CN101517070A - Methods of selecting stem cells and uses thereof - Google Patents

Abstract

The present invention discloses a method of selecting stem cells from a heterogeneous population of cells. The method comprises contacting the population of cells with an apoptosis inducing agent under conditions which are apoptotic to non-stem cells and non-apoptotic to stem cells, thereby selecting the stem cells from the heterogeneous population of cells. The selected stem cells may then be used for a variety of applications including transplantation and differentiation.

Description

Select method of stem cell and uses thereof

Invention field and background

The present invention relates to method of selecting stem cell and uses thereof.

Stem cell has the peculiar property of reconstituted cell colony in vivo.Typically, stem cell is divided into two big classes: adult stem cell and embryonic stem cell.The importance of the technology relevant with stem cell (two kinds of sources of adult and/or embryo) amplification has obtained the proof of the many clinical preceding and clinical applications of these cells in the multiple disease of treatment.

Different with all existing treatments of the medicine that depends on surgical intervention or adjusting physiologically active, stem cell provides the replacement of the tissue of dysfunction or degeneration.Use stem cell, replace the prognosis that treatment can significantly change many diseases of not controlling at present, recover the function of impaired organ and revise inborn metabolic disturbance and deficiency.

The adult stem cell of recent findings derived from bone marrow can produce non-hemopoietic tissue, and this hints that these cells may have the bigger differentiation potential than previous supposition, and treats application for it and opened frontier [Petersen .Science such as B.E. 1999; 284:1168-1170; Brazelton .Science2000 such as T.R.; 290:1775-1779; Krause .Cell such as D.S. 2001; 105,369-377].

Research shown the stem cell in Cord blood source can repair brain injury and in wind-induced nerve injury [.Cell Transplant.2002 such as Lu D; 11:275-81], and can function and form be incorporated into the animal hearts tissue [Proc.Natl.Acad.Sci.USA 2001 for Orlic, D. etc.; 98:10344-9].

One of clinical application the earliest of stem cell is exactly to be used for carrying out bone marrow transplantation the patient with hematologic malignancies, and wherein the hemopoietic stem cell in donor bone marrow source is administered to the recipient after radiation that sufficient dosage is provided for described recipient and/or chemotherapy.Malignant cell is not only melted in this treatment, and melts non-malignant cell.Endogenous hematopoietic cell seldom survives marrow radiation clearly, and matrix is subjected to major injury.The result of ablative damage is, donor hematopoiesis is done and progenitor cell (HSPC) finds its approach that enters host's marrow, and its inoculation also moves into host's marrow, to rebuild immune hemopoietic system.The generally source of hemopoietic stem cell and progenitor cell comprises marrow, Cord blood and mobilizes cell to peripheral blood.

Except the treatment hematologic malignancies, situation about do, ancestral and immunocyte being used for the treatment of solid tumor.Therefore, for example, use the autologous hematopoietic cellular transplant to carry out treatment of solid tumors at mammary cancer [Peppercorn etc., 2005, Cancer 104:1580-1589], colorectal carcinoma [Leff etc., J Clin Oncol 1986 in conjunction with high dosageization/radiotherapy; 4:1586-1591], lung cancer [Ziske etc., Anticancer Res 2002; 22:3723-3726], nasopharyngeal carcinoma [Chen etc., Jpn J Clin Oncol 2003; 33:331-335] and cancer [Gratwohl etc., the Ann Oncol 2004 of other types; 15:653-660] in carried out extensive studies.

1 type transmembrane protein/mucoprotein CD34 of adhesion molecule saliva causes using CD34 as the evaluation of hemopoietic stem cell mark ⁺Cell select as the active method of concentrating hematopoietic stem cells [Civin etc., JImmunol 1984; 133:157-165].Specifically, though confirmed that myelomonocyte contains the 1-4%CD34 that has an appointment ⁺Cell still, is used these cells rather than has been eliminated CD34 to the baboon of accepting lethal exposure ⁺The marrow of cell causes hematopoietic reconstitution [Berenson etc., J Clin Invest 1988; 81:951-955].Similarly, CD133 has been considered to that hematopoiesis is done and mark [KobariL etc., the J Hematother Stem Cell Res.2001 of progenitor cell; 10:273-281].Especially, the incidence of stem cell in marrow is much lower, is about 0.2-0.5%.

Above-mentioned separation of C D34 ⁺Or CD133 ⁺The method of cell obtains comprising that the lymph hematopoiesis in all pedigrees and stage is done and the doing and the mixed cell population of progenitor cell of progenitor cell and precursor cell in some in late period.This is disadvantageous, because shown only separation of C D34 ⁺Primary cell is favourable [Askenasy N. etc., Current Stem Cell Research and Therapy 2006 in the cell colony; 1:85-94].In addition, described positive select procedure also has some shortcomings, comprises CD34 ⁺There is material (as antibody and/or magnetic bead) on the cell and removes the damage that these material pair cells cause.

In addition, nearest evidence shows that the expression of CD34 on cytolemma is always active not relevant with stem cell.Be presented at philtrum and exist lacked CD34 and express, but have the ability of rebuilding completely height immobilized stem cell colony [Dao etc., Leukemia 2000; 14:773-776].Repeated to show, compared the multipotency differentiation potential of hemopoietic progenitor cell limited [Jang YY etc., Nat Cell Biol.2004 with the stem cell in Cord blood source with the adult marrow that lacks these phenotype marks; 6:532-539].

Therefore, people pay close attention to the discovery additive method always, to substitute or to replenish existing separation and concentration stem cell and original progenitor cells colony.

Do with progenitor cell and need under the extreme condition of injured and inflammation, carry out the differentiation task usually.In this process, the expression and the activation that form the death receptor of hematopoietic cell are endowed multiple function, and the particularly negative adjusting of noble cells it be unclear that but death receptor involves in nearly HSPC function phases.Hematopoietic reconstitution cell flourishing mechanism in described destruction environment merits attention especially, because it can be used for improving the efficient of immigration.

There are 40 different ligand-receptor systems of surpassing to be considered to belong to tumour necrosis factor (TNF) superfamily at present.Most of tnf ligand, the most outstanding is that Fas-part (FasL) apoptosis induction ligand (TRAIL) relevant with tumour necrosis factor is as the embrane-associated protein synthetic, discharges soluble form by proteolysis then.Different cell types are stored in FasL by different physiological stimulations activation the time in the excretory vesica.Express after several minutes, FasL is promptly downcut from cell surface by matrix metalloproteinase, and accumulation is soluble molecule.Described solvable and form membrane have different functions in apoptosis and immunomodulatory.Apoptosis is mainly mediated in conjunction with FasL by film, and the biology of the solvable isotype (sFasL) of FasL is complicated, and comprises apoptosis, anti-apoptosis and chemotactic activity.Anti-apoptosis (s) FasL combines with described form membrane competition Fas, and is the chemokine of neutrophilic granulocyte.

The Fas acceptor hematopoiesis do and progenitor cell (HSPC) in expression be variable, and break up with pedigree and to change.The prematurity CD34 that has shown tire liver, Cord blood (UCB) and adult marrow (BM) source ⁺CD38 ^-People's cell subsets is expressed low (but can detect) this acceptor of level and the TNF acceptor of other mediated apoptosis [Niho etc., Curr Opin Hematol.1998; 5:163-165].

In the hematopoietic cell atomization, the Fas expression of receptor serves as far-end differentiation negative conditioning agent [Gaur U, Aggarwal BB, the Biochem Pharmacol.2003 of all pedigrees in propagation and differentiated progenitor cells; 66:1403-1408; Greil R etc., Crit Rev Immunol.2003; 23:301-322].The described enhancing of having observed Fas in the hemopoietic progenitor cell of cultivating is expressed, and described enhancing express with the cells in vivo exposing cell factor, amplification and operation indirectly after vigor impaired that decline takes place is relevant with the clone.

So far, thought the HSPC susceptibility that TNF superfamily receptors and part participate under the various experiment conditions to be increased apoptotic signal.Suppose that the overexpression among the HSPC that is subjected to wounding signal of Fas behind swaging graft has promoted the enforcement of apoptosis in donorcells, and participate in suppressing the donorcells activity.People CD34 ⁺HSPC and mouse c-ki ⁺Lin ^-SCA-1 ⁺(KLS) between HSPC and TNF-α in the junctor Fas expression of receptor of incubation and increase relevant, and cause going back to the nest (homing) and moving into [Bryder D, the J Exp Med 2001 of defective; 194:941-952; Dybedal I, Blood.2003; 102:118-126].All these deleterious effects are all effectively induced by the activation anti-Fas antibody and are reversed by sealing anti-Fas antibody and solvable Fas-part.Prove mutually with the negative interaction of Fas acceptor in HSPC moves into, the hypoplastic bone marrow that graft versus host disease (GVH disease) causes is by injection FasL deficient cells improve [Iwasaki T etc., Cell Immtmol.1999; 197:30-38].

Civin etc. [No. the 20040131599th, U. S. application] have instructed by express recombinant FasL gene in donor hematopoietic stem cell and have suppressed the method for Mammals recipient to the immunne response of donor hematopoietic stem cell graft.The active effect of FasL in this environment ascribe to the reactive T lymphocyte that kills and wounds the host with the reactive T lymphocyte that improves isotype exclusion and kill and wound donor to improve graft versus host disease (GVH disease).

Shirwan etc. [No. the 20040018170th, U. S. application] have instructed the method for the treatment of the illness that is eased by the apoptosis activated lymphocytes.Specifically, Shirwan etc. disclose and have used for example stable tetramer of FasL of protein, to strengthen the efficient of activation death receptor in activated lymphocytes.

Two kinds of methods all use the part of death receptor to eliminate reactive immunocyte [Cohen JJ, Duke RC.Ann Rev Immunol.1992 by the necrocytosis of activation inductive; 10:267-293].

But, no matter be Civin etc., or Shirwan etc. does not all mention or mentions indirectly, and stem cell before transplanting is selected or the purifying of stem cell colony.

Josefsen etc., [Exp Hematol.1999; 27:1451-1459] instructed by adding soluble Fas L increase CD34 ⁺CD38 ^-The clone of cell viability and enhancing cytokine induction is taken place.Use CD34 ⁺⁺CD38 ^-Fetal liver cell has obtained similar result [Barcena etc., Exp Hematol.1999,27:1428-1439].In this case, soluble Fas L is used to suppress activation (trimerizing) mediated Apoptosis [Askenasy N etc., the Blood.2005 by the Fas acceptor; 105:1396-404], the Fas acceptor is being implanted into people curee [Saheki K etc., Br J Haematol.2000; 109:447-452] and heteroplastic mouse model [Dybedal I etc., Blood.2003; 102:118-126] remarkable fractional people cell in express.But, do not propose to use Fas-L as the reagent of selecting stem cell.

Summary of the invention

According to an aspect of the present invention, the method of selecting stem cell from foreign cell colony is provided, described method comprises that making described cell colony and inducer of apoptosis is being apoptosis for non-stem cell, and be to contact under the condition of non-apoptosis stem cell, thereby from described foreign cell colony, select stem cell.

According to another aspect of the present invention, provide the method for the stem cell transplantation of selecting being gone into the host, described method comprises:

(a) making the stem cell of foreign cell colony and inducer of apoptosis is being apoptosis for non-stem cell, and is to contact under the condition of non-apoptosis stem cell, thereby selects stem cell; With

(b) host is gone in the stem cell transplantation of described selection, thereby transplant the stem cell of described selection.

According to the further feature in the preferred embodiment of describing, described method further is included in step (a) is separated described selection afterwards before with step (b) stem cell.

According to a further aspect of the invention, provide the method for differentiated stem cells, described method comprises:

(a) making the stem cell of foreign cell colony and inducer of apoptosis is being apoptosis for non-stem cell, and is to contact under the condition of non-apoptosis stem cell, thereby selects stem cell; With

(b) induce the differentiation of the stem cell of described selection, thus differentiated stem cells.

According to the further feature in the preferred embodiment of the invention described below, described stem cell is selected from the group of being made up of cord blood stem cell, mobilization back peripheral hematopoietic stem cells, bone marrow stem cell and neural stem cell.

According to the further feature in the preferred embodiment of describing, described stem cell is a bone marrow stem cell.

According to the further feature in the preferred embodiment of describing, described bone marrow stem cell is a hemopoietic stem cell.

According to the further feature in the preferred embodiment of describing, described method is modified described stem cell before further being included in described contact, to generate the stem cell of modifying.

According to the further feature in the preferred embodiment of describing, described method further is included in the described contact described stem cell of purifying before, to generate the stem cell of purifying.

According to the further feature in the preferred embodiment of describing, described method further is included in the described stem cell of increasing before the described contact, to generate the stem cell of amplification.

According to the further feature in the preferred embodiment of describing, described bone marrow stem cell is a mescenchymal stem cell.

According to the further feature in the preferred embodiment of describing, described stem cell is an adult stem cell.

According to the further feature in the preferred embodiment of describing, described stem cell is an embryonic stem cell.

According to the further feature in the preferred embodiment of describing, described inducer of apoptosis is selected from the group of being made up of TNF-α, FasL, Trail and Tweak.

According to the further feature in the preferred embodiment of describing, described inducer of apoptosis is FasL.

According to the further feature in the preferred embodiment of describing, described FasL is coupled to the surface.

According to the further feature in the preferred embodiment of describing, described FasL can not cut.

According to the further feature in the preferred embodiment of describing, described method further is included in and raises apoptosis receptor expression in the described foreign cell colony before the described contact.

According to the further feature in the preferred embodiment of describing, described apoptosis acceptor is selected from the acceptor group of being made up of Fas acceptor, TNF-α acceptor, Tweak acceptor and Trail acceptor.

According to the further feature in the preferred embodiment of describing, the up-regulated expression of described apoptosis acceptor contacts realization by making described foreign cell colony with interferon-gamma or TNF-α.

According to the further feature in the preferred embodiment of describing, described foreign cell colony does not comprise the T lymphocyte of immune activation.

According to the further feature in the preferred embodiment of describing, described foreign cell colony comprises the pedigree positive cell.

According to the further feature in the preferred embodiment of describing, described pedigree positive cell is selected from the group of being made up of granulocyte, scavenger cell, natural killer cell, erythroblast, antigen presenting cell, medullary cell, lymphoidocyte and megalokaryocyte.

According to the further feature in the preferred embodiment of describing, described foreign cell colony comprises the malignant cell of apoptotic sensitivity.

According to the further feature in the preferred embodiment of describing, described method further is included in described contact separate stem cells afterwards.

According to the further feature in the preferred embodiment of describing, described stem cell is that the host is from body.

According to the further feature in the preferred embodiment of describing, described stem cell is that the host is isogenic.

According to the further feature in the preferred embodiment of describing, described stem cell is that the host is heterogenic.

According to the further feature in the preferred embodiment of describing, described stem cell is host's xenogeneic.

According to the further feature in the preferred embodiment of describing, describedly induce differentiation to realize by expressing gene product in described stem cell.

According to the further feature in the preferred embodiment of describing, described gene product is a polypeptide.

According to the further feature in the preferred embodiment of describing, described gene product is polynucleotide.

The present invention has successfully solved the shortcoming of existing known configurations by providing based on the novel method of stem cell to the selection stem cell of the insensitivity of apoptotic signal.

Except as otherwise noted, all technology used herein are identical with the general implication of understanding of those skilled in the art with scientific terminology.Though method similar or equivalent to methods described herein and material also can be used for implementing or check the present invention, suitable method and material are as mentioned below.If any conflict, then be as the criterion with patent specification (comprising definition).In addition, described material, method and embodiment only are illustrative, are not intended to limit.

