CN1756836B - Modulation of stem and progenitor cell differentiation, assays, and uses thereof - Google Patents

Modulation of stem and progenitor cell differentiation, assays, and uses thereof Download PDF

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CN1756836B
CN1756836B CN03813627.9A CN03813627A CN1756836B CN 1756836 B CN1756836 B CN 1756836B CN 03813627 A CN03813627 A CN 03813627A CN 1756836 B CN1756836 B CN 1756836B
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cell
differentiation
progenitor cell
compound
progenitor
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CN1756836A (en
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R·J·哈里里
D·I·斯蒂尔林
K·W·H·陈
L·A·穆托-德帕塞瓦尔
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Celgene Corp
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Celgene Corp
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Abstract

The present invention relates to methods of modulating mammalian stem cell and progenitor cell differentiation. The methods of the invention can be employed to regulate and control the differentiation and maturation of mammalian, particularly human stem cells along specific cell and tissue lineages. The methods of the invention relate to the use of certain small organic molecules to modulate the differentiation of stem or progenitor cell populations along specific cell and tissue lineages, and in particular, to the differentiation of embryonic-like stem cells originating from a postpartum placenta or for the differentiation of early progenitor cells to a granulocytic lineage. Finally, the invention relates to the use of such differentiated stem or progenitor cells in transplantation and other medical treatments.

Description

Adjusting, evaluation and the application thereof of stem cell and progenitor cell differentiation
The application requires the U.S. Provisional Application 60/372348 of submission on April 12nd, 2002, the right of priority of the U.S. Provisional Application 60/437350 that the U.S. Provisional Application 60/437348 that on December 31st, 2002 submitted to and on December 31st, 2002 submit to is quoted the full content of each piece at this.
1. introduce
The present invention relates to regulate the method for mammalian stem cell and/or progenitor cell differentiation.Method of the present invention can be used for regulating and controlling Mammals, especially people's stem cell along specific cells and differentiation and the maturation of organizing pedigree.Method of the present invention relates to uses some little organic molecule to regulate stem cell or progenitor cell along specific cells with organize the differentiation of pedigree, especially come from the placenta in postpartum embryonic-like stem cell differentiation or along specific differentiation pathway, the particularly adjusting of the early stage hemopoietic progenitor cell of granulocyte differentiation pathway.The invention still further relates to and use these organic molecules to regulate the particular lineage progenitor cell, as CD34 +, CD45 +And CD133 +The differentiation of progenitor cell.The invention still further relates to the time aspect that progenitor cell is grown, and based on these time-related external models.The invention further relates to these adjusted cells in prevention and methods of treatment, be included in the application in the pharmaceutical composition of this cell and/or little organic compound.At last, the present invention relates to the application of cell in transplanting and other therapeutic treatments of this differentiation.
2. background of invention
The evaluation of people's stem cell and progenitor cell, separate and propagation on important concern is arranged.Stem cell is the all-round or multipotency precursor cell that can produce multiple mature cell pedigree, and precursor cell is the cell that can produce specific cells pedigree cell.These abilities are as for the essential cytodifferentiation of organ pipe and tissue development and the basis of specialization.
Recently provide new clinical tool with the marrow-reconstitution after being used for because of disease, being exposed to toxic chemical substance and/or the excision of radiating marrow and/or replenish transplanting success on stem cell and the progenitor cell.Further the existence of evidence proof stem cell can be used for rebuilding many, even be not whole tissues and repair function on physiological situation and physiology and the anatomy.The application of stem cell in organizational project, gene therapy transmission and cell therapy also just developing by leaps and bounds.
Many dissimilar mammalian stem cells and ancestral stem cell have obtained identifying.For example, known have embryonic stem cell, embryonic genital cell, adult stem cell or committed stem cell or a progenitor cell.The also not separated and evaluation of some stem cell, but allowing under the condition of restricted portion differentiation, to obtain cultivation.Yet surplus next basic problem is exactly that control and regulate stem cell and progenitor cell is very difficult as the differentiation of hemopoietic progenitor cell.At present, the method for existing these cytodifferentiation of adjusting is jejune and uncontrollable, thereby cell is divided into the unwanted cells type in the unwanted time.And the productive rate of cellular product is generally lower.
