Patent us20170086453 – methods for obtaining stem cells – google patents anoxic brain injury prognosis

The term “extracting mononuclear cells”, as use herein, refers to in vitro separation of mononuclear cells from other type of cells present in the harvested tissue. Standard methods to carry out extraction of mononuclear cells are well known by one skilled in the art. For example, one can use the following methods: mechanical dissociation of the tissue (i.E. The tissue is minced into small pieces) followed by enzymatic digestion (using proteolytic enzymes such as pronase, trypsin, dispase, collagenase and/or an association of several enzymes (chazaud et al., exp. Cell. Res., 258(2): 237-44, 2000)) and/or or non-enzymatic procedures (ca2+ chelation) and/or an association of mechanical dissociation and enzymatic digestion combined to immunomagnetic cell sorting or FACS-cell sorting (arnold L, et al., J.Anoxic brain injury prognosis


exp. Med., 204(5):1057-69, 2007) and/or association of mechanical dissociation, enzymatic or non-enzymatic digestion followed by fluorescence-activated cell sorting (FACS) after multiple immunostainings using appropriate markers (for human muscle stem cells CD56+CD45−). When cells are in suspension such as in blood or in bone marrow, one can use ficoll paque plus (amersham biosciences) density gradient or adhesion steps (friedenstein A J, calci tissue int, 56:S17, 1995), as well as immunomagnetic or FACS cell sorting (prospective isolation of mesenchymal stem cells from multiple mammalian species using cross-reacting anti human monoclonal antibodies).

Methods for engineering vectors (viral and non-viral vectors), especially expression vectors, and for transforming, transfecting or transducing mammalian stem cells with a vector, especially a vector of expression, comprising at least one polynucleotide sequence of interest so that said cells express a polynucleotide sequence of interest (called transgenic stem cells) are well known in the art, and are for instance described by cossu and colleagues (dellavalle A, sampaolesi M, tonlorenzi R, tagliafico E, sacchetti B, perani L, innocenzi A, galvez B G, messina G, morosetti R, li S, belicchi M, peretti G, wright W E, torrente Y, ferrari S, bianco P, cossu G pericytes of human skeletal muscle are myogenic precursors distinct from satellite cells.Anoxic brain injury prognosis nat cell biol. 2007 march; 9(3):255-67. Epub 2007 feb. 11) or chamberlain (li S, kimura E, ng R, fall B M, meuse L, reyes M, faulkner J A, chamberlain J S. A highly functional mini-dystrophin/GFP fusion gene for cell and gene therapy studies of duchenne muscular dystrophy. Hum mol genet. 2006 may 15; 15(10):1610-22. Epub 2006 apr. 4).

The hematopoietic system malfunction or disease to be treated by the method may be selected from the group comprising:

• leukemias and lymphomas, including acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, juvenile myelomonocytic leukemia, hodgkin’s lymphoma, non-hodgkin’s lymphoma;

• multiple myeloma and other plasma cell disorders;

anoxic brain injury prognosis

• severe aplastic anemia and other marrow failure states, including severe aplastic anemia, fanconi anemia, paroxysmal nocturnal hemoglobinuria (PNH), pure red cell aplasia, amegakaryocytosis/congenital thrombocytopenia;

• SCID and other inherited immune system disorders, including severe combined immunodeficiency (SCID, all sub-types), wiskott-aldrich syndrome;

• hemoglobinopathies, including beta thalassemia major, sickle cell disease;

• hurler’s syndrome and other inherited metabolic disorders, including hurler’s syndrome (MPS-IH), adrenoleukodystrophy, metachromatic leukodystrophy;

• myelodysplastic and myeloproliferative disorders, including refractory anemia (all types), chronic myelomonocytic leukemia, agnogenic myeloid metaplasia (myelofibrosis);

anoxic brain injury prognosis

• familial erythrophagocytic lymphohistiocytosis and other histiocytic disorders.

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 (1980±212 GFP +/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 103,000±8494 GFP +/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 460±80 GFP +/TA, n=5 mice).Anoxic brain injury prognosis 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•

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.Anoxic brain injury prognosis 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).Anoxic brain injury prognosis 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.

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.Anoxic brain injury prognosis 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.

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.Anoxic brain injury prognosis 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®.Anoxic brain injury prognosis 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.).

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.Anoxic brain injury prognosis using, 2, 3 and 4 days post mortem BM, blood cells were readily and fully reconstituted by BM progenitors in lethally irradiated recipients (n5 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.Anoxic brain injury prognosis to 4.F.).

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).Anoxic brain injury prognosis 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.Anoxic brain injury prognosis these results demonstrate that cells 30 maintained without oxygen had at least the same transplantation capacity than those maintained with oxygen.