muscle stem cells are characterized by a lack of detectable expression of Myogenin gene, while muscle stem cells extracted immediately after death (which is considered as representing cells present in a living subject) do express myogenin.

[0237] To assess the functional potential of surviving satellite cells, clonal analysis of cells sorted from Tg:Pax7-nGFP mice was performed. The percentage of clonogenicity (i.e. percentage of cell forming colonies after FACS in a 96 well plate) was not significantly different between day 0 and day 4 post mortem (20% vs. 16.3%) but this value drops dramatically at day 8 post mortem (1.6%)(FIG. 3.C.). This suggests strongly that satellite cells are able to resist to stress from the environment after organismal death. All colonies were myogenic as assessed by contracting myotube formation after differentiation. Although potential to form colonies was equivalent between day 0 and day 4 post mortem, we observed that exit from cell quiescence, assessed by scoring the first division after plating, was longer in post mortem (29 hours) sorted cells in comparison to alive animals (21 hours) (see FIG. 3.D.). After the first division cells divided every 7 hours and synchronously in the tested culture conditions in both situations indicating that cells recovered their correct cell cycle time after culture from dead animals.

[0238] To characterize further the sub-population of resisting cells in a hostile environment, their energizing state was measured by assessing the mitochondrial number and activity, as well as ATP level in SCs 4 days post mortem. To do this, SCs were isolated by FACS from Tg:Pax7-nGFP mice from day 0, day 4 and day 8 animals post mortem. Staining with Mitotracker allowed assessing the number of active mitochondria in the different conditions by flow cytometry. The number of active mitochondria was significantly diminished in post mortem samples compared to live control adult animals. Interestingly, as shown in FIG. 3.E., no significant difference in this value was observed between day 4 and 8 post mortem. The level of ATP in the 3 conditions was also monitored (alive-day 0-, day 4 and 8 post mortem). Cells were isolated as indicated above. For all 3 conditions, 20,000 cells were used and this number was double-checked by direct counting with a Malassez Chamber. The quantity of ATP was determined by fluorescence using luciferase activity as readout. Like mitochondrial activity, the quantity of ATP dropped dramatically in day 4 and day 8 post mortem in comparison to the day 0 alive controls (FIG. 3.F.).

[0239] Taken together these results reveal a direct correlation between the energizing threshold of the cell, presumably to ensure essential basal cellular activity and maintain viablilty, and the capacity to resist to a hostile environment. Cells exhibiting values below this threshold are not viable. These readouts provide insights into the mechanisms that allow these stem cells to survive after organismal death, and they provide a powerful tool to be used in diagnostic and therapeutical purposes.

[0240] To determine if stem cells have the functional capacity to regenerate a tissue after transplantation, SCs were extracted 4 days post mortem and engrafted into preinjured regenerating skeletal muscles of immunocompromised Rag1/2.sup./:.sub.C.sup./ recipient mice. Donor mice were double transgenic Tg:Pax7-nGFP::Tg:CAG-PLAP (PLAP, human alkaline phosphatase) mice to prospectively isolate satellite cells by FACS using GFP. The ubiquitous reporter PLAP permits to follow the fate of the engrafted cells.

[0241] In all the cases a significant contribution to regenerating skeletal muscle by the donor population was observed. After engrafting 10,000 SCs extracted from day 4 post-mortem mice, a mean of 300 PLAP-expressing myofibers were obtained. This result is similar to what is observed using control freshly isolated satellite cells.

Example V. Assessment of the Viability and Engraftment Potential of Haematopoietic Stem Cells Isolated from Mice Post Mortem