In addition, obtain sufficient amount be used for the treatment of or the human stem cell of research purpose still has problem.Partly and since in blood or tissue the limited quantity of the stem cell found or progenitor cell and with obtain the relevant remarkable discomfort of marrow aspirate, the stem cell or the progenitor cell populations that are separated into normal presence in the body tissue have technical difficulty, and the expense costliness.Generally speaking, be used for the treatment of or the stem cell of research purpose or progenitor cell effort very normally, comprise from substituting source results sufficient amount, for example, from donor or patient's harvested cell or tissue, vitro culture and/or propagate cell, dissociated cell etc., comprise obtaining these cells among the preborn especially from embryo or fetal tissue as for stem cell, risen to the misgivings of religion and ethics.Extensively people embryo who assert or the fetus conviction that just constitutes independent life has been facilitated at cell in source like this and has been used for all purposes, comprises forbidding of government on the purposes of medical research.Thereby the substituting source that does not need to use the cell that obtains from embryo or fetus just is desirably in further developing the stem cell clinical application.Yet, the substituting source of available stem cell or progenitor cell, particularly human stem cell or progenitor cell is seldom arranged, therefore, supply is limited.
(WO00/73421 such as Hu, be entitled as " separation of people's amniotic epithelial cells, the method for cryopreservation and therepic use ", be disclosed in 2000-12-07) separation, cultivation of the people's amniotic epithelial cells that comes from when childbirth placenta disclosed, for the cryopreservation that uses in the future or induce differentiation.Described according to Hu etc., divide the puerperium to obtain a placenta immediately and separate amnion, for example by dissecting from chorion.Amniotic epithelial cells separates from amnion according to the cell separation technology of standard.The cell of the disclosure can be cultivated in various substratum, propagation, refrigeration or quilt are induced differentiation in cultivation.It is multipotential hemopoietic stem cell (also may be pluripotential hemopoietic stem cell) that Hu etc. disclose amniotic epithelial cells, and can be divided into epithelium, as cornea face tissue or vagina tissue.Yet the shortcoming of this method is a labour intensive, and the productive rate of stem cell is very low.
The in-vitro multiplication method of existing cell colony also is labor-intensive.For example, (Emerson etc. such as Emerson, U.S. Patent number 6326198, be entitled as " being used for the duplicating of stem cell in the body, the optimization that hemopoietic progenitor cell is cultivated and metabolism, GM-CSF secretion and/or IL-6 excretory method and the component that strengthens people's stroma cell ", be published in 2001-12-04) method of optimization of human stem cell division and/or artificial blood progenitor cell and the culture medium condition of vitro culture disclosed.According to disclosed method, come from the human stem cell of marrow or the progenitor cell liquid medium within and cultivate, it can be replaced, preferred perfusion culture, continuously or periodically, with the speed of 1 milliliter of substratum of every milliliter of culture, every about 24 hours to 48 hours is the cycle.Under physiologically acceptable condition, keep when cultivating, remove meta-bolites, the nutrient of supplement consumed.
(Kraus etc. such as Kraus, U.S. Patent number 6338942, be entitled as " selective proliferative of targeted cell population ", be published in 2002-01-15) disclose predetermined targeted cell population and optionally bred, come from the initial standard specimen cell of Cord blood or peripheral blood ground by in growth medium, introducing, cause the targeted cell population division, and in growth medium, use the selection component exposing cell, comprise the binding molecule (for example single colony antibody of CD34 cell) that the cell colony of being scheduled to (as the CD34 cell) is had specific avidity, in growth medium, to select predetermined targeted cell population from other cells.
Rodgers etc. (U.S. Patent number 6335195 is entitled as " method that promotes hematopoiesis and mesenchymal cell propagation and differentiation " and is published in 2002-01-01) disclose hematopoiesis and mesenchymal cell vitro culture and by proangiotensin, angiotensin I (AI), AI analogue, AI fragment and its analogue, Angiotensin II (AII), AII analogue, AII fragment and its analogue or AIIAT are being arranged 2 Type 2 receptor stimulants are united the existence special cell proliferation and the induced differentiation method of pedigree of growth down separately or with other somatomedins and cytokine.This stem cell source is in marrow, peripheral blood or Cord blood.Yet as discussed above, the shortcoming of this method is that the method for this external evoked stem cells hyperplasia and differentiation is consuming time, has also caused the low yield of stem cell.
Stem cell and progenitor cell have the potential that is used for the treatment of various disease conditions, comprise malignant tumour, inborn errors of metabolism, hemoglobinopathy and immune deficiency.Relate to a utilization of the stem cell that comes from Cord blood or placenta or a main field of research and be used for marrow and other relevant transplantations for using these cells produce small amounts of cells.Yet up to now, the no one proposes to produce a large amount of stem cells or progenitor cell, as people CD34 +Or CD133 +The method of progenitor cell.Especially, a large amount of latter cells will use that the methods of treatment of progenitor cell becomes easily.At this, method disclosed by the invention relates to this demand.