The accompanying drawing summary

This paper has described the present invention by way of example in conjunction with the accompanying drawings.At length specify figure for this paper, should emphasize, shown certain content is just given an example and for the preferred embodiments of the invention illustrative is discussed, its purpose that is suggested provides and is considered to content the most useful and description easy to understand principle of the present invention and notion aspect.In this, do not attempt to show CONSTRUCTED SPECIFICATION of the present invention in greater detail, this of accompanying drawing is described making that how implementing various ways of the present invention in practice is tangible to those skilled in the art than basic comprehension the present invention is necessary.

In the accompanying drawings:

Figure 1A-H explanation Fas/FasL interactional function participates in homogenic hematopoietic cell and moves into.(Myelaoblated) (950 rad) GFP mouse (CD45.2 of A. clear marrow ⁺GFP ⁺) transplanted from homogenic wild-type (CD45.1 ⁺GFP ^-) and Fas-defective (lpr) donor (CD45.2 ⁺GFP ^-) 5x10 ⁵Individual lin ^-1 of BMC; 1 mixture.It is chimeric to measure hematopoiesis after transplanting 3 weeks in peripheral blood, to compare wt (CD45.1) and lpr (CD45.2) donorcells (GFP ^-) immigration (n=16).B. with 5x10 ⁵Individual lin ^-BMC is isograft to go into (850 rad) recipient (CD45.1 → CD45.2) of sublethal exposure.When the lpr Transplanted cells being gone into wild-type (wt) recipient (n=8) and when lpr recipient (n=10) is gone in the wt Transplanted cells, the immigration during 3 weeks is a defective.Defective immigration in gld recipient (n=11) is effectively reversed (n=9) by dystopy FasL albumen by the expression of biotinylation on the donorcells surface to the C.gld cell with the wt cell in wt recipient (n=8).The isograft bag of D.wt cell is by the chimeric real diagnosis difference of FasL albumen 3 week back peripheral blood.E.FasL is being implanted into the homogenic lpr recipient (5x10 of CD45.1 → CD45.2) ⁵Individual lin ^-Be expressed in for 3 whens week to immigration do not make significant difference (n=6) among the BMC.F. will be from 10 of GFP donor ⁷Individual complete BMC is implanted into clearly, and the lpr recipient of marrow causes 6 week of transplanting back donor (GFP ⁺) chimeric fully (n=8) in marrow.The chimeric matrix culture of G.GFP/lpr is excluding CD45 ⁺And CD11c ⁺Mainly lpr (GFP behind the cell ^-) host's phenotype.Data represented 5 cultures of transplanting mouse.H. GFP/lpr mosaic (Fas completely ⁺GFP ⁺BMC and Fas ^-GFP ^-Matrix) serve as the lin that wraps quilt from the natural and FasL of homogenic CD45.1 mouse (n=6) ^-The recipient of BMC.Similar to the graft in the lpr mouse, the chimeric influence that not expressed by FasL shows donorcells FasL target matrix.

The proteic ectopic expression of Fig. 2 A-G explanation FasL has improved the heterogenote immigration.A. (Radiation-conditioned) recessive allele (H2K of radiation conditioning ^d→ H2K ^b) recipient's (850 rad) injected 10 ⁶Individual natural lin ^-BMC and 10 ⁶Individual lin ^-The splenocyte of BMC or FasL albumen bag quilt, control group is transplanted 2x10 ⁶Individual natural lin ^-BMC (n=5).Transplant 3 weeks of back and in peripheral blood, determine the chimeric level of donor.B. Streptavidin-FasL chimeric protein passes through the biotinylation active adsorption in the BMC surface.Use anti-FasL monoclonal antibody in flow cytometry, to detect described albumen.C. the proteic expression of dystopy FasL has improved (850 rad) recipient (B6=H2K that is implanted into sublethal exposure ^b) recessive allele (BALB/c=H2K ^d) immigration of cell.D. transplant operation and lin FasL bag quilt ^-Begin in the mouse of BMC 16 weeks after transplanting to form chimeric completely (n=10).E. transplant (H2K first ^d→ H2K ^b) 14 weeks of back, described gomphosis mouse serves as the (H2K to inferior sublethal exposure ^b) host's (n=5) the donor of wBMC.Transplanting the back measured chimeric in peripheral blood in 14 weeks.F. transplanted back 7 days, and used 5 days MLR to measure assessment and injected 8x10 ⁶The recessive allele lin of individual natural or FasL bag quilt ^-BMC (H2K ^d) B6 mouse (H2K ^b) splenocyte (Spelenocytes) (n=6).B10.BR mouse (H2K ^k) splenocyte serve as third party's antigen.G. use the MLR assessment to inject 8x10 ⁶Spleen (the H2K of the mouse of the recessive allele splenocyte of individual natural or FasL bag quilt ^d→ H2K ^b) (n=5).

The expression of Fig. 3 A-H explanation death receptor in the donorcells that marrow is gone back to the nest.A. complete BMC expresses low-level TNF family death receptor, and～30% lin-BMC is the TRAIL-R2 positive.B. irrelevant with Transplanted cells, residual medullary cell is expressed low-level death receptor (n=5) in full-body exposure (850 rad) back.C.Fas, TNF-R1 and TNF-R2 significantly raise to the homogenic host's of irradiation the donorcells of marrow successfully going back to the nest.Mean value ± the SD of the expression that data represented transplanting is back 48 hours (n=5).D. in initial several days after transplanting, the expression of death receptor in donorcells increases (n=4) day by day.E. will be with the Lin of CFSE preliminary making ^-BMC (＞90% is pure) is implanted into the homogenic recipient (850 rad TBI) of irradiation, the CFSE dilution (n=7) of the cell that 48 hours post analysis marrow is gone back to the nest.About 25% cell cycle is quick, as CFSE dilution (CFSE ^Secretly) determine.F. death receptor is mainly at Rapid Cycle cell (CFSE ^Secretly) middle rise (n=5), and the round-robin cell has low fractional expression (CFSE at a slow speed ^Bright).G. about 1/5th donor lin ^-BMC expresses pedigree mark, the early stage differentiation of indication (n=11) in back 48 hours of transplanting.H. death receptor mainly raises (n=8) in the noble cells in early days.

The dynamic expression of Fig. 4 A-G explanation Fas acceptor and part.A. use 850 rad full-body exposures (TBI) conditionings injected in mice 1-2x10 ⁷Individual recessive allele lin ^-BMC (H2K ^d→ H2K ^b).After 48 hours, the marrow of results recipient mouse, and serve as expression (n=8) with reference to definite Fas and FasL with donor-host source and pedigree marker expression.B.Fas and FasL are by identical donorcells coexpression jointly.Shown in reading characterize a plurality of experiments (n=29).C. analyze natural lin by RT-PCR ^-Existence with the mRNA of coding Fas and FasL among complete (w) BMC.3 independent experiments of data represented demonstration analog result.D-G. determine lin by flow cytometry ^-BMC is isograft (after CD45.1 → CD45.2) goes into to shine mouse (850 rad TBI), Fas and FasL are parallel to the expression (n=5) of these molecules in the parenchyma of corresponding organ at (D) marrow, (E) lung, (F) spleen and (G) expression pattern in the liver.

Fig. 5 A-H explanation death receptor hematopoiesis do and progenitor cell in expression.A-B. with HSPC mark (A) Sca-1 and (B) c-kit be reference, at isograft CFSE ⁺Lin ^-Behind the BMC 2 and 6 days, analyze the expression (n=8) of the death receptor of the cell that marrow goes back to the nest.The expression of HSPC mark is expressed as the mark of death receptor positive cell.C. be defined as lin ^-Sca-1 ⁺C-kit ⁺Go back to the nest to all candidate's hematopoiesis HSPC of marrow of the homogenic host of irradiation and all express death receptor (n=8).D.lin ^-Sca-1 ⁺C-kit ⁺The representative reading that TNF-R1 expresses among the HSPC.Though E. find～30% c-kit ⁺Cell expressing Fas, but the major part of this subclass and nearly all lin ^-Sca-1 ⁺And lin ^-Sca-1 ⁺C-kit ⁺Cell is all expressed FasL (n=7).F. the CFSE that goes back to the nest of marrow ⁺Lin ^-The Sca-1 of cell ⁺And c-kit ⁺The representative reading that Fas expresses in the subclass.G. eluriate separation small volume cell by under the 25ml/min flow velocity, carrying out adverse current, analyze its pedigree marker expression, and eliminate pedigree to produce Fr25lin ^-Cell (LTR)＞90% lineage negative subclass.Data represented 7 independently experiments.H. eluriate the back and collect the STR cell in position away from rotor (rotor off).The host of homogenic irradiation is gone in LTR and STR Transplanted cells, and (CD45.2 → CD45.1) gathers in the crops it two days later and is used to analyze Fas and FasL expresses.The mean value of data represented 5 independent experiments.

Fig. 6 A-H explanation hematopoietic reconstitution cell is to the resistance of apoptosis.It is A. isograft that (cell that CD45.1 → CD45.2) results marrow is gone back to the nest after 2 days makes it stand FasL albumen (250ng/ml) apoptosis and attacks 18 hours (n=6).Be parallel to pedigree marker expression, death (7AAD) and apoptosis (annexin-V), determine the donor and the host source of described cell.The cell served as control of incubation in (no FasL) substratum.B. be reference with the Fas expression of receptor, by the gate donorcells, with lineage negative (lin ^-) and the pedigree positive (lin ⁺) mark measurement apoptosis death (annexin-V) (n=5).The Fr25lin that C. will eluriate with marrow was gone back to the nest in the 2nd day ^-(LTR) and STR cell incubation in supporting substratum, and at external use 250ng/ml FasL albumen attack 18 hours (n=5).D. be expressed as reference with Fas, in three independent experiments with marrow was gone back to the nest in the 2nd day LTR and the death of STR cell measurement apoptosis.E. after they are exposed to external 250ng/ml FasL albumen apoptosis and attack, Fas, the TNF that determines the 2nd day marrow is gone back to the nest and the apoptosis death of TRAIL receptor positive cell.F. (850 rad) recipient to sublethal exposure transplants 5x10 ⁵Individual fresh (n=10) or with (n=12) homogenic cell of 24 hours of FasL albumen preincubation (CD45.1 → CD45.2).Determine the chimeric of peripheral blood lymphocyte by flow cytometry after 3 weeks.G. do not influence short-term and long-term move into (n=8) with the preincubation of FasL albumen.H. transplant 1.5x10 ⁵Individual from homogenic (CD45.2 → CD45.1) and recessive allele (H2k ^b→ H2k ^d) survival rate of mouse (950 rad) of clear marrow of the natural and pretreated BMC of FasL of donor (n=20).

Fig. 7 A-K explanation hemopoietic stem cell is to the susceptibility of Fas mediated Apoptosis.5x10 ⁶Individual cell/ml is the incubation different time sections in the α-MEM substratum that replenishes StemPro nutritious supplementary, 2mM L-glutaminate, 50 μ M, 2 beta-mercaptoethanols.A. complete BMC is containing and is not containing in the proteic substratum of 250ng/mlFasL incubation 24 hours.Mix by 7AAD and annexin-V respectively and determine necrocytosis and apoptosis, use mixtures of antibodies to determine the pedigree mark.Data have been summarized 5 independent experiments.B. response is stood the analysis announcement of the BMC pedigree of apoptosis with the proteic incubation of 250ng/ml FasL, all main subclass of the positive BMC of pedigree are all to the apoptosis susceptible: granulocyte (GR-1), scavenger cell (Mac-1), class red corpuscle (Ter), bone-marrow-derived lymphocyte (B220) and T lymphocyte (CD5).Account for the percent value of whole wBMC colony in data represented 6 independent experiments.C. apoptosis (annexin ⁺) and survivaling cell (annexin ^-) being defined as the function of Fas expression of receptor, described Fas expression of receptor is with the pedigree marker expression (lin in the natural complete BMC in 24 hours (n=7 independent incubation) backs of incubation (containing and do not contain 250ng/ml FasL albumen) ^-And lin ⁺) be reference.Fas when on behalf of incubation, horizontal stripe begin expresses.D.Lin ^-BMC was adding and is not adding under 10ng/ml STEM CELL FACTOR (SCF), 100ng/ml thrombopoietin (TPO) and the proteic condition of 75ng/ml FasL incubation 24 hours.With the Fas expression of receptor is that apoptosis (annexin is measured in reference (the individual independently incubation of n=3) ⁺) and vigor (annexin ^-).E-J.10 ⁷Individual wBMC incubation 3 days in replenishing the substratum of 50ng/ml TNF-α (TNF), and replenish 250ng/ml FasL albumen (FasL) and their combination (TNF+FasL) the last day.With Fas and TNF receptor expression serves as with reference to mix definite apoptosis (the individual independently incubation of n=4) by annexin.The lin of E. in substratum, surviving behind the incubation ^-And lin ⁺The quantity of cell.F. the apoptosis per-cent of the receptor positive cell of incubation in the substratum.G. the lin of in the substratum that replenishes SCF and TPO, surviving behind the incubation ^-And lin ⁺The quantity of cell.H. with SCF and TPO incubation after the apoptosis per-cent of receptor positive cell.I. the lin of in the substratum that replenishes SCF, TPO and interleukin-(IL)-3, surviving behind the incubation ^-And lin ⁺The quantity of cell.J. with SCF, TPO and IL-3 incubation after the apoptosis per-cent of receptor positive cell.K.wBMC is incubation lin after 5 days in substratum and in the substratum that replenishes SCF, TPO and IL-3 ^-The increase of cell per-cent (the individual independently incubation of n=3).

The double nutrition and the apoptosis function of Fig. 8 A-F explanation death receptor.3x10 ⁴Individual cell is coated with dull and stereotyped Dulbecco substratum (IMDM) in 1.2% methylcellulose gum that contains 20% foetal calf serum, 1% bovine serum albumin, 0.1mM 2 beta-mercaptoethanols, 10u/ml recombinant human erythropoietin (EPO), 20ng/ml recombined small-mouse (rm) STEM CELL FACTOR (SCF), 10ng/ml rm interleukin 3 (IL-3) and 10ng/ml granulocyte (granulogyte)-macrophage colony stimulating factor (rmGM-CSF) and Iscove improvement.A. complete BMC (wBMC) and lin ^-BMC and FasL oligomer be incubation in semi-solid methylcellulose gum culture, causes lin ^-Active (clonogenic activity) dose-dependently of product clone of BMC increases.Under high protein concentration (＞1 μ g/ml), produce the active unexpected decay of clone and be similar to observed phenomenon among the complete BMC.Data have been summarized 8 independent experiments.B. under identical culture condition, high density (＞250ng/ml) TNF α stimulates lin ^-The activity of BMC and do not influence the activity (n=5) of wBMC.C. from wBMC and the lin of Fas-defective (lpr) mouse ^-The product of BMC clone is active and dead to exist insensitive (n=5) to FasL is proteic.D. suppress Caspase-3 and suppress the product clone active (n=5) that Caspase-8 has recovered complete BMC with Z-DEVD-fmk with Z-IETD-fmk.E. Caspase-3 suppresses (DEVD) does not influence the protein induced lin of 500ng/ml FasL ^-Clone the enhancing that takes place among the BMC, and reduced the apoptosis death under the deleterious protein concentration.F. the inhibition of Caspase 3 (DEVD) has significantly increased by eluriating isolating short-term repopulation cell (short-term repopulating cell) clone's generation (n=5) (STC).