[0242] To determine if this extreme resistance to post mortem conditions is only the case for skeletal muscle stem cells, or another stem cell population behaves in a similar manner, hematopoietic stem cells were studied. At daily intervals post mortem, the bone marrow (BM) of Tg:CAG-GFP mouse femur was flushed (two limbs) and kept at 4 C. BM transplantations were performed in lethally irradiated C57BL6 recipient mice. The engraftment potential of transplanted BM progenitors was assessed by the percentage of GFP+ leukocytes found in the circulating blood. Using, 2, 3 and 4 days post mortem BM, blood cells were readily and fully reconstituted by BM progenitors in lethally irradiated recipients (n>5 in each case). Viability was ensured with all the animals that received a BM transplantation except when using BM from 4 days post mortem where viability was 60% (FIG. 4.A.). In all these animals GFP+ cells represented more than 70% of leukocytes, a result normally found in controls (donors) corresponding to 100% chimerism (FIG. 4.B.) After serial transplantation, same results were obtained except at day 4 where only 50% chimerism was found. In all these experiments 100% of animals survive. In all the cases, GFP+ leukocytes were found in all lineages: lymphocytes B, lymphocytes T, granulocytes, or monocytes as assessed by flow cytometry using B220, CD5, Gr1 or CD11b expression, respectively (FIGS. 4.C. to 4.F.).

[0243] To determine if the cells that were extracted from post mortem BM contained long term hematopoietic stem cells, a serial transplantation was performed with the grafted BM. GFP+ cells from the bone marrow of previously grafted animals were isolated by FACS 2 months after the first transplantation and re-grafted in lethally irradiated recipients. In all the cases, independently of the source of the initial BM (day 2, 3 or 4 post mortem), all of the animals were viable with 100% chimerism, and we obtained GFP+ leukocytes in all the different lineages.

Example VI. Anoxia and Low Temperature Enhance the Viability and Transplantation Potential of Stem Cells Isolated Post Mortem

[0244] To investigate the mechanism which confers the observed resistance of stem cells, the hostile environment occurring after death in a tissue i.e. hypoxia followed by anoxia was modeled. Culture conditions was established with a device usually used to culturing anaerobic bacteria (GenBag Chambers). The cells were maintained at 4 C. for various time intervals in the absence oxygen (less than 0.1% according to manufacturer). SCs isolated by FACS from Tg:Pax7-nGFP mice were maintained at 4 C. for 4, 7, 14 and 21 days in the absence oxygen (less than 0.1% according to manufacturer). Strikingly, it was observed that SCs better survived in a complete anoxic environment than in normoxic (20%) environment at 20 4 C. (see FIG. 5). As an example, after 4 days at 4 C., 82.3% of SCs were lost in the normoxia condition compared to 28.9% in anoxia and after 7 days at 4 C., 99.7% of cells were lost in normoxia compared to 97.7% in anoxia (FIG. 5.). The functional capacity of these cells was maintained after several days without oxygen as assessed by their ability to grow and differentiate when cultured under 25 normal conditions. To test the functional capacity of these cells in greater detail, SCs from Tg:Pax7-nGFP::Tg:CAG-PLAP donor mice were isolated by FACS and maintained at 4 C. with or without oxygen until 4 days. After this period, the cells were transplanted them by intramuscular injection into pre-injured TA muscles of C57BL6 recipient mice. These results demonstrate that cells 30 maintained without oxygen had at least the same transplantation capacity than those maintained with oxygen.

[0245] Similar experiments were performed with hematopoietic stem cells isolated from mice post mortem and stored in the presence or absence of oxygen. BM cells were extracted from two femurs of Tg:CAG-GFP animals, kept at 4 C. for 1, 2, 3, or 4 days, and transplanted into lethally irradiated C57BL6 recipient mice (n5). Cell mortality in such conditions was important i.e. 63+/7% of cell loss compare to immediately after extraction after one day in anoxia, 79+/3% after two days in anoxia, 98+/1 after 3 days in anoxia and 96+/1 after 4 days in anoxia (FIG. 6A). Even with this important cell loss, number of cells was sufficient enough to graft into irradiated recipients and blood chimerism was slightly 100% (FIG. 6.B. black bars) and all hematopoietic lineages were reconstituted (FIGS. 6.C. to 6.F.). Serial transplantations of BM cells isolated after the first round of transplantation demonstrated that the HSCs had a long term capacity repopulate the lineage (FIG. 6.B. grey bars).

[0246] In summary, the inventors have shown in animal model that transplantation of skeletal muscle and hematopoietic stem cells obtained from cadavers by the method of the invention, or enriched from biological sample when maintained in the absence of oxygen are functional and contribute to the regeneration of their respective tissues.