Retinene derivative is as vitamin A and vitamin A acid (RA), the known differentiation that influences stem cell.For example, vitamin A acid has been used to suppress the propagation (Nadkarni etc. of irregular orientation (chronic lymphocytic leukemia) hemopoietic stem cell, 1984, Tumori 70:503-505) and in preceding marrow leukemia cell, induce the differentiation and the forfeiture (Melchner etc. of self potential, 1985, Blood 66 (6): 1469-1472).Vitamin A acid also further is used to induce the sexual cell precursor (Damjanov etc. of conversion, 1993, Labor.Investig, 68 (2): 220-232), placenta cells precursor (Yan etc., 2001, Devel.Biol.235:422-432), reach the differentiation of endotheliocyte precursor (Hatzopoulos etc., 1998, Development 125:1457-1468).Yet the effect of retinene derivative in differentiation very understood, and it can be used as the regulating measure of control differentiation of stem cells and uses.
Folacin, the effect in the hemopoietic stem cell differentiation has obtained research as aminopterin and methotrexate (methotrexate).Folacin is used for acute lymphocytoblast anaemia and other blood proliferative disorders and cancers as chemotherapy reagent, and show that it influences the differentiation (DeLoia etc. of stem cell by killing certain class stem cell colony off, 1998, Human Reproduction13 (4): 1063-1069), therefore, can not become the effective tool of the differentiation that is used to regulate a large amount of stem cells that deliver medicine to the patient.
Some cytokines, as IL-1, IL-2, IL-3, IL-6, IL7, IL-11, with some albumen such as erythropoietin, Kit part, M-CSF and GM-CSF, also be shown and instructed stem cell to be divided into particular cell types (Dushnik-Levinson etc. by hematopoietic lineage, 1995, Biol.Neonate67:77-83), still, these processes are not fully understood and still too coarse and out of true and can not be as the regulating measure of control differentiation of stem cells.
Up to now, the no one describes compound, the application of immunomodulator compounds described as follows in stem cell or precursor cell differentiation.Especially, the no one proves such change composition adjusting progenitor cell, and as the CD34+ progenitor cell, away from the purposes of dendritic cell pedigree differentiation, this differentiation is the useful ability that is used to improve the transplantation immunity tolerance.And the no one describes compound described herein in the purposes of breeding in order to produce the medical composition that contains this cell in the progenitor cell populations.The progenitor cell culture of this propagation is useful in the raising of treatment graft versus host disease (GVH disease) and immunotolerance.Because control stem cell and precursor cell differentiation can be produced effective cell colony in treatment, therefore, for control and adjusting marrow sample dendritic cell pedigree cell, or early progenitor cell, have as the ability of the differentiation of people CD34+ or CD133+ progenitor cell required, with control dendritic cell and/or granulocytic generation.
3. summary of the invention
The invention provides the method for regulating Mammals, particularly human stem cell or progenitor cell differentiation.Specifically, method of the present invention can be used for regulating and controlling human stem cell along specific cell and differentiation and the maturation of organizing pedigree.The present invention includes influences the immunomodulatory of this adjusting and control small organic molecule, more preferably amino isoindoline compounds, the especially Actimid that replaces TMOr Revimid TMThe purposes of compound.The invention further relates to the progenitor cell that delivers medicine to, to regulate differentiation from particular approach at specific these compounds of period.
Method of the present invention comprises that stem cell or progenitor cell are divided into the regulation and control of specific cells pedigree, these cell lineages include but not limited to, a matter, hematopoiesis, living fat, living liver, living pedigree neural, collagen, that give birth to cartilage, angiogenic, myogenic, living cartilage or living bone.In specific embodiment, method of the present invention comprises that differentiation of stem cells is the regulation and control of hematopoietic lineage cell.
The present invention comprises that also committed cell becomes the regulation and control of particular cell types, and described cell type is for example mesenchymal cell, hematopoietic cell, adipocyte, liver cell, neuroblast, spongioblast, chondrocyte, endotheliocyte (EC) progenitor cell, myocyte, chondrocyte or scleroblast.In specific embodiment, the present invention includes directed hemopoietic progenitor cell and be divided into red corpuscle, thrombocyte or white corpuscle (white cell), as neutrophilic granulocyte, monocyte, scavenger cell, eosinophilic granulocyte, basophilic leukocyte, mastocyte, B cell, T cell or plasmacytic regulation and control.