The function of Fig. 9 A-F explanation death receptor under the 5FU inductive stress hematopoiesis (stress hematopoiesis).To injected in mice 10 μ g/g 5FU, and their medullary cell of results is used for analyzing (n=6) after 1,3 and 5 day.A. the expression of the characteristic cell surface marker of candidate mouse HSPC Sca-1 and c-kit.The expression of B.Fas and FasL.C. natural B MC and to use in the cell of 5FU after 5 days with the pedigree marker expression be the expression of the Fas and the FasL of reference.D. natural B MC and use cell incubation (5x10 in the α-MEM substratum of additional StemPro nutritious supplementary, 2mM L-glutaminate, 50 μ M, 2 beta-mercaptoethanols that 5FU gather in the crops after 5 days ⁶Individual cell/ml) 24 hours is attacked with 250ng/ml FasL albumen then.By being that the annexin-V of reference mixes definite apoptosis (n=5) with the pedigree marker expression.E. use BMC that 5FU gathers in the crops after 1,3 and 5 days incubation 24 hours in substratum, to determine Fas ⁺Vigor (annexin in the cellular component ^-) and apoptosis (annexin ⁺).E. the cell of incubation carries out the apoptosis attack with 250ng/ml FasL albumen under the same conditions.F. with apoptosis (annexin ⁺) the Fas expression of receptor is drawn, summarized data from 5 mouse.

Preferred embodiment is described

The present invention is the method for selecting stem cell and remove foreign cell colony from non-stem cell.

But the principle of system of selection of the present invention and operation reference example and appended description and be better understood.

Before in detail explaining at least one embodiment of the present invention, should understand the detailed content that application of the present invention is not limited to propose in the following description or embodiment partly gives an example.The present invention can have other embodiments or implement in a different manner or execution.Equally, wording and the term that should understand this paper employing are for purpose of description, and should not be considered as restriction.

The variety of applications of stem cell in the numerous diseases of treatment shows that a large amount of described cells of discriminated union purifying are very important.

In hemopoietic system, stem cell is typically according to its cell surface phenotype CD34 for example ⁺Differentiate.Yet competent evidence shows that the expression of CD34 on cytolemma is always active not relevant with stem cell.Other strategies that have been used for detection and purification of hematopoietic stem cells (HSC) are the dyeing patterns according to fluorescence dye.Dyeing reduction with living body fluorescent dyestuff Hoechst 33342 (with the regional bonded bisbenzimidazole that is rich in VITAMIN B4-thymus pyrimidine of DNA ditch) and rhodamine 123 (it preferentially accumulates in the active wire plastochondria) is used for for a long time at flow cytometry experiment enrichment HSC always.

Use above-mentioned select procedure to trend towards selecting the stem cell or the early progenitor cell of preference hematopoiesis phenotype.

In the immigration potential of research hematopoietic cell, the inventor is the early detection rise (Fig. 3 A-B) that death receptor is expressed in the donorcells after transplanting, and it is subjected to inducing owing to the factor that radiation injury discharges (chemokine and cytokine) at least in part.

The inventor thinks that the inductive death receptor has an active effect in early days what hematopoietic cell moved into, because they notice immigration potential decline (Figure 1A-B) of hemopoietic stem cell in Fas-defective (lpr) and FasL-defective (gld) mouse.In addition, the inventor shows that homogenic (Fig. 1 C-D) of dystopy FasL proteic instantaneous displaying the improvement simultaneously hemopoietic stem cell and recessive allele (Fig. 2 A-E) cell move into.

In addition, the inventor shows to have only primary progenitor cell to raise death receptor soon after transplanting, and keeps this expression (Fig. 5 A-H) in ensuing a couple of days.Show high-caliber death receptor even it should be noted that these cells, they still keep the resistance (Fig. 6 A-B) to apoptotic signal.

The inventor reaches a conclusion from these results, and in the progenitor cell with hematopoietic reconstitution potential, the death receptor of expressing on it is the mediated apoptosis signal not.The far-end stage of differentiation and in somatocyte the dead same receptor of mediation, most of primary hematopoiesis do and progenitor cell in mediate trophic signals.

When enforcement is of the present invention, that the inventor shows is natural (is non-modification, for example express the apoptosis amboceptor) medullary cell and short apoptosis ligand such as FasL preincubation stimulates the expression (Fig. 7 C) of Fas, and a big chunk that further shows these cells is to apoptosis insensitive (Fig. 7 D).

The present invention is intended to this naturally occurring phenomenon is used for selecting stem cell with enrichment foreign cell colony.Because having shown, the inventor has only primary progenitor cell to short apoptotic signal immunity, therefore, the cell colony that the present invention produces separates the cell colony that produces than by the expression according to cell sign thing such as CD34 and has higher plasticity-, and thus with CD34 ⁺The cell difference can the preference hematopoietic lineage.Therefore, the inventor has proposed the functions of use characteristic and selected dried and progenitor cell: these cells are insensitive to the apoptotic signal that transmits by the cell surface death receptor.

The present invention can be used for providing stem cell between the junctor in-group, it is used in hematopoietic cell transplantation and produces the application of the stem cell aspect that can be used for the cytogene treatment that is fit to genetic manipulation.Other application can include but not limited to that adoptive immunotherapy, multiple disease are as the interior differentiation of treatment, body of for example β-hemoglobinopathy (hemoglobinopathia) and the indirect in-vivo tissue engineering under the stem cell transplantation under the commentaries on classics differentiation background and differentiation and the commentaries on classics differentiation background.

Therefore, according to an aspect of the present invention, provide from foreign cell colony select stem cell between method in the junctor.Described method comprises that making described cell colony and inducer of apoptosis is being apoptosis for non-stem cell, and is to contact under the condition of non-apoptosis stem cell.

As used herein, term " selection " refers to distinguish as the method for defined stem cell of the present invention and non-stem cell hereinafter.Since method of the present invention cause inevitably non-stem cell death (as hereinafter further as described in), therefore described chosen process causes the enrichment of stem cell of the present invention by removing non-stem cell.

Idiom " stem cell " refers to express (or can by abduction delivering) apoptosis amboceptor but the cell of the non-differentiation of end eventually of anti-apoptotic signal as used herein.Therefore, be included in the definition of described stem cell down be early progenitor cell, it breaks up more than stem cell to a certain extent, according to inventor's demonstration, though its expression apoptosis acceptor, but it has resistance to apoptotic signal.Can identify according to functional performance according to the stem cell that the inventive method is selected.Thereby for example, the inventor has shown that the stem cell of selecting according to the inventive method comprises and compare enhanced according to the stem cell that other prior art approach are selected and has moved into characteristic.

Stem cell of the present invention can derive from Cord blood, peripheral blood, marrow (for example mescenchymal stem cell, hemopoietic stem cell) or any adult tissue (including but not limited to brain, liver and muscle).Described in addition stem cell is embryonic stem cell and its derivative also.

Embryonic stem cell and its restoration methods are known in this area, referring to for example Trounson AO (Reprod Fertil Dev 2001; 13:523), Roach ML (Methods Mol Biol 2002; 185:1) and SmithAG (Annu Rev Cell DevBiol 2001; 17:435).Adult stem cell is the stem cell that derives from adult tissue, and also is known in this area.Separate or the method for enrichment adult stem cell referring to for example, Miraglia .Blood such as S. 1997; 90:5013; Uchida .Proc.Natl.Acad.Sci.USA such as N. 2000; 97:14720; Simmons .Blood such as PJ. 1991; 78:55; Prockop DJCytotherapy 2001; 3:393 .Fetal Diagn Ther 2002 such as Bohmer RM; 17:83) and .Bone Marrow Transplant 1998 such as Rowley SD; 21:1253; Stem Cell Biology (stem cell biological) Daniel R.Marshak and Richard L.Gardner compile, publisher: ColdSpring Harbor Laboratory Press, (2001) and Hematopoietic Stem CellTransplantation (hematopoietic stem cell transplantation), Anthony D.Ho and Richard Champlin compile, publisher: Marcel Dekker (2000).

According to this aspect of the invention, described stem cell is selected from foreign cell colony.

As used herein, idiom " foreign cell colony " refers to the mixture of at least two types cell (a type is stem cell as defined above, and another kind is the cell of apoptotic sensitivity).Described foreign cell colony can derive from any single or multiple organism, preferably Mammals and more preferably people.

According to an embodiment, described foreign cell colony comprises the mixture of pedigree positive cell and stem cell.In the case, the inventive method can be used for carrying out the pedigree removing.

As used herein, idiom " pedigree positive cell " points to the cell of specific cells pedigree orientation, as committed progenitor and other cells that further breaks up.Typically the pedigree positive cell is expressed the mark of pedigree differentiation, and the example includes but not limited to CD3, CD61, CD19, CD33, CD14, CD15 and/or CD4.

Can be according to the present invention the example of the pedigree positive cell removed of system of selection include but not limited to B and T lymphocyte (sophisticated and immature), granulocyte, scavenger cell, natural killer cell, erythroblast, antigen presenting cell, medullary cell, lymphoidocyte and megalokaryocyte.Preferably, described T lymphocyte is not immune activation the T lymphocyte is a TXi Baoshouti activated T lymphocyte.

According to another embodiment, described foreign cell colony comprises the mixture of the malignant cell of stem cell and apoptotic sensitivity.Therefore, the inventive method can be used for removing the malignant cell of described heterogeneous population.

Before system of selection of the present invention, can use the stem cell of the described foreign cell of following technology enrichment known in the art colony: (wherein said stem cell is defined as CD34 by FACS ⁺Or any other mark), by discharging rhodamine 123 and Hoescht, removing by the adverse current centrifugal elutriation with by pedigree, to obtain little paotoblastic colony.

Described foreign cell colony can be included in tissue (for example marrow) or its part, coacervate, the single cell suspension or as the part of primary culture or cell sample, if their short apoptosis agent that is the present invention can and.

Described foreign cell colony can improve before system of selection of the present invention, but preferred described improved, process does not influence the resistance of described stem cell to apoptosis agent, and their possibly can't be selected according to the inventive method more at that rate.Therefore, for example, described foreign cell colony can carry out genetic improvement to express molecule (s) of interest.

Alternatively or additionally, described foreign cell colony can increase.Preferably, the culture condition of the described foreign cell colony that is used to increase causes the net increase of stem cell of the present invention, and can not cause described stem cell to become to apoptotic sensitivity.

The method of the stem cell of culturing in vivo different tissue sources is known in field of cell culture indirectly.To this, referring to for example textbook " Culture of Animal Cells-A Manual of BasicTechnique " (Zooblast culture medium present technique handbook), Freshney, Wiley-Liss, N.Y. (1994), the third edition, its instruction is incorporated by reference this paper.The special methods of culturing stem cells is known in the art under the condition of not breaking up allowing cell amplification, referring to No. the 20050181504th, 20050265980,20050276793 and 20050124003, U.S. Patent application for example, it all is incorporated by reference this paper.Should be understood that described expansion of stem cells also can realization in select procedure process of the present invention and/or after the select procedure of the present invention.

As described, system of selection of the present invention is based on stem cell and demonstrates the discovery to the resistance of short apoptosis agent higher than non-stem cell.Therefore, can be by killing and wounding the stem cell that contacts enrichment of cell colony under the condition of non-stem cell with short apoptosis agent.

As used herein, idiom " short apoptosis agent " refers to promote the reagent (for example chemical preparations or polypeptide) of apoptosis.

Can include but not limited to TNF-α, FasL, Trail (Apo2 part) and Tweak (Apo3 part) according to the exemplary short apoptosis agent that the present invention uses.Described short apoptosis agent can be recombinant polypeptide, biological chemistry synthetic or from the cell extract purifying.Reorganization TNF-α, FasL, Trail and Tweak all can be from company such as R﹠amp; (Minnneapolis MN) obtains with Abnova Corporation (Taiwan) is commercial D Systems.One skilled in the art will appreciate that the medicament that has many enhancing apoptosis.Described dose comprises bisindole maleimide-8 and ouabain (quabain).If desired, these agent can be united use with the short apoptosis agent of the present invention.

In this respect the preferred embodiment according to the present invention, being used to select the short apoptosis agent of stem cell is FasL.

As used herein, term FasL refers to can be in conjunction with at least one active part of Fas acceptor and apoptosis-induced FasL polypeptide.Preferably FasL is for example people of Mammals.The exemplary peptide sequence of people FasL is as described in the GenBank AAC50124.Therefore, according to this aspect of the invention, described FasL can be the little organic molecule agonist of the biological activity peptide derivant of Fas ligand polypeptide, the biological activity class peptide that derives from the Fas ligand polypeptide or Fas ligand activity.Described Fas ligand polypeptide can be bioactive Fas ligand polypeptide such as Fas ligand polypeptide variant, Fas ligand polypeptide derivative, the Fas ligand polypeptide of improvement or the Fas ligand polypeptide of brachymemma.

In this respect the embodiment according to the present invention, described FasL is coupled to surface (for example cytolemma), so that it can make Fas acceptor trimerizing, thereby strengthens its activatory efficient.FasL can be positioned at other surfaces as for example liposome, maybe can use the FasL of Streptavidin coupling to be connected to the biotinylation pearl.

FasL and described surface can be cut maybe can not cut, though according to present preferred embodiment of the present invention, described FasL can not cut, so that keep the trimerizing of Fas acceptor.Only the example of the people Fas part of the naturally occurring non-cutting of expressing with film combining form is as described in the Gen Bank AAG60017.1.No. 6951919 instruction of United States Patent (USP) is owing to being difficult for the Fas part that proteolysis has the enhanced apoptosis activity.

In this respect another embodiment according to the present invention, described FasL is free polypeptide (promptly not being coupled to the surface), but exists with four dimerization states (promptly insoluble), so that it can induce the trimerizing of Fas acceptor, thus apoptosis-induced.The example of described polypeptide is known in this area, referring to for example No. the 20040018170th, patent application (being incorporated by reference this paper).In addition, shown that the FasL that connects Streptavidin can produce the tetramer, and therefore served as short apoptosis ligand.

Should be understood that the short apoptosis agent of the present invention can with dyestuff such as the Hoechst or the similar dye coupling of initiatively discharging from stem cell.Described dyestuff can commercially widely obtain, for example Invitrogen, MolecularProbes.Like this, stem cell of the present invention can not be subjected to any negatively influencing of described short apoptosis agent.

The short apoptosis agent of the present invention can be enough to induce the time of the apoptosis of non-stem cell with described foreign cell population exposed.Typically, starting the used time of apoptosis is about 1 hour, though preferably begin to wait for about 12-18 hour before back, the described select procedure of execution at apoptosis.Induce the most effective FasL concentration of non-stem cell apoptosis can use analyzed in vitro to determine, and can be dependent on the type of the cell that exists in the accurate preparation of FasL and the described heterogeneous population.

Alternatively, the short apoptosis polypeptide of the present invention can be expressed in heterogeneous population of the present invention.

Therefore, the present invention further provides the short apoptosis polypeptide expression construct of coding, it is used in the foreign cell of the present invention colony and expresses described polypeptide.For example, the polynucleotide sequence of part that derives from whole or the selection of Mammals FasL albumen clone, coding full-length proteins can be used for producing the recombinant forms of FasL polypeptide.The example of the nucleotide sequence of encoding wild type people FasL is as described in the GenBank U1182.1.Coding only with the example of the nucleotide sequence of film combining form people Fas part that express, naturally occurring non-cutting as described in the GenBank AF288573.

Nucleic acid construct of the present invention (being also referred to as " expression vector " at this paper) typically comprises gives other sequences that this carrier is adapted at prokaryotic cell prokaryocyte, eukaryotic cell or preferably duplicates and integrate in the two (for example, shuttle vectors).In addition, typical cloning vector also can contain and transcribes with translation initiation sequence, transcribes and translation termination and polyadenylation signal.

Eukaryotic promoter typically contains two class recognition sequences, TATA box and upstream promoter element.The TATA box is positioned at 25-30 base pair place, transcription initiation site upstream, and it is synthetic that it is considered to participate in guide RNA polysaccharase starting rna.Other upstream promoter elements are determined the speed of transcription initiation.