In another embodiment, method of the present invention relates to regulates stem cell to hematopoietic lineage cell, especially, and CD34 +, CD133 +And CD45 +The differentiation of hematopoietic lineage, and produce the prevention that contains this cell or treat the method that goes up useful pharmaceutical composition.In another embodiment, method of the present invention relates to the differentiation of adjusting early progenitor cell to dendritic cell pedigree or granulocyte pedigree, endotheliocyte pedigree or cardiomyocyte lineage cell.
In another embodiment, the invention provides and regulate progenitor cell, especially the method for dendritic cell pedigree or granulocyte pedigree, endotheliocyte pedigree, neurocyte pedigree or cardiomyocyte lineage cell differentiation to hematopoietic cell lineage.In specific embodiment, described progenitor cell is CD34 +Or CD133 +Cell.These regulation and control are by finishing with compound contact progenitor cell of the present invention in culturing process.In one embodiment, described compound is a TNF-alpha active supressor.In a more particular embodiment, described compound is an immunomodulator compounds as herein described, or Thalidomide, or more preferably, is the isoindoline compounds of amino replacement.In specific embodiment further, described compound is Actimid TMOr Revimid TM
In another specific embodiment, method of the present invention comprises that progenitor cell is divided into the inhibition of dendritic cell.In another specific embodiment, the invention provides the method that is adjusted in the progenitor cell differentiation in the culturing process in initial six days, its cultivation is used to produce the multiplication culture thing of this progenitor cell.In another embodiment, method of the present invention comprises early progenitor cell is developed into granulocytic promotion that it be effective that this granulocyte may infect for opposing.Granulocyte pedigree committed progenitor (CD15 +Being increased in cell) alleviates in neutrocytopenia and its infection complication subsequently and has the potential purposes, and this illness has been represented the most general dose-limiting toxicity of cancer chemotherapy.In another embodiment, method of the present invention can be used for suppressing the differentiation of dendritic cell, and it is effective for the effect that relaxes graft versus host disease (GVH disease).
Progenitor cell of the present invention, after compound adjusting of the present invention, for transplanting is effectively (being the recovery of hematopoiesis), and can be used as the renewable source that substitutes cell and tissue (as pancreas, heart, liver, kidney, liver, brain, lung, bladder, intestines or muscle cell) and be used for the treatment of regenerating medicine in normal aging, damage or disease such as heart trouble, apoplexy, Parkinson's disease and the alzheimer's disease.This cell also is useful in the biochemical route in the born of the same parents of the effect of measuring mediation compound of the present invention.These cells also are useful for new medicine and the toxin of screening, for example, are used for determining the potential of cancer therapy drug, understand the cause of inborn defect etc.
Method of the present invention can be used for suppressing specifically the generation of red blood cell or red corpuscle colony (BFU-E and CFU-E), and leukocyte increasing and thrombocyte colony form the generation of (CFU-GM) and the output that improves total colony forming unit simultaneously.Method of the present invention not only can be used for regulating stem cell and progenitor cell such as CD34 +The differentiation of progenitor cell also can be used for stimulating colony forming efficiency, moves into speed and provides important help to hematopoietic stem cell transplantation by improving bone marrow graft.
Any mammalian stem cell except that human embryo stem cell can use according to method of the present invention, includes but not limited to, from Cord blood, placenta and remove extraembryonic other of the people isolating stem cell of originating.Stem cell can separate from any mammal species, for example, and rat, mouse, rabbit, cavy, dog, cat, pig, sheep, ox, horse, monkey etc., more preferably, the people.Stem cell can comprise pluripotent cell, that is, have and break up diversity, energy self completely and keep dormancy or the immobilized cell in tissue.Stem cell can also comprise multipotency cell or committed progenitor.In a preferred embodiment, that the present invention utilizes is alive, the stem cell of immobilized, versatility, it is present in or results from subsequently the embryo of foot phase, in other words, this cell can be along with the childbirth and the placenta of success are given birth to, bloodletting and placenta perfusion and obtain reclaiming, cause the nearly generation and the recovery of 1,000,000,000 karyocytes, its karyocyte produces 50 to 10,000 ten thousand multipotency or multipotent stem cells.This cell is referred to herein as people's placenta stem-cell or embryonic-like stem cell.
In a specific embodiment of the present invention, cell, for example marrow or postpartum dabbling placenta endogenous cell, include but not limited to embryonic-like stem cell, progenitor cell such as CD34 +Or CD133 +Cell, pluripotent cell or multipotential cell are exposed to compound of the present invention and are subjected to inducing differentiation.This endogenous cell can be in external breeding.In another embodiment, endogenous cell can be collected from placenta and substratum and obtain, and cultivates under external suitable condition, and cultivates for some time to the cell type or the pedigree that are enough to by expectation and induce differentiation.