Enhancer element can stimulate transcribing up to 1,000 times from homology that is connected or allogeneic promoter.Enhanser all has activity when being placed in transcription initiation site downstream or upstream.Many enhancer elements that derive from virus have host range widely, and have activity in multiple tissue.For example, SV40 early gene enhanser is suitable for many cell types.Be suitable for other enhancers/promoters combination of the present invention comprise derive from polyomavirus, people or murine cytomegalovirus (CMV), from those of the long time-histories tumor-necrosis factor glycoproteins (long term repeat) of miscellaneous retroviruses such as murine leukemia virus, mouse or Rous sarcoma virus and HIV.Referring to Enhancers and Eukaryotic Expression (enhanser and eukaryotic expression), Cold Spring Harbor Press, Cold Spring Harbor, N.Y.1983, it is incorporated by reference this paper.

When construction of expression vector, preferably the distance with its transcription initiation site under its natural background is identical apart from the distance of allos transcription initiation site for promotor.But, as known in the art, can adapt to this is not lost promoter function apart from necessarily changing.

Described expression vector also can add the polyadenylation sequence to increase the efficient of short apoptosis polypeptide mRNA translation.Accurate and effective polyadenylation needs two different sequential elements: the sequence that is rich in GU or U and the Hexanucleotide sequence A AUAAA that is positioned at the high conservative at upstream 11-30 Nucleotide place that are positioned at downstream, described polyadenylation site.Be suitable for termination of the present invention and polyadenylation signal and comprise those that derive from SV40.

Except the element of having described, expression vector of the present invention can typically contain to be had a mind to increase clone's expression of nucleic acids level or is beneficial to other special element of differentiating the cell that carries recombinant DNA.For example, many animal viruss contain the dna sequence dna that promotes that the extrachromosome of viral genome in the cell type of permission duplicates.As long as provide the suitable factor by gene that carry on the described plasmid or the host cell gene group, the plasmid that carries these virus replication can dissociate and duplicate.

Described carrier can comprise or not comprise the eucaryon replicon.If there is the eucaryon replicon, then can use the appropriate selection mark described carrier that in eukaryotic cell, increases.If described carrier does not comprise the eucaryon replicon, then can not dissociate and duplicate.Alternatively, described recombinant DNA is integrated into the genome of the cell of being processed, and described promotor instructs required expression of nucleic acids therein.

Expression vector of the present invention can further comprise and allow for example to translate a plurality of proteic extra polynucleotide sequences from single mRNA, the sequence of the genome conformity of promotor chimeric polyeptides as internal ribosome entry site (IRES) and as described in being used for.

The example of mammalian expression vector includes but not limited to can be available from pcDNA3, the pcDNA3.1 of Invitrogen (+/-), pGL3, pZeoSV2 (+/-), pSeeTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1, pSinRep5, DH26S, DHBB, pNMT1, pNMT41, pNMT81, can be available from the pCI of Promega, can be available from pMbac, pPbac, pBK-RSV and the pBK-CMV of Strategene, can be available from the pTRES of Clontech and their derivative.

Also can use the expression vector that contains from eucaryon virus as retroviral regulatory element.The SV40 carrier comprises pSVT7 and pMT2.The carrier that derives from bovine papilloma virus comprises pBV-1MTHA, and the carrier that derives from Epstein-Barr virus comprises pHEBO and p2O5.Other exemplary carrier comprise pMSG, pAV009/A ⁺, pMTO10/A ⁺, pMAMneo-5, baculovirus pDSVE and allow albumen in SV-40 early promoter, SV-40 late promoter, metallothionein promoter, MuMTV promotor, rous sarcoma virus promoter, polyhedrin promotor or be presented at any other carrier of expressing under the guidance of other promotors of effective expression in the eukaryotic cell.

Virus is the infector of the very specialization of hiding host defense mechanism of having evolved out in many cases.Typically, specific cell type and the breeding therein of virus infection.The targeting specific of virus vector utilizes its natural specificity of the pre-cell type of determining of target specifically, thereby introduces recombination to the cell that infects.Therefore, the type of the carrier of the present invention's use will depend on the cell transformed type.Suitably in the limit of power of those of ordinary skill, so this paper does not provide the general description of selecting precaution according to the ability of cell transformed type selecting suitable carriers.For example, I type human T-cell leukemia virus (HTLV-I) target medullary cell can be used, Liang CY etc. can be used, (ArchVirol.2004; 149:51-60) the allogeneic promoter target nephrocyte that exists in the baculovirus Autographa californica multicapsid nucleopolyhedrosisvirus nucleopolyhedrosis virus (AcMNPV) of Miao Shuing.

Recombinant viral vector can be used for the expression in vivo of short apoptosis polypeptide, because they provide the advantage as side direction infection and targeting specific.It is for example retroviral life cycle inherent that side direction infects, and it is the process that the cell of single infection produces many progeny virus particles, and described virus particle is sprouted and infected flanking cell.The result is that big area is infected rapidly, and wherein major part is not by protovirus particle primary infection.This infection with the vertical-type that infector is wherein only propagated by filial generation is opposite.Also can produce can not lateral propagation virus vector.If required purpose is that specified gene is imported the only targeted cells of local amount, this characteristic comes in handy.

Diverse ways can be used for expression vector of the present invention is imported stem cell.Described method is generally referring to Sambrook etc., Molecular Cloning:A Laboratory Manual (molecular cloning laboratory manual), Cold Springs Harbor Laboratory, New York (1989,1992), Ausubel etc., CurrentProtocols in Molecular Biology (molecular biology updated plan), John Wiley and Sons, Baltimore, Md. (1989), Chang etc., Somatic Gene Therapy (somatic gene therapy), CRC Press, Ann Arbor, Mich. (1995), Vega etc., Gene Targeting (gene target), CRC Press, Ann Arbor Mich. (1995), Vectors:A Survey ofMolecular Cloning Vectors and Their Uses (carrier: molecular cloning vector and its purposes summary), Butterworths, [Biotechniques1986 such as Boston Mass. (1988) and Gilboa; 4:504-512], described method comprises for example stable or transient transfection, fat transfection, electroporation and use recombining virus carrier infection.In addition, relevant positive system of selection, referring to United States Patent (USP) the 5th, 464,764 and 5,487, No. 992.

Import relative additive method of nucleic acid such as fat transfection and electroporation by virus infection and have a plurality of advantages, reason is because the infectious essence of virus can obtain higher transfection efficiency.

The present invention also is expected at the apoptosis acceptor (for example Fas acceptor, TNF-α acceptor, Tweak acceptor and Trail acceptor) that raises before the select procedure of the present invention in the described foreign cell colony.Can amplify response like this to apoptotic signal, stem cell heliotropism direction of the present invention, and the non-stem cell of the present invention is to negative direction.

The method of upregulation of apoptosis acceptor is known in this area, as cell is contacted with interferon-gamma or TNF-α.Described reagent can be from company such as Sigma Aldrich and the commercial acquisition of Promokine.Preferably described cell contacts 3 hours to 5 days any time with these reagent.Therefore, the illustrative methods that stem cell is selected can comprise makes described foreign cell colony and TNF-α incubation 2 days with rise Fas, subsequently with Fas-L incubation 1 day to kill and wound non-stem cell.The further example that in this respect stem cell is selected according to the present invention can comprise makes described foreign cell colony and interferon-gamma incubation 3 days with rise TNF-α acceptor, subsequently with TNF-α incubation 1 day to kill and wound non-stem cell.

Should be understood that can with strengthen the resistance of stem cell of the present invention before described short apoptosis agent contacts to apoptotic signal.For example, (FADD of brachymemma for example is referring to .Cell Immunol 2001 such as for example Wu can to form the dominant negative mutant forms of the relevant death domain (FADD) of component such as Fas to the dominant negative that the armoring cell of FasL of the present invention imports the Fas approach; 208:137-47) maybe can block another molecule of Fa approach such as the death domain sample interleukin-1 ' beta ' converting emzyme arrestin (FLIP) that Fas is correlated with, avoid the death of FasL inductive to protect described armoring cell.The relevant further example that how to increase stem cell to the resistance of short apoptosis agent is referring to [No. the 20040131599th, U. S. applications] such as Civin.

Can be applicable to the various clinical situation according to stem cell in the junctor between the inventive method selection.Some have been listed below.

Transplanted cells: hematopoietic cell transplantation has become the treatment of many heredity or malignant disease and has selected.Though early stage transplanting program uses complete marrow (BM) colony, use the stem cell (CD34 that determines more recently ⁺Cell) enrichment colony.Except marrow, described cell can derive from other source as mobilize to the bone marrow stem cell of peripheral blood (PB) and umbilical cord blood (CB).Compare with BM, transplant the time that has shortened pancytopenia, and reduced infection and risk of bleeding with the PB cell.

Described donor and recipient can be body or different individualities one by one, respectively for example from body or allotransplantation.When implementing allotransplantation, as known in the art, should take to reduce the system of transplant rejection and/or graft versus host disease (GVH disease).Described system is implemented in human therapy at present.The cell colony of selecting according to the inventive method provides remarkable removings T lymphocyte, and it can be used for recessive allele and the monoploid transplanting background that is harmonious, with the reduction graft versus host disease (GVH disease).

State-of-the-art system is referring to publication Slavin S. etc., for example, and J Clin Immunol 2002; 22:64 and J Hematother Stem Cell Res 2002; 11:265 .Blood 2002 such as Gur H.; 99:4174 and Martelli MF etc., Semin Hematol 2002; 39:48, it is incorporated by reference this paper.

For the recipient's who suffers from malignant disease autotransplantation, the contaminative tumour cell in the body infusion often causes the recurrence of described disease.The selection and the non-pernicious stem cell of increasing will reduce the loading capacity of tumour cell in the final graft.

The antenatal diagnosis of rare cell hereditary defect: antenatal diagnosis is included in intrauterine and collects pregnant woman's embryonic cell and analyze its hereditary defect.Preferred, the noninvasive method of collecting embryonic cell comprise separation infiltration periphery parent round-robin embryo tool nucleated red blood cell precursor.Yet because the quantity of these cells is very rare, therefore further application of the present invention is to select described cell according to methods described herein before analyzing.Therefore, the invention provides the method that selection is applied to the embryonic stem cell of antenatal diagnosis.

Gene therapy: for the long-term gene therapy of success, high-frequency genetic improvement to have stable integration to the genetically modified stem cell in its genome be necessary requirement.In the BM tissue, though most of cell is circulation progenitor cell and precursor cell, stem cell only constitutes the small portion of cell colony, and the great majority in them are in the acyclic state of immobilized.Based on the carrier cell fission that need enliven of virus (for example, retrovirus) so that transgenosis is integrated into host genome.Therefore, the gene transfering efficiency to fresh BM stem cell is very low.It will provide the possibility of its genetic improvement of increase amplification and purifying stem cell colony and adjusting in indirect intravital fissional ability.

Therefore, can improve the cell of the present invention's selection to express gene product mentioned above.

As used herein, idiom " gene product " finger protein, peptide and function RNA molecule (being polynucleotide).Usually, the gene product of nucleic acid molecule encoding is to supply with the gene product of curee's needs.The example of described gene product comprises normal albumen, peptide, glycoprotein and the lipoprotein that produces of organ by the curee who accepts.For example, can replace the gene product of supplying with pancreas defective organ by gene and comprise Regular Insulin, amylase, proteolytic enzyme, lipase, trypsinogen, chymotrypsinogen, carboxypeptidase, rnase, deoxyribonuclease, triacylglycerol (triaclyglycerol) lipase, phospholipase A ₂, elastoser and amylase; The normal gene product that produces of liver comprises that thrombin such as blood coagulation factor VIII and factors IX, UDP glucuronyl transferase (transferae), ornithine change formamyl enzyme (transcarbanoylase) and cytopigment p450 enzyme and the adenosine deaminase that is used to process serum adenosine or endocytosis low-density lipoprotein; The gene product that thymus gland produces comprises serum thymic factor, Thymus humoral factor, thymopoietins and thymosin; The gene product that the digestive tube cell produces comprises tert-Amyloxycarbonyltetragastrin, secretin, cholecystokinin, somatostatin, serotonin (serotinin) and P material.

Alternatively, the gene product inducing cell of described coding is expressed required gene product (for example, the genetic material encoding transcription factor that is imported, it induces transcribing of the gene product of supplying with the curee).

In another embodiment, recombination can provide heterologous protein, for example, is non-natural to the cell of expressing this heterologous protein.For example, can provide various people MHC components to be supported in the immigration among the people recipient to inhuman cell.Alternatively, described transgenosis suppresses the expression or the effect of the donor mhc gene product of microorganism (microorgan) explant normal expression.

Non-hematopoiesis do and progenitor cell between select in the junctor:

Other purposes of the technology that this paper proposes comprise can be used for that non-hematopoiesis is done and progenitor cell between select in the junctor, described cell comprises for example neural stem cell, oligodendrocyte progenitor cells and similar cell.Can make its expressing gene product (polypeptide or polynucleotide products) by the described stem cell of transfection, force described cell in a junctor, to break up along particular path.Alternatively or additionally, can induce selected cytodifferentiation by in suitable substratum, cultivating.

The myelin disease forms the important people's sacred disease do not controlled so far of a class.Progress in the animal model, the particularly transplanted cells of oligodendrocyte pedigree causes the myelinization again (remyelination) of remarkable focus, and has obtained the physiology evidence of functional rehabilitation.Treatment in the future can comprise transplanting simultaneously and promote endogenous reparation, and these two kinds of methods can combine with the indirect in-vivo procedures of donor tissue.

United States Patent (USP) the 5th, 486 illustrates that isolating human mesenchymal stem cell can be divided into for No. 359 and surpasses a kind of types of organization's (for example bone, cartilage, muscle or bone marrow matrix), and the method for human mesenchymal stem cell in separation, purifying and the amplification cultivation thing is provided.

United States Patent (USP) the 5th, 736, be provided at that pedigree directional induction in the external or junctor is isolating, the method for the human mesenchymal stem cell of amplification cultivation for No. 396, described method comprises makes mescenchymal stem cell contact with the biologically active factors that effective induced dry-cell is divided into selected pedigree.Further disclose the pedigree inductive mescenchymal stem cell that comprises amplification cultivation and imported primary, with the method for regeneration or reparation mescenchymal tissue from the body host.

United States Patent (USP) the 4th, 642 provides the composition of repairing cartilage and bone defective for No. 120.These compositions provide with former state or the gel form that embeds natural or artificial bone.Described gel comprises the cell of some type.Cell is directed in the cell in cartilaginous precursor cell or any mesenchyme source, and it can be converted to potentially and be functional chondrocyte, typically forms inducible factor by comprising with Fibrinogen, protease inhibitor and zymoplasm bonded cartilage.

United States Patent (USP) the 5th, 654, No. 186 explanation haematogenous mesenchymal cells cultivate and body in breeding under the condition (in confirmation) as animal model, and can from blood, move into injury site with formation skin.

United States Patent (USP) the 5th, 716 has disclosed the method for the skin of the injured or burn of in animal or human regeneration for No. 411.This method comprises the step that begins to cover with collagen glycosaminoglycan (GC) matrix wound, is beneficial to the infiltration of mesenchymal cell and the blood vessel health tissues below in the GC matrix of transplanting.Subsequently, will from take from the animal or human not injury site epidermal growth and the epithelium autograft sheet (sheet) of the cultivation that comes is applied to body surface.The transplanting that obtains has fabulous inclusion rate and has the outward appearance of normal skin, growth, maturation and differentiation.

United States Patent (USP) the 5th, 716 provides for No. 616 treatment to suffer from the recipient's of the disease, illness or the illness that are characterised in that bone, cartilage or lung defective method.Described method comprises that intravenously uses separation from stroma cell normal, homogenic individuality, or intravenously is used described isolated cells after revising the hereditary defect of separating in described recipient's stroma cell.The method that gene is imported recipient's individuality is also disclosed.Described method comprises the marrow sample of the homogenic donor that obtains the individual or coupling of described recipient, and separates attached cell from described sample.After separating is applied to recipient's individuality with gene transfection donor attached cell and intravenously.Also disclose the composition that comprises isolating stroma cell, described isolating stroma cell comprises the foreign gene that may be operably coupled to the adjusting sequence.

In above-mentioned each example, all use non-hematopoiesis to do and the progenitor cell disappearance of organ or the outside cell source of damaged cell as a supplement.Described use needs the treatment of being advised to be successfully applied to the progenitor cell composition of doing of purifying.Because the urgent demand of the dried and progenitor cell populations of this a pair of large-scale purification, the inventive method and application have solved the important leftover problem of disclosed any method (critical niche) in the above-mentioned United States Patent (USP).

Between in the junctor and other examples of using in the body:

Other application dried and progenitor cells amplification comprise that skin regeneration, liver regeneration, anathrepsis and stimulation of bone growth are to be applied to osteoporosis.

Be expected in the time limit of this patent, will develop and many relevant short apoptosis agent, the scope of term inducer of apoptosis is intended to comprise in advance all these type of new technologies.

As used herein, term " about " refers to ± 10%.

Check after the following example that other targets of the present invention, advantage and new feature will be significantly to those of ordinary skills, described embodiment meaning is not in restriction.In addition, above-described and hereinafter claimed each different embodiments of the present invention of claim part and the aspect experiment support that obtains the following example.

Embodiment

With reference now to the following example,, it illustrates the present invention with description above with non-limiting way.

Usually, the experimental arrangement of term used herein and the present invention employing comprises molecule, biological chemistry, microbiology and recombinant DNA technology.Described technology throws a flood of light in the literature.Referring to, " Molecular Cloning:A laboratory Mannal " (molecular cloning laboratory manual) Sambrook etc. for example, (1989); " Current Protocols in Molecular Biology " (molecular biology updated plan) I-III volume, Ausubel, R.M. compiles (1994); Ausubel etc., " Current Protocolsin Molecular Biology " (molecular biology updated plan), John Wiley and Sons, Baltimore, Maryland (1989); Perbal, " A Practical Guide to Molecular Cloning " (molecular cloning practical guide), John Wiley ﹠amp; Sons, New York (1988); Watson etc., " Recombinant DNA " (recombinant DNA), Scientific American Books, New York; Volumes such as Birren, " Genome Analysis:A Laboratory Manual Series " (genome analysis laboratory manual book series) 1-4 volume, Cold Spring Harbor Laboratory Press, New York (1998); As United States Patent (USP) the 4th, 666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272, No. 057 described method; " Cell Biology:A Laboratory Handbook " (Cell Biology Experiment handbook) I-III volume, Cellis, J.E. compiles (1994); " Current Protocols inImmunology " (immunology updated plan) I-III volume, Coligan J.E. compiles (1994); Volumes such as Stites, " Basic and Clinical Immunology " (basis and clinical immunology), (the 8th edition), Appleton ﹠amp; Lange, Norwalk, CT (1994); Mishell and Shiigi compile, " SelectedMethods in Cellular Immunology " (system of selection in the cellular immunology), W.H.Freeman and Co., New York (1980); The available immunoassay are existing in patent and scientific literature extensively to be described, referring to for example, and United States Patent (USP) the 3rd, 791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281, No. 521; " Oligonucleotide Synthesis " (oligonucleotide is synthetic), Gait, M.J. compiles (1984); " NucleicAcid Hybridization " (nucleic acid hybridization), Hames, B.D. and Higgins S.J. compile (1985); " Transcription and Translation " (transcribe and translate), Hames, B.D. and Higgins S.J. compile (1984); " Animal Cell Culture " (animal cell culture), Freshney, R.I. compiles (1986); " Immobilized Cells and Enzymes " (immobilized cell and enzyme), IRL Press, (1986); " APractical Guide to Molecular Cloning " (molecular cloning practical guide) Perbal, B., (1984) and " Methods in Enzymology " 1-317 volume, Academic Press; " PCRProtocols:A Guide To Methods And Applications " (the PCR scheme: the methods and applications guide), Academic Press, San Diego, CA (1990); Marshak etc., " Strategies forProtein Purification and Characterization-A Laboratory Course Manual " (protein purification and evaluation strategy experiment curriculum guide), CSHL Press (1996); It all is incorporated by reference this paper, as described at this paper fully.Other general reference run through presents to be provided.Think that program wherein is known in this area, and provide them to help reader.The all information that wherein comprises is incorporated by reference this paper.

General material and method

Animal prepares and transplants: the mouse of using in this research is C57B1/6J (B6, H2b, CD45.2), B6.SJL-Ptprc ^aPepc ^b/ BoyJ (H2K ^b, CD45.1), B6.MRL-Faslpr/J (lpr, H2K ^b, CD45.2), B6Smn.C3-Tnfsf6gld/J (gld, H2k ^b, CD45.2) and C57BL/6-TgN (ACTbEGFP) 1Osb (GFP, H2k ^b), available from Jackson Laboratories.Mouse is raised in the barrier facility.By using x-ray irradiation instrument (RadSource 2000) to carry out Asia deadly (850 rad) and cause death (950 rad) full-body exposure conditioning recipient with the speed of 106 rads/min.Mouse carries out conditioning in 18-24 hour before transplanting.The clear marrow dosage and the toxicity of x-ray irradiation significantly are different from gamma-irradiation.For transplanting, the cell that will be suspended in 0.2ml phosphate buffered saline (PBS) injects lateral tail vein.

Cellular segregation, evaluation and dyeing: under aseptic condition, gather in the crops complete medullary cell (wBMC) in phosphate buffered saline (PBS, Beit Haemek) from femur (femurs) and shin bone.To lineage negative (lin ^-) immunomagnetic isolation of BMC, with the specific rat anti-mouse monoclonal antibody of CD5, B220, TER-119, Mac-1, Gr-1 and the NK1.1 of cell and saturation capacity (mAb) 4 ℃ of incubations 45 minutes.Except that Ter-119 and NK1.1 (eBioscience), all antibody are all available from the Hybridoma Cell Culture thing.With the cell of antibody sandwich with the PBS washed twice that contains 1% foetal calf serum (FCS, Biological Industries), then with the ratio incubation (Dynal) of the sheep anti rat IgG that is coupled to the M-450 magnetic bead in 4 magnetic beads of each cell.Collect the lin of not coupling by being exposed to magnetic field ^-BMC, use at the fluorescein isothiocyanate (isothyocyanate) of pedigree mark (FITC) mixture of the mAb of mark (eBioscience and BD Pharmingen) assess isolating efficient once more by flow cytometry.In order to obtain higher purity (＞95%), can repeat immunomagnetic isolation under the certain situation.

Use the Beckman whizzer, the J-6 rotor separates long-term (LTR) and short-term (STR) hematopoietic reconstitution cell by the adverse current centrifugal elutriation.Results from the wBMC of femur and shin bone at 15,25 (Fr25), 29 and the flow velocity of 33ml/min, 3000rpm and away from carrying out sorting under the condition of rotor (STR).By 4 ℃ with at TER119 (eBioscience) incubation of AA-4, CD5, GR-1, Mac-1, B220 (from hybridoma cell line) rat anti-mouse mAb and purifying the Fr25 cell is carried out pedigree and removes, to obtain LTR colony.(BD Pharmingen eBioscience) assesses the pedigree elimination efficiency of LTR cell once more by flow cytometry to use the mixture of the mAb of fluorochrome label.

For the dyeing of using the cell inner dye, with cell and 2.5 μ M5-(with-6-)-Fluoresceincarboxylic acid oxalic acid succinimide ester (CFSE, Molecular Probes) incubation 20 minutes, washing are also resuspended.

Use the proteic apoptosis of FasL to attack: to show before that Streptavidin-FasL chimeric protein was to Fas ⁺The effective apoptotic signal of cell transduction [.Immunity 2002 such as Yolcu ES; 17:795-808].With cell incubation (5x10 in the α-MEM substratum that replenishes StemPro nutritious supplementary (Stem Cell Technologies), 2mM L-glutaminate, 50 μ M2 β-ME ⁶Individual cell/ml) 24 hours.In some cases, described culture medium supplemented 10ng/ml STEM CELL FACTOR (SCF) and 100ng/ml thrombopoietin (TPO).All supplement are all available from PeproTech.By adding 75-250ng/ml Streptavidin-FasL chimeric protein attack cells, kept 18-24 hour, then by flow cytometry apoptosis and death.

The organ results: after intracardiac perfusion contains the cold PBS of 30ml of 100 units heparin, results spleen, lung and liver.With described tissue slice and handle: lung 37 ℃ down with 380u/ml collagen type v enzyme (Sigma) digestion 60 minutes, liver at 37 ℃ down with 1500u/ml collagenase digesting 20 minutes.All tissues comprise that spleen filters cell suspending liquid PBS washed twice with 40 μ m mesh.

With the FasL protein adsorption in cell surface: with cell at room temperature, be to suspend 30 minutes in the 5 μ M freshly prepared EZ-Link Sulfo-NHS-LC-vitamin Hs (Pierce) of PBS.After the PBS washed twice, described cell with in Streptavidin-FasL chimeric protein (100ng albumen/10 of PBS ⁶Individual cell) incubation.Use the anti-goat IgG (R﹠amp of secondary pig; D Systems) anti-Streptavidin mAb of the elementary goat of counterstaining (Zymed) and anti-FasL antibody (clone MFL-4, BD Pharmingen) are by flow cytometry assessment adsorption efficiency.Determine the logarithmic value of positive staining, use the painted control cells of isotype control antibodies to carry out normalization method.

Flow cytometry: measure and use Vantage SE flow cytometer (Becton Dickinson) to carry out.Explanation (Cedarlane) according to the manufacturer separates tool nuclear peripheral blood and medullary cell by the Fick gradient centrifugation.Cell washs in PBS, with the elementary mAb of mark 4 ℃ of incubations 45 minutes or with the secondary mAb counterstaining of fluorochrome label.Use at minor antigen CD45.1 (clone A20, eBioscience) and CD45.2 (clone 104, monoclonal antibody eBioscience) is determined the chimeric per-cent that accounts for donor and host's peripheral blood lymphocyte (PBL) of the donor in the isograft.

Determine with the amino actinomycin D of 5 μ g/ml 7-(7-AAD, Sigma) and necrocytosis and apoptosis in the cell of annexin-V (IQ product, Groningen, The Netherlands) incubation.

Use primary label mAb to differentiate acceptor and part: Fas (CD95) clone 15A7 (eBioscience), TNF-R1 (CD120a) clone HM104 (Serotec), TNF-R2 (CD120b) clone TR75-89 (Serotec), Trail-R2 (DR5) clone MD5-1 (eBioscience) and FasL clone MFLA (BD Pharmingen).

The cell surface marker of the stem cell of supposing is differentiated and is Sca-1 (Ly-6A) clone D7 (eBioscience) and c-kit (CD 117) clone 2B8 (eBioscience).Use is coupled to FITC, phycoerythrin (PE), allophycocyanin (APC) and peridinin chlorophyll a-albumen, and (PerCP, Streptavidin BDPhamingen) carries out counterstaining to biotinylated antibody.

Sxemiquantitative RT-PCR.Use EZ-RNA II to extract reagent or RNeasy micro-column (Qiagen, Hilden, Germany) the total RNA of extraction from cell.Described RNA and pd (T) _12-18Primer one is used from reverse transcription reaction.Use following primer that group is carried out PCR step: mouse FAS-forward 5 ' GCCTTGGTTGTTGACCA (SEQ ID NO:1), reverse 5 ' GTACCAGCACAGGAGCA (SEQ ID NO:2) produces the 300bp fragment; Mouse FAS-part-forward 5 ' ACCGCCATCACAACCA (SEQ ID NO:3), reverse 5 ' TCAACCTCTTCTCCTCCA (SEQ ID NO:4) produces the 500bp fragment.The primer of beta-actin is as internal contrast and will express normalization method.

External colony forming unit (CFU) is measured.3x10 ⁴Individual cell is coated with dull and stereotyped Dulbecco substratum (IMDM) in 1.2% methylcellulose gum that contains 20%FBS, 1%BSA, 0.1mM 2 β-ME, 10u/ml recombinant human erythropoietin (EPO), 20ng/ml recombined small-mouse (rm) SCF, 10ng/ml rm interleukin 3 (IL-3) and 10ng/ml rmGM-CSF (PeproTech) and Iscove improvement.Counting surpasses the colony of 50 cells (CFU-C) after 7-10 days.With 200-1, the increase concentration in the 500ng/ml scope is added Streptavidin-FasL chimeric protein, or by biotinylation it is adsorbed in cell surface [.Immunity 2002 such as Yolcu ES; 17:795-808; .Circulation2003 such as Askenasy N; 107:1525-1531; Pearl-Yafe M etc., Stem Cells 2007; In the distribution].The human soluble FasL of the reorganization of interpolation 5ng/ml concentration (SuperFasL, Alexis).Respectively by adding Z-DEVD-fmk and Z-IETD-fmk (R﹠amp; D Systems) suppresses Caspase 3 and 8.

Statistical study.To each experimental program, data all are expressed as mean value ± standard deviation.Assess the result's of each experimental group repeatability by the linear regression of twice measurement.Use the difference between the check of the Scheffe t-afterwards assessment experimental program, in p＜0.05 time, think significant difference.

Embodiment 1

The physiological activation of Fas during hematopoietic cell moves into

The result

Ascribe to the interactional harmful result of Fas/FasL in the hematopoietic cell show hematopoiesis do and progenitor cell (HSPC) graft environment in negatively influencing.This interactional activity of preventing shows that Fas-defective HSPC is owing to the active adjusting of donorcells that Fas is mediated and/or prevent the insensitive immigration advantage that has.In order to check this possibility, use Fas-defective (lpr) and FasL-defective (gld) mouse in the inventor the is homogenic transplanted thing, with avoid participating in the immunologic mechanism of transplant rejection and graft versus host disease (GVH disease) (GVHD) and this molecule of emanating to the effect in the migration process in early days.Will be from wild-type (CD45.1) or Fas-defective (lpr, CD45.2) 10 of donor ⁶Individual lin ^-BMC is implanted into (950 rad) the homogenic GFP recipient (CD45.2) of marrow clearly, transplants the back and obtain donor chimeric (n=5) completely 3 weeks in peripheral blood.By will be from the 5x10 of wild-type (wt) and lpr donor ⁵Individual lin ^-BMC is implanted into clearly, and the homogenic GFP recipient (n=16) of marrow carries out the competitive experiment that moves into.Under these conditions, mosaic presents 59 ± 5%CD45.1 and 35 ± 4%lpr chimeric (Figure 1A) when 3 weeks, transplant the back and 14 weeks still kept this difference.These data show that the Fas/FasL interaction has active effect in the commitment that hematopoietic cell moves into, and no evidence shows active the preventing of donorcells of Fas mediation.On the contrary, defective moves into the support effect that shows donorcells Fas.

Further at the 5x10 of (850 rad) recipient who is implanted into sublethal exposure to realize that mixing is chimeric ⁵Individual lin ^-Analyze in the BMC isograft and move into (Figure 1B).First general observations is that the lpr cell is in wt recipient and the defective immigration of wt cell in lpr host (p＜0.05) on the contrary.Ironically, not only when donorcells is Fas-defective (lpr), and when the wt cell being inculcated to the lpr recipient, immigration is defective (Figure 1B), shows that surpassing a kind of mechanism relates to Fas/FasL and interact.In addition, when the gld cell being inculcated to the wt recipient and inculcating the wt cell to the gld recipient, moving into also is (Fig. 1 C) of defective.The integrated interpretation of these data is pointed to the immigration of the cell disappearance 20-25% that lacks Fas acceptor and part.

Embodiment 2

The proteic instantaneous displaying of dystopy FasL has improved homogenic cell and has moved into

The result

For whether the immigration defective of determining the gld cell can be recovered by FasL, use FasL albumen bag by donorcells by biotinylation.FasL albumen has recovered their immigration defectives (p＜0.001) (Fig. 1 C) in homogenic wt recipient in the expression of donor gld cell surface.Observed similar effect (p＜0.001) when the gld recipient is gone in the wt Transplanted cells of expressing FasL, shown that the donor FasL-host Fas that moves in the cell interacts and tangible autocrine Fas/FasL activity.The adjusting that moves into ascribes the specific effector of FasL to, rather than the long-time effect of cell surface protein decoration.Decorate wt-BMC by biotinylation with FasL albumen and improved early stage immigration (Fig. 1 D) among (p＜0.005) homogenic wt recipient, begin to form that donor is chimeric completely mouse 16 weeks after transplanting.These results show that immigration BMC is insensitive to FasL inductive apoptosis, and this proteic expression has improved the short-term immigration and do not damaged long-term repopulation cell.

Embodiment 3

The elementary target of donorcells FasL is the bone marrow matrix in the isograft

The result

In order to determine that dystopy FasL albumen promotes that whether the effect that moves into works by the Fas signal transmission among the host, goes into Fas-defective lpr recipient (n=8) with the Transplanted cells of wild-type mice.Immigration advantage forfeiture by realizing at cell allogeneic engraft surface expression FasL albumen shows that described mechanism relates to the competitive Fas signal pipeline (Fig. 1 E) among the host.These results prove that further it is the specific effector of FasL that the enhanced of the cell that FasL decorates moves into, rather than the illusion of indirect cells in vivo operation.

The lin that FasL decorates ^-The rejection activity of BMC can work in the whole body level, and the active host immune system that stops of wherein said rejection destroys the preceding BMC that goes back to the nest, or works in marrow, and wherein said rejection is active can remove the residual host cell of surviving in irradiation.In isograft, the expection immunomodulatory is not the correlated variables of migration process.Can be comprised immunocyte, HSPC and bone marrow matrix by the residual host cell of donorcells FasL target.The evidence that non-immunologic mechanism participates in the immigration buttressing effect of FasL makes the inventor seek the specific cell target of cell in host's (matrix or the residual host HSPC and immunocyte of surviving) that FasL decorates in irradiation.The inventor is by will be from 10 of GFP donor ⁷Individual complete BMC is implanted into (950 rad TBI) lpr recipient of marrow clearly, has produced to have Fas ^-Matrix and Fas ⁺The gomphosis mouse of hematopoietic cell.Transplant 6 weeks of back, these mouse show donor hematopoiesis completely chimeric (Fig. 1 F) in peripheral blood and marrow.In order to determine that bone marrow matrix is lpr host's phenotype, the marrow aspirate is coated in the extended culture.Exclude CD11c ⁺And CD45 ⁺After the cell, the main phenotype of the cell of growing in the culture is lpr host (GFP ^-) source (Fig. 1 G).(850 rad) mosaic of sublethal exposure serves as the recipient of the homogenic CD45.1 Transplanted cells of secondary.Transplant lin natural and FasL bag quilt ^-The chimeric level of hematopoiesis behind the BMC similar (Fig. 1 H), and to the lpr recipient in the forfeiture similar (Fig. 1 E) of immigration advantage of FasL mediation.By getting rid of, these data show that the elementary target of donorcells FasL is a mouse matrix, rather than to the rejection activity of hematopoietic cell residual in host's marrow.

Embodiment 4

The ectopic expression of FasL strengthens the immigration of recessive allele hematopoietic cell

The result

For the donorcells of determining FasL is expressed in the influence of whole body level, by biotinylation at splenocyte and lin ^-The surface expression FasL chimeric protein of BMC.With these cells and 10 ⁶Individual natural lin ^-BMC is implanted into the recessive allele host (H2K of irradiation together ^d→ H2K ^b).Natural lin with twice quantity ^-BMC compares, the immigration level by FasL albumen at lin ^-Expression on the BMC significantly improves (p＜0.001), and is further improved (p＜0.001) (Fig. 2 A) by its expression on donor spleen cells.These data show that the inhibition allogeneic is replied the donor hematopoietic cell is moved into is useful.But whether the still unclear cell that moves into is at lin ^-In the subclass of the natural or FasL bag quilt of BMC.

In order to determine whether the lin of FasL in all immigrations ^-Expression on the BMC cell all can influence immigration, and described albumen is adsorbed to donorcells surface (Fig. 2 B) with almost completely efficient.Transplant 1.5x10 ⁶The recessive allele lin that individual FasL decorates ^-BMC obtained comparing the higher chimeric level of donor hematopoiesis (p＜0.001) (Fig. 2 C) with the cell of not improvement when 3 weeks.All mouse all began to form chimeric completely after transplanting in 16 weeks, showed that the proteic instantaneous displaying of described dystopy does not influence the chimeric final foundation of lasting hematopoiesis (Fig. 2 D).For whether the early stage immigration of checking improvement causes the disappearance of stem cell, carried out in proper order and transplanted.Will be from complete chimeric lin ^-The host that BMC is implanted into the clear marrow of secondary shows immigration no significant difference (Fig. 2 E).Therefore, the expression of FasL albumen on donorcells tolerated well, and the short-term of having improved them moves into, and can not influence their long-term hematopoietic reconstitution potential.

Embodiment 5

The proteic expression of FasL is sealed alloreactivity in Fas dependency mode

The possible mechanism of supporting heterogenote to move into is to cross expression FasL albumen inhibition allogeneic by the activation inductive necrocytosis (AICD) of host immune cell to reply.For the direct evidence of immunosuppressive action that FasL is provided, the cell peritoneal injection of described albumen bag quilt is gone into the recessive allele host.In mixed lymphocyte reacion (MLR), analyze the reaction of recipient's splenocyte after 7 days.The lin that FasL decorates ^-BMC and splenocyte specificity sealing alloreactivity are replied, but to antigenic reply be kept perfectly (Fig. 2 F-G) of third party.The splenocyte that FasL decorates is suppressing to compare lin aspect the alloreactivity t cell response ^-BMC is more effective.This may be because the full-time antigen presenting cell of ubiquity in the spleen, can effectively activate alloreactivity T cell and make its sensitivity of killing and wounding to the FasL mediation by AICD (3,35,36).Take all factors into consideration, these digital proofs FasL participates in the hematopoietic cell migration process very early, and its part is to suppress the alloimmunity of graft is replied mediation by whole body.This homogenic donorcells with the previous report of the inventor has better go back to the nest (1) than recessive allele donorcells consistent.

Embodiment 6

Death receptor is raised in donorcells

The result

Assessed results and removed (lin from the complete BMC (wBMC) and the pedigree of the natural tool nuclear of natural C57B1/6 and BALB/c mouse ^-) Fas, TNF and TRAIL receptor level among the BMC.Outstanding difference is lin ^-TNF in the subclass compares with wBMC with the TRAIL acceptor has stronger expression (Fig. 3 A).To transplant back death receptor change of Expression in order monitoring, to use lin ^-BMC, reason is in the wBMC graft, the primary progenitor cell is compared preferentially go back to the nest (1) with mature cell more.With lin ^-BMC is implanted into the homogenic recipient of irradiation (850 rad) (CD45.1 → CD45.2) afterwards, gather in the crops and analyze marrow by gate donor and host cell.Their expression variation littler (Fig. 3 B) is compared in the residual host cell demonstration of survival (after 48 hours) with the distribution of these acceptors in natural B MC in irradiation, marrow (BM) donorcells of going back to the nest showed Fas and the two kinds of TNF acceptors (p＜0.001) that significantly raise in the 2nd day, and this relative less increase with the TRAIL-R2 acceptor is (Fig. 2 C) on the contrary.Subsequently, death receptor is expressed in the donorcells (Fig. 2 D) that increases to 60-75% after 6 days.Abreast, residual host BMC showed the increase of following substances appropriateness in back 6 days in transplanting: Fas to 14.5 ± 3%, TNF-R2 to 22.5 ± 2.5% and TRAIL 19.5 ± 0.5% (p＜0.001 is compared with baseline value).Under these transplanting conditions, mouse forms～50% donors chimeric (n=15) in 3 week backs, and begins to form that donor is chimeric completely after 16 weeks.Take all factors into consideration, these data presentation are transplanted the early stage acute expression of death receptor in donorcells in back.

Embodiment 7

Death receptor is expressed the dependency to circulation and differentiation

The result

The expression of death receptor can be by division and early stage induction, because some donorcellses promptly participate in circulation (1) as far back as going back to the nest to marrow.Therefore, serve as with reference to the expression of TNF family receptors in donorcells during the nearly seeded process of monitoring (proximal seeding process) with CFSE dilution and pedigree marker expression.Use born of the same parents' inner dye CFSE preliminary making donorcells before transplanting.About 1/3rd cell shows significant CFSE dilution, indicates the cell fission (Fig. 3 E) of going back to the nest to host's marrow.Acceptor mainly is expressed in and transplants back 24 hours maintenance CFSE ^BrightCell compare CFSE ^SecretlyThe cell (p＜0.001) (Fig. 3 F) of part.Observed similar uneven distribution (Fig. 3 G) in the cell of expression pedigree mark in initial 48 hours after transplanting.Transplant the 1in that BM goes back to the nest in back 2 days ⁺The increase of BMC from 5 ± 1.7% to 20 ± 2% is attended by the expression (Fig. 3 H) of death receptor.These data indicate described receptor expression main with go back to the nest to and be inoculated in the early stage cell cycle behind host's marrow and break up relevant.

Embodiment 8

Fas/FasL behind transcription activating coexpression in donorcells

The result

In view of the acute expression of death receptor, the inventor attempts to determine whether to express equally similar part.Film binding partner unique in the TNF superfamily is FasL.With recessive allele lin ^-BMC is implanted into the host (H2K of irradiation ^d→ H2K ^b) two days afterwards, cell that results marrow is gone back to the nest and the expression of analyzing Fas acceptor and part.Though residual host BMC shows the rise of expression of these molecules of appropriateness, donorcells shows that significant Fas and FasL raise (Fig. 4 A) in a few hours to the marrow going back to the nest.The composing type co expression of Fas acceptor and part (Fig. 4 B) may be the result that the cytokine environment that is twisted and round-robin medullary cell raise these molecules in the marrow of irradiation.

In order to determine whether the increase that Fas and FasL express is regulated in the rna transcription level, and donorcells has been carried out RT-PCR analysis (before transplanting).The mRNA (Fig. 4 C) of the mRNA of the coding Fas of the medullary cell expression conspicuous level that tool nuclear is complete and the coding FasL of trace.The lin that the mRNA transcript of two kinds of molecules is being used to transplant ^-All can't detect among the BMC.Therefore, the appearance on the cell that moves into of Fas and FasL albumen is at the transcriptional level inductive.

Embodiment 8

Dynamic expression with Fas/FasL in the cell that moves into after the different substrates interaction

The result

The expression in hematopoietic cell of Fas acceptor and its part changes the response various environmental factors and (4-14,23) take place the differentiation incident.Bone marrow matrix is to move into unique microenvironment that the site is provided for the HSPC that determines.Go back to the nest to the donorcells of homogenic host's Different Organs (CD45.1 → CD45.2) in order to check the feature of the cell whether rise that Fas and FasL express only go back to the nest for marrow, to have analyzed.Acceptor and its part raise to the donorcells of marrow (Fig. 4 D) and lung (Fig. 4 E) going back to the nest.Different is, goes back to the nest mainly to raise its FasL to the immigration cell of spleen (Fig. 4 F) and liver (Fig. 4 G) and express.These molecule transient expressions are in going back to the nest to the donorcells (reaching peak level in back 24 hours in transplanting) of lung and liver, and their carrying out property are expressed in the cell that marrow and spleen are gone back to the nest.Abreast, radiation injury induces these molecules to express (Fig. 4 D-G) in the parenchyma of these organs.The essence of Different Organs and move into the property followed rise in the cell and show that the expression of Fas and FasL partly is subjected to inducing owing to the factor (chemokine and cytokine) that radiation injury discharges.But other inducibility stimulating factors that Fas and FasL express are different in Different Organs.

Embodiment 9

Death receptor is subjected to the bone marrow matrix conditioning and induces with bone marrow matrix is interactional

The result

In order to determine the factor of the outside cell that influence is expressed, inculcating to unirradiated homogenic (CD45.1 → CD45.2) monitor acceptor behind the recipient.Under these conditions, low about 40-65% among the host of the expression ratio of death receptor and FasL irradiation, the release of indication matrix damage back chemokine and cytokine is the partly cause that death receptor is expressed.In experiment subsequently, with lin ^-BMC incubation 6 hours in the junctor in the middle of the femur is determined expression level by flow cytometry.This of short duration incubation program is expressed Fas and is increased to 11.8 ± 3.2% (p＜0.001) from 3.5 ± 1.2%, make FasL increase to 12.2 ± 2.8% (p＜0.005) from 6 ± 1.5%, pro cell-matrix being interacted is defined as the important inducible factor of these molecules.

Embodiment 10

Death receptor the candidate send that blood is done and progenitor cell in expression

The result

Whether character and these cells of having studied the cell that raises death receptor belong to the progenitor cell subclass with hematopoietic reconstitution potential.Stem cell accounts for complete BMC's～and 0.5%, thereby they are at lin ^-Incidence in the cell is little, has the BMC subclass of significant functional heterogeneity based on common generation of the separable programming of phenotype.Use two kinds of separable programming enrichment hemopoietic stem cells and progenitor cell.

A. phenotypic evaluation does and progenitor cell

Mouse HSPC mainly belongs to phenotype and is defined as lin ^-Sca-1 ⁺C-kt ⁺Subclass [37,38].Transplanting CFSE ⁺Lin ^-Measure the distribution of death receptor in the donorcells subclass that the BM that 2 (n=7) and 6 days (n=5) express these marks behind the donorcells goes back to the nest.After 48 hours, death receptor mainly is expressed in Sca-1 ⁺Cell (Fig. 5 A) and the outstanding c-kit that is expressed in ^-Cell (Fig. 5 B).Though Sca-1 ⁺Mark (CFSE ⁺Lin ^-The donorcells that BM-goes back to the nest 11.5 ± 2%) and death receptor express that (Fig. 5 A) is not significant to be changed, but c-kit ⁺Subclass from the 2nd day 32 ± 5% reduce to the 6th day 20 ± 3% (Fig. 5 B).The minimizing of this quantity is accompanied by the c-kit that expresses described acceptor ⁺The relative increase of cell fraction (for Fas, TNF-R2 and TRAIL, p＜0.05).The c-kit of TNF receptor negative ⁺The mark of cell and the minimizing of absolute quantity are unexpected, but because these receptor-mediated apoptosis of technician's expectability.

To the CFSE that forms by primary candidate HSPC ⁺Lin ^-Sca-1 ⁺C-kit ⁺The further analysis of cell subclass discloses all death receptors and expressed (Fig. 5 C) in back 48 hours in transplanting.The example that TNF-R1 expresses is shown in Fig. 5 D.Similarly, in fact the most corresponding all CFSE with repopulation HSPC ⁺Lin ^-Sca-1 ⁺C-kit ⁺Cell is the Fas and the FasL positive (Fig. 5 E-F).At lin ^-C-kit ⁺In the cell subclass ,～30% expresses Fas, and～85% expresses FasL (Fig. 5 E).Take all factors into consideration, these data indicate primary mouse progenitor cell to raise death receptor soon after transplanting, and keep this expression in ensuing a couple of days.

B. the expression in short-term and long-term hematopoietic reconstitution cell

For purifying has different long-term (LTR) and two kinds of cell colonys of short-term hematopoietic reconstitution (STR) potential, use separable programming based on density.Remove processing by pedigree and eluriate the minicell of collecting under the flow velocity, to produce the component (Fr25lin of enrichment LTR cell at 25ml/min ^-) (Fig. 5 G), big STR subclass then when the BMC fractionation finishes in collecting away from rotor-position.Most of Fr25lin that transplants ^-The mouse of cell is dead (8/11) in the common observed death time section of the mouse of the clear marrow of not accepting cellular transplant.The mouse of transplanting the STR cell survives the time period of several weeks, and it is chimeric that major part wherein (5/8) fails to set up persistent donator type.Different is that the mouse of transplanting two kinds of cell colonys (LTR+STR) shows persistent immigration and competent hemoposieis (not shown) in series is transplanted.These data with to the early stage of these cell subclass and late period hematopoietic reconstitution potential previous report consistent. ³⁵

The analysis of the cell of fresh elutriation is disclosed FasL at 27 ± 4% Fr25lin ^-Express in the cell (Fig. 5 H), the lymphocytic flow cytometry of polluting is presented at lin ^-Expression among the BMC.This shows that FasL forms expression in the component of enrichment LTR stem cell.Next (CD45.2 → CD45.1), results are used for analyzing two days later two subclass to be implanted into (850 rad) homogenic recipient of irradiation.The Fr25lin that marrow is gone back to the nest ^-Show that significant Fas and FasL raise, and have higher levels of expression (p＜0.001) (Fig. 5 H) with the STR cell to the marrow in the STR progenitor cell going back to the nest.Therefore, the primary subclass that has the stem cell of long-term and short-term hematopoietic reconstitution potential and progenitor cell respectively is at the early expression death receptor to the marrow of going back to the nest.

Embodiment 11

The anti-Fas mediated Apoptosis of the cell that marrow is gone back to the nest

The result

The remarkable rise of Fas expression of receptor shows that donorcells becomes to apoptotic sensitivity.In order to check this possibility, transplant homogenic lin to the mouse (850 rad) of radiation conditioning ^-BMC (CD45.1 → CD45.2), the cell of going back to the nest from femur bone marrow results BM after 2 days.These cells are exposed to the proteic apoptosis of 250ng/ml FasL attack 18 hours under the situation of supportive chemokine of external shortage and serum to strengthen the susceptibility of cell to apoptosis.FasL attacks announcement marrow residential cell apoptosis is had significant resistance (Fig. 6 A).In the residual host BMC of radiation survival, expection is because low-level Fas expresses to the resistance of FasL inductive apoptosis.Different is that the donorcells that about 45% BM goes back to the nest was the Fas positive in back 48 hours in transplanting.With Fas and pedigree marker expression is that the apoptosis of reference is measured (Fig. 6 B) and proved most of Fas ⁺The donorcells that BM goes back to the nest is insensitive to FasL inductive apoptosis.

Identical apoptosis is attacked and is applied to transplant the LTR of elutriation and the cell (Fig. 6 C) that the BM after the STR subclass goes back to the nest.BM went back to the nest in the 2nd day cell shows FasL albumen relative insensitivity, and compares with the cell subclass of natural elutriation, and apoptosis significantly reduces (p＜0.001).The apoptotic cell death that is expressed as reference with Fas is measured (Fig. 6 D) announcement LTR and the concentrated Fas of two sons of STR ⁺Cell is all insensitive to FasL inductive apoptosis.Fas ⁺LTR cell and Fas ⁺The STR cell is compared has the stronger resistance to FasL inductive apoptosis.Carry out similar experiment by cells contacting TNF-α and the TRAIL that the 2nd day BM gone back to the nest, produced consistent lin ^-BMC is to the resistance of similar receptor-mediated apoptosis.

Embodiment 12

The acceptor string holds together the influence of (cross talk) pair cell apoptosis susceptibility

The previous TNF that studies show that induces the Fas acceptor, its ascribed to the vigor of hematopoietic cell and function have highly deleterious result (15-22,28-30).All donorcellses (Fig. 3 C) of the early stage rise indication～30% of TNF acceptor and the lin of 89-93% ^-Sca-1 ⁺C-kit ⁺Cell was expressed the acceptor (Fig. 5 C) of TNF and FasL after 2 days.Therefore, with the reference that is expressed as of TNF superfamily receptors, monitor after 1-2 days results and use FasL albumen to carry out the apoptosis of the cell that BM that apoptosis attacks goes back to the nest.In order to increase general susceptibility, incubation cell in the substratum that does not contain supplement and chemokine to apoptosis.Dead mark in the receptor positive subclass does not have significant difference, and FasL not in the cell of TNF and Fas acceptor stained positive apoptosis-induced (Fig. 6 E).These data show that the inducibility between the death receptor is crosstalked and the perception of particular subset to FasL inductive apoptosis has nothing to do.This insensitivity may be to be caused the Fas mediated Apoptosis is insensitive by the cell that lacks acceptor coexpression or some subclass.In view of all death receptors of primary HSPC coexpression, it is insensitive to apoptosis that these data are pointed to cells, rather than lack the acceptor that the perception apoptosis triggers.

Embodiment 13

Remove the cell of apoptotic sensitivity and can not eliminate the hematopoietic reconstitution potential of progenitor cell and stem cell

Next the inventor inquires whether the doing with progenitor cell of hematopoietic reconstitution of the persistent and short-term of the mouse of being responsible for clear marrow resides in the apoptotic sensitivity or apoptosis resistance subclass of BMC.To go into inferior (850 rad) homogenic (CD45.1 → CD45.2 recipient (embodiment 14 that vide infra) that causes death and nurse one's health with the Transplanted cells preincubation of FasL albumen external.(Fig. 6 F) chimeric on level terms during 3 weeks, and all recipients begin to form complete donor chimeric (Fig. 6 G) 14 weeks after transplanting.Use the donor of chimeric medullary cell as (950 rad) recipient (CD45.2) of the clear marrow of secondary.All secondary recipients (n=7) show after week that at 12-16 donor is chimeric completely.Take all factors into consideration, these data indication short-terms and long-term hematopoietic reconstitution cell all are not subjected to of short durationly to be exposed to that short apoptosis FasL is proteic influences.Suppose that progenitor cell resides in lin ^-Component is by making lin before transplanting ^-BMC is exposed to FasL and carries out identical experiment.Transplant with substratum (66 ± 5.8%) and with the lin of FasL albumen (65 ± 4.1%) incubation after 24 hours ^-The similar chimeric level (n=6) that BMC obtains provides progenitor cell to the insensitive direct evidence of apoptosis.

In order to check with short apoptosis ligand preincubation whether improve the transplanting result, inculcated small amounts of cells.Significantly, find that this incubation time period induces Fas to express and apoptosis-induced (embodiment 14 that vide infra) in ripe BMC.Succour the lin of homogenic host (950 rad) needs of clear marrow ^-The quantity limitation of cell is about 2x10 ⁵Therefore individual cell inculcates 1.5x10 to mouse ⁵Individual BMC (Fig. 6 H).It is isograft that (4 weeks of back of CD45.2 → CD45.1) are higher than the survival rate (9/20) that (14/20) is implanted in the mouse of the BMC of preincubation in (not conforming to FasL) substratum with the recipient's of the cell of FasL preincubation survival rate.At allograft (H2K ^b→ H2K ^d) in observed similar survival rate difference, the survival rate of transplanting the mouse of the pretreated and contrast BMC of FasL is respectively 10/20 and 6/20.Therefore, improved the radio-protective quality that moves into cell with the preincubation of FasL albumen.Next the inventor has assessed FasL whether the activation of hemopoietic progenitor cell has been eliminated the stem cell with long-term reconstruction potential.The chimeric medullary cell of 12 week results behind first set grafting is inculcated homogenic recipient (CD45.1) to the clear marrow of secondary with it.Half of femur entocyte is implanted into each secondary recipient, obtains after 16 weeks that donor is chimeric completely, indication with short apoptosis ligand incubation after kept the self of stem cell.

Embodiment 14

The nature bone myelocyte is to the susceptibility of apoptosis

The result

In a series of experiments, checked of the behavior of nature bone myelocyte in the external response apoptotic stimulus factor.WBMC and FasL albumen incubation caused in 24 hours～40% apoptosis death (Fig. 7 A), and wherein the main mark of apoptotic cell is contained in lin ⁺Subclass.Further analyze and disclose all main BMC pedigrees all to Fas mediated Apoptosis sensitivity (Fig. 7 B).From low death receptor incidence (Fig. 3 A), Fas's is expressed in irriate among the natural wBMC (Fig. 7 C) under these incubation conditions.But, remarkable fractional lin ^-Fas ⁺Cell is insensitive to the FasL mediated Apoptosis.Incubation lin under the same conditions ^-BMC has confirmed the rise of Fas, and further shows about 50% lin ^-Fas ⁺Cell is to apoptosis insensitive (Fig. 7 D).Unexpectedly, FasL more effectively induces Fas than the combination of 10ng/ml STEM CELL FACTOR (SCF) and 100ng/ml thrombopoietin (TPO).

Subsequently incubation was carried out 1,3 and 5 day, and added the somatomedin that SCF+TPO or SCF+TPO+IL-3 are survived in culture as the hematopoiesis support cell.A series of pilot study indication lacks significant effect with TNF-α as the cell that apoptosis triggers agent preincubation.In addition and since TNF-α induce Fas express in and supposition function in the perception apoptosis, with cell reagent incubation 3 days therewith, and during last 24 hours of incubation, add FasL.The absolute quantity of cell descends, but lin ^-The mark of BMC obtains keeping (Fig. 7 E) to a great extent.With Fas and TNF receptor expression serves as with reference to apoptosis is analyzed (Fig. 7 F).Incubation in the substratum shows TNF-R2 ⁺Cell and Fas ⁺And TNF-R1 ⁺Cell is compared has higher apoptosis rate (p＜0.001).Exist TNF-α to Fas in the substratum ⁺And TNF-R2 ⁺Cell has less influence, and has reduced TNF-R1 ⁺The apoptosis of cell (p＜0.001) shows that this acceptor participates in the stimulation rather than the inhibition of cell.In the 3rd day of incubation, be exposed to FasL and significantly increased apoptosis rate (p＜0.001) and irrelevant with the preincubation of TNF-α.These vitro datas show that the Fas mediated Apoptosis is to express the joint effect approach of apoptosis in the cell of TNF superfamily receptors and determine that TNF-α has the effect of appropriateness relatively in the perception of these cells.

Incubation pair cell quantity (Fig. 7 G) and apoptotic cell mark (Fig. 7 H) have less influence under the situation of SFC+TPO existing.But exist under the situation of SCF+TPO+IL-3, the quantity of survivaling cell significantly increases, particularly at lin ^-In the subclass (Fig. 7 I).This is accompanied by the reduction (Fig. 7 J) of receptor positive cell to the susceptibility of apoptosis.Further analyze and show that the stem cell markers Sca-1 of expression supposition and the cell of c-kit mainly are positioned at the survival component of cell.Similarly, in the contrast incubation that is to support to carry out in the substratum, 10 ± 2% LTR cell and 22 ± 4% STR cell are annexin-V stained positive (Fig. 6 C).Add the STR subclass apoptosis that FasL albumen causes 40% fresh elutriation, and the LTR subclass shows that less apoptosis increases.Significantly, during this incubation time period, two kinds of cells of LTR and STR raise its Fas respectively and express to 18 ± 5% and 42 ± 6%.With TNF incubation 5 days and after adding FasL in the end one day, survival lin ^-The quantity of BMC increases by 2.3 times, and the death of indication apoptosis accounts for lin ⁺The remarkable mark of BMC (Fig. 7 K).These data exhibitings from the different methods of the initial undifferentiated cellular component of marrow inoculum enrichment.

Embodiment 15

Receptor-mediated apoptosis of Fas and TNF and non-apoptotic signal

Under physiological condition, the stem cell with hematopoietic reconstitution potential constitutes the very little part that a large amount of progenitor cells (being in different differential periods) include with progenitor cell.In the far-end stage of differentiation, death receptor has vital role in the size of regulating the amplification clone.These acceptors have been confirmed as all hematopoietic lineages negative regulon (7-14) of end differentiation eventually.It is early stage to transplant the back, at donorcells apoptosis is lacked under the situation of susceptibility, and the rapid rise of death receptor has proposed query to the effect of these acceptors.Therefore, the inventor has assessed under the minimal stimulation condition, increases FasL and TNF-α step by step to wBMC and lin ^-BMC produces the active regulating effect of clone.The activity of wBMC is prevented (Fig. 8 A) day by day along with the increase of FasL concentration, when having TNF-α then unaffected to a great extent (Fig. 8 B).Lack the apoptotic signal consistent (referring to embodiment 13) that TNF-α triggers among these data and the natural B MC.Significantly opposite, lin ^-The product clone activity of BMC increases gradually, increases by 45% and 70% respectively when～500ng/ml FasL and TNF-α concentration.When adding soluble Fas L oligomer to substratum and by biotinylation protein adsorption all having been observed this behavior (Fig. 8 A) during to cell surface.FasL becomes to lin when the threshold concentration of～1 μ g/ml ^-BMC is poisonous.Consistent to the no remarkable effect of colony formation in mouse (17) and people HPSC (5,18) with the activation Fas antibody (Jo2) of previous report, the solubility superFasL of 5ng/ml concentration fails to weaken lin ^-The clone of BMC is taken place.Take all factors into consideration, these data show that Fas acceptor trimerizing is that the growth signals transduction is essential.

WBMC and lin ^-The relative product clone of BMC is active show apoptotic sensitivity with insensitive cell subclass in the property followed nutrition and apoptotic signal transmission by the Fas acceptor.In the wBMC culture, dead cell can suppress the product clone activity of progenitor cell.In order to check this possibility, apoptosis wBMC and 500ng/ml FasL albumen are added into lin together ^-The BMC culture.As survival lin ^-The ratio of BMC and dead BMC is 1: 1 o'clock, and FasL inductive enhanced the clone be eliminated, and shows that the effect of vigor colony of a large amount of cells forms.

Near-end and far-end stage in the apoptosis cascade have been carried out further assessment.In order to determine FasL, results have been carried out from the cell of Fas-defective (lpr) mouse produced clone's mensuration by influencing cell with the Fas receptors bind.The lpr sudden change makes wBMC and lin respectively ^-BMC is to proteic apoptosis of FasL and nutritional effect insensitive (Fig. 8 C), and the active adjusting of indication progenitor cell connects the specificity mediation by the Fas acceptor.The clone who has observed similar decline behind described cell and the closure TNF-R1 antibody incubation is taken place, show that this acceptor compares preferential participation trophic signals transduction with TNF-R2.

At the far-end of apoptosis cascade, suppress Caspase 3 and 8 to reduce the apoptosis death in the culture to greatest extent.Suppress Caspase 3 (DEVD) and 8 (IETD) have all reduced apoptosis and improved wBMC under all FasL concentration product clone active (Fig. 8 D).After suppressing these Caspases, at lin ^-During measuring, the product clone of BMC observed consistent result (Fig. 8 E).Adding not remarkably influenced of Caspase inhibitor clone under the situation that does not have FasL takes place.It should be noted that suppressing the Caspase activity does not eliminate 500ng/ml FasL to lin ^-The stimulatory effect of BMC, the nutrition (tropism) of indication Fas mediation is mediated by the Caspase-8 activatory near-end factor.

It is irrelevant with the existence of FasL that the analysis of the cell colony eluriated is shown that the nothing of LTR cell is produced clone's active (5 colonies and four parts of blank cultures are arranged in a culture), as expection and the previous report [34,35] to these cells.Significantly different is active increase by 26 ± 6% (p＜0.005) (Fig. 8 F) of the product clone of STR cell when having the 500ng/ml FasL albumen of super lethal concentration.1.5 it is active that the FasL of μ g/g concentration eliminates the product clone of these progenitor cells fully.In order to assess the balance between receptor-mediated nutrition of Fas and the apoptotic signal, the inhibition of joint effect Caspase-3 (DEVD) makes the clone's effect of promoting production of FasL further be enhanced to 75 ± 5% (p＜0.001 is with natural the comparing with culture of no Caspase-3 inhibition).Take all factors into consideration, these data indicate the subclass of early stage hematopoietic reconstitution cell (STR) to receive dual apoptosis and trophic signals by the Fas acceptor.

Embodiment 16

Progenitor cell in stress hematopoiesis to the resistance of apoptosis

The result

The problem whether mechanism that the HSPC of the natural or derived from bone marrow transplanted has proposed the death receptor mediation to the insensitivity of apoptotic signal works under stress the hematopoiesis condition.5 FU 5 fluorouracil (5FU) is poisonous to the progenitor cell of Rapid Cycle, and they kill and wound the synchronous stimulation of inducing to original hemopoietic progenitor cell in the marrow.In order to activate and synchronous hemopoietic progenitor cell, to injected in mice 150 μ g/g 5 FU 5 fluorouracils (5FU), their medullary cell of results after 1,3 and 5 day.After 5 days, the incidence that Sca-1 expresses increases to 29 ± 2% (p＜0.001 is 2 ± 1% among the contrast natural B MC), and the incidence that c-kit expresses increases to 12 ± 2% (p＜0.05 is 8 ± 1.5% among the contrast natural B MC), and Sca-1 ⁺C-kit ⁺Mark increase to 5 ± 0.9% (p＜0.001 is 0.4 ± 0.1 among the contrast natural B MC) (Fig. 9 A).The rise (p＜0.001) (Fig. 9 B) that these change with Fas acceptor and its part is included under the steady state conditions lin with these molecules of low expression level ^-The mark of BMC (Fig. 9 C).TNF and TRAIL acceptor have been observed the similar variation of less degree.In a junctor, under the incubation conditions, attack the 18 hours other time period of cell of results this moment with FasL albumen.Lin ^-And lin ⁺Subclass with FasL albumen incubation after all show with results and compare significantly higher vigor (Fig. 9 D) from the BMC of natural mouse.In order to show the relation between Fas expression and the apoptosis, after the interior incubation of a junctor is injected the cell of gathering in the crops in 1,3 and 5 day behind the 5FU, by the quantitative described acceptor of flow cytometry.With the necrocytosis fraction representation is the function that Fas expresses, and shows several characteristic (Fig. 9 E-F).The first, after using 5FU, being expressed in of Fas increase in time.The second, compare with the cell (Fig. 9 E) of incubation in substratum, be exposed to FasL (Fig. 9 F) in the iuntercellular junctor and further strengthened described receptor expression.The 3rd, most of Fas ⁺BMC is insensitive to FasL inductive apoptosis after 5FU handles.Take all factors into consideration, the progenitor cell during the indication of these data forms is to the rise of FasL inductive Fas acceptor susceptible more, and do not follow the perception to apoptosis.These data indication death receptors, particularly Fas are brought into play trophism in the physiological function of hemopoietic system under stressed condition.

Discuss

Different with most of somatocyte, stem cell often need be carried out the differentiation task under the extreme condition of injured and inflammation.Adult stem cell is participated in tissue repair, particularly breaks up and adopt the versatility requirement of the functional performance of damaged tissue, proposed these cells are survived under adverse environment and the query of the ability that works.Known program is that hematopoiesis is done and progenitor cell (HSPC) transplanting, and it carries out after causing the aggressive chemotherapy of the major injury of marrow and radiation usually.Endogenous hematopoietic cell seldom survives marrow radiation clearly, and matrix is subjected to major injury.The consequence of ablative damage is, donor HSPC finds its approach that enters host's marrow, and its inoculation also moves into host's marrow, to rebuild immune hemopoietic system.In this process, in remarkable fractional donorcells, find the rapid rise of death receptor.Previous work thinks that death receptor plays deleterious effect in the hematopoietic cell function, and this is from the notion of acceptor in the inhibition function employing in the far-end stage of immune hemopoietic system differentiation.On the contrary, the inventor has found the active effect of death receptor in the stem cell function.The hematopoietic reconstitution cell especially merits attention by which kind of mechanism prosperity in this breaking-up environment, because it can be used for improving the efficient of immigration.

In a word, the inventor shows:

1. under steady state conditions, death receptor with low expression level in medullary cell.Described acceptor comprises at hemopoietic under the condition of transplanting and sharply raising.

2. do and the initial subset of progenitor cell is expressed all death receptors.

3. in the progenitor cell with hematopoietic reconstitution potential, death receptor is the mediated apoptosis signal not.

Fen Hua the far-end stage and in somatocyte the dead same receptor of mediation most of primary hematopoiesis do and progenitor cell in mediate trophic signals.

5. use dead signal to eliminate short-term hematopoietic reconstitution potential that non-stem cell do not influence progenitor cell and the long-term reconstruction potential of stem cell.

6. the expression of death receptor transcriptional level is subjected to inducing of a plurality of inherences and external factors.These comprise factor response injured, with the release of matrix phase mutual effect, circulation and differentiation.

7.Fas-the composing type of part and enhancement type are expressed by eliminating alloimmunity and are replied and the immigration of non-immunologic mechanism increase hematopoietic cell.

8. death receptor is effectively removed non-stem cell at indirect intravital abduction delivering and activation by apoptotic signal.

Take all factors into consideration, these data point to TNF superfamily death receptor do with progenitor cell in different binding modes.Under physiological condition, induce HSPC activatory signal also to induce the expression of death receptor.This can pass through the synchronous active activation illustration of progenitor cell of 5FU, and it is with the expression gradually of Fas acceptor.But the activatory progenitor cell is insensitive to the Fas mediated Apoptosis to a great extent: Fas ⁺The mark of cell increase 3-4 doubly with 5 days after the overwhelming majority of described cell relevant to the resistance of FasL inductive apoptosis.These data show that Fas is raised (may be because the cytokine environment that the death of marrow inner cell causes is twisted) in early days after using 5FU, and play a role to promote the growth of hemopoietic progenitor cell.The Fas acceptor only continues its function (7-14) as the negative regulon of progenitor cell in forming in the far-end stage of differentiation characteristic.

In transplanting background, think that following plot participates in the Fas/FasL signal pipeline of the commitment of hematopoietic cell immigration.Donorcells is gone back to the nest to marrow, inflammatory environment and induce the expression of acceptor and part with the interaction of matrix.The expression of Fas makes donorcells be converted into its active environmental factor of response regulation, hematopoietic cell is moved into produce supportive rather than harmful effect.The progenitor cell of eliminating differentiation more by apoptosis has increased the chance that more primary is done and progenitor cell moves into.This can be regarded as inoculating and early stage migration process in the mechanism of HSPC enrichment, continued enrichment process more early and ascribed HSPC to and compare better go back to the nest (1) with committed progenitor.Resistance development to the death of Fas mediated Apoptosis is the functional performance of those donorcellses that successfully move into.This characteristic is given former progenitor cell and is used the part of TNF superfamily to strike back immunoreactive ability (32-34).

The expression of TNF superfamily death receptor may be the physiological responses of immune hematopoietic cell after meeting with adverse environment or being exposed to stressed condition.Be endowed the maintenance that guarantees the critical elements in this development system with progenitor cell of doing of these protectiveness factors.In the apoptosis resistance progenitor cell, these acceptors with help to raise that former progenitor cell breaks up is relevant with the trophic signals transmission of breed.This mechanism has not only been protected the most important element stem cell of this development system, and participates in their activation under the extreme condition of damage.Previous research has proved that high-caliber anti-apoptosis factor (comprises FLIP, Bc1-2, survivin and uniquely expresses that Caspase-8L) the protection hemopoietic progenitor cell is avoided apoptosis (5,6).Data indication of the present invention, trophic signals transmission produce the main path from the relevant apoptotic signal transmission of the death receptor of Caspase-8 activation near-end.

Should be understood that for clarity and some feature of the present invention of describing in the embodiment environment that separates also can make up in single embodiment and provides.Conversely, the different characteristics of describing in single embodiment environment for simple and clear purpose of the present invention also can be individually or is closed with any suitable subgroup and to provide.

Though invention has been described in conjunction with its specific embodiments, many apparently to substitute, modify and change those skilled in the art be tangible.Therefore, the invention is intended to comprise that all that fall in claims spirit and the broad scope substitute, modify and variation.All publications, patent and patent application of mentioning in this specification sheets and GenBank accession number are here integrated by reference and are incorporated into this specification sheets, reach as pointing out that clearly and individually each concrete publication, patent and patent application and GenBank accession number are incorporated by reference the degree of this paper.In addition, quote among the application or any reference of determining all should not be construed as and admits that described reference can be used as prior art of the present invention.

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Claims (52)

1. method of from foreign cell colony, selecting stem cell, described method comprise make described cell colony and inducer of apoptosis for non-stem cell be apoptosis and be to contact under the condition of non-apoptosis stem cell, thereby from described foreign cell colony, select stem cell.

2. the method for claim 1, wherein said stem cell are selected from by cord blood stem cell, the group of mobilizing back peripheral hematopoietic stem cells, bone marrow stem cell and neural stem cell to form.

3. method as claimed in claim 2, wherein said stem cell is a bone marrow stem cell.

4. method as claimed in claim 3, wherein said bone marrow stem cell is a hemopoietic stem cell.

5. the method for claim 1, it modifies described stem cell before further being included in described contact, to generate the stem cell of modifying.

6. the method for claim 1, it further is included in the described stem cell of purifying before the described contact, to generate the stem cell of purifying.

7. the method for claim 1, it further is included in the described stem cell of increasing before the described contact, to generate the stem cell of amplification.

8. method as claimed in claim 3, wherein said bone marrow stem cell is a mescenchymal stem cell.

9. the method for claim 1, wherein said stem cell is an adult stem cell.

10. the method for claim 1, wherein said stem cell is an embryonic stem cell.

11. the method for claim 1, wherein said inducer of apoptosis is selected from the group of being made up of TNF-α, FasL, Trail and Tweak.

12. method as claimed in claim 11, wherein said inducer of apoptosis is FasL.

13. method as claimed in claim 12, wherein said FasL is coupled to the surface.

14. method as claimed in claim 13, wherein said FasL can not cut.

15. the method for claim 1, it further is included in and raises apoptosis receptor expression in the described foreign cell colony before the described contact.

16. method as claimed in claim 15, wherein said apoptosis acceptor are selected from the acceptor group of being made up of Fas acceptor, TNF-α acceptor, Tweak acceptor and Trail acceptor.

17. method as claimed in claim 15, the described apoptosis receptor expression of wherein said rise contacts realization by making described foreign cell colony with interferon-gamma or TNF-α.

18. the method for claim 1, wherein said foreign cell colony does not comprise the T lymphocyte of immune activation.

19. the method for claim 1, wherein said foreign cell colony comprises the pedigree positive cell.

20. method as claimed in claim 19, wherein said pedigree positive cell is selected from the group of being made up of granulocyte, scavenger cell, natural killer cell, erythroblast, antigen presenting cell, medullary cell, lymphoidocyte and megalokaryocyte.

21. the method for claim 1, wherein said foreign cell colony comprises the malignant cell of apoptotic sensitivity.

22. the method for claim 1, it separates described stem cell after further being included in described contact.

23. host's method is gone in the stem cell transplantation with selection, described method comprises:

(a) stem cell that makes foreign cell colony and inducer of apoptosis for non-stem cell be apoptosis and be to contact under the condition of non-apoptosis stem cell, thereby select stem cell; With

(b) host is gone in the stem cell transplantation of described selection, thereby transplant the stem cell of described selection.

24. method as claimed in claim 23, it further is included in step (a) is separated described selection afterwards before with step (b) stem cell.

25. method as claimed in claim 23, wherein said stem cell are selected from the group of being made up of cord blood stem cell, mobilization back peripheral hematopoietic stem cells, bone marrow stem cell and neural stem cell.

26. method as claimed in claim 25, wherein said stem cell is a bone marrow stem cell.

27. method as claimed in claim 26, wherein said bone marrow stem cell is a hemopoietic stem cell.

28. method as claimed in claim 23, it modifies described stem cell before further being included in described contact, to generate the stem cell of modifying.

29. method as claimed in claim 23, it further is included in the described contact described stem cell of purifying before, to generate the stem cell of purifying.

30. method as claimed in claim 23, it further is included in before the described contact or the described period of contact described stem cell of increasing, to generate the stem cell of amplification.

31. method as claimed in claim 26, wherein said bone marrow stem cell is a mescenchymal stem cell.

32. method as claimed in claim 23, wherein said stem cell is an adult stem cell.

33. method as claimed in claim 23, wherein said stem cell is an embryonic stem cell.

34. method as claimed in claim 23, wherein said inducer of apoptosis is selected from the group of being made up of TNF-α, FasL, Trail and Tweak.

35. method as claimed in claim 34, wherein said inducer of apoptosis is FasL.

36. method as claimed in claim 35, wherein said FasL is coupled to the surface.

37. method as claimed in claim 36, wherein said FasL can not cut.

38. method as claimed in claim 23, it further is included in and raises apoptosis receptor expression in the described foreign cell colony before the described contact.

39. method as claimed in claim 38, wherein said apoptosis acceptor are selected from the group of being made up of Fas acceptor, TNF-α acceptor, Tweak acceptor and Trail acceptor.

40. method as claimed in claim 38, the described apoptosis receptor expression of wherein said rise contacts realization by making described foreign cell colony with interferon-gamma or TNF-α.

41. method as claimed in claim 23, wherein said foreign cell colony does not comprise the T lymphocyte of immune activation.

42. method as claimed in claim 23, wherein said foreign cell colony comprises the pedigree positive cell.

43. method as claimed in claim 42, wherein said pedigree positive cell is selected from the group of being made up of granulocyte, scavenger cell, natural killer cell, erythroblast, antigen presenting cell, medullary cell, lymphoidocyte, class red corpuscle and megalokaryocyte.

44. method as claimed in claim 23, wherein said foreign cell colony comprises the malignant cell of apoptotic sensitivity.

45. method as claimed in claim 23, wherein said stem cell are that the host is from body.

46. method as claimed in claim 23, wherein said stem cell are that the host is isogenic.

47. method as claimed in claim 23, wherein said stem cell are that the host is heterogenic.

48. method as claimed in claim 23, wherein said stem cell are host's xenogeneic.

49. the method for a differentiated stem cells, described method comprises:

(a) stem cell that makes foreign cell colony and inducer of apoptosis for non-stem cell be apoptosis and be to contact under the condition of non-apoptosis stem cell, thereby select stem cell; With

(b) induce the differentiation of the stem cell of described selection, thus differentiated stem cells.

50. method as claimed in claim 49, wherein said induce the differentiation realize by expressing gene product in described stem cell.

51. method as claimed in claim 50, wherein said gene product is a polypeptide.

52. method as claimed in claim 50, wherein said gene product is polynucleotide.

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