In another embodiment of the invention, stem cell or progenitor cell are derived from other source as Cord blood, peripheral blood or adult blood, and are exposed in the compound of the present invention quilt and are induced differentiation.In a preferred embodiment, differentiation is carried out for some time under external appropriate condition, and the described time is enough to induce to desired clone or cell type differentiation.Compound of the present invention is used for by adding, and produces in position or mode that other any permission stem cells or progenitor cell contact with compound of the present invention is used for breaking up medium/substratum.
The time that has been found that compound administration of the present invention is to CD34 +The differentiation of progenitor cell has profound influence.Therefore, in one embodiment of the invention, CD34 +Progenitor cell is divided into dendritic cell and comprises that by a kind of the method that makes progenitor cell contact compound of the present invention when cultivating in first day obtains postponing or suppressing.In another embodiment, from CD34 +The CD1a that progenitor cell comes +Cells whose development is reduced or hinders by a kind of method that makes described progenitor cell contact compound of the present invention when first day cultivates that comprises.In another embodiment, come from CD34 +The CD1a of progenitor cell +The persistence of cell colony contacts this compound by the described progenitor cell of cultivation in the presence of no compound of the present invention and is improved after six days.
The present invention also comprises adjusting early progenitor cell such as people CD34 +And CD133 +The method of cytodifferentiation, comprise make progenitor cell propagation with the differentiation phase between different time contact with one or more compounds of the present invention.Therefore, in one embodiment, the present invention includes a kind of method of regulating progenitor cell differentiation, comprise described cell was only contacted with one or more compounds of the present invention cultivating in first day.In another embodiment, described cell contacts with dose with described compound any given day in first day to the 12nd day that cultivates.In another embodiment, described cell is at 0 to 12 day, comprise 0 day with 12 days different number of days in contact twice with described compound at least.Still in another embodiment, described cell propagation and/or differentiation contact with one or more compounds period one day twice, once a day or every once a day.In another embodiment, described contact is carried out external.Still in another embodiment, described contact is carried out in curee's body.In a more particular embodiment, described curee is people, inhuman Mammals, birds or Reptilia.
Generally speaking, the endogenous that can cultivate in the dabbling placenta in postpartum or exogenous stem cell or progenitor cell are exposed to compound of the present invention and can be incubated at dabbling placentogenesis in postpartum at these cells, or preferably, can take place from placenta, reclaim or remove the back at cell in vitro.
The present invention includes compound and grow the purposes of conditioning agent as stem cell and/or progenitor cell with TNF-alpha active.In a specific embodiment, this compound is immunomodulator compounds, IMiDs as is known TMThe compound of class includes but not limited to thalidomide analogs, Actimid TM(CelgeneCorp., Warren, NJ) and Revimid TM(CelgeneCorp., Warren, NJ).
The present invention also comprises through pretreated stem cell or progenitor cell and being used for the treatment of and prophylactic transplanting.In one embodiment, a patient who need to transplant before transplanting, middle and/or transplant the medicine that the back bacterium gives compound.
The present invention further comprises by the progenitor cell of method generation of the present invention or the purposes of specific cell type.In other words, the present invention includes the purposes of the white corpuscle, granulocyte or the dendritic cell that are made by the hemopoietic progenitor cell differentiation, wherein said progenitor cell differentiation is regulated or regulation and control with compound of the present invention.
In another embodiment, the present invention includes control or the regulation and control of stem cell, by delivering medicine to its required patient with stem cell and micromolecular compound of the present invention in vivo.
In one embodiment, the invention provides and comprise CD34 +Or CD133 +The pharmaceutical composition of progenitor cell and pharmaceutically acceptable carrier, wherein said progenitor cell were promoting under the condition of described progenitor cell proliferation and differentiation and compound of the present invention, the compound contact of especially transplanting the TNF-alpha active in preceding 6 days that cultivate.In a specific embodiment, pharmaceutical composition comprises the cell of cultivating back collection and cryopreservation in six days.In another specific embodiment, the cell in the pharmaceutical composition is CD34 +CD38 -CD34 -Or CD34 +CD38 -CD34 +Cell.In another specific embodiment, with the compound of cells contacting be immunomodulator compounds of the present invention, or Thalidomide or thalidomide analogs.In another specific embodiment, with the compound of cells contacting be Actimid TMOr Revimid TM
In another embodiment, the present invention also provides the method for pharmaceutical compositions, comprises with the compound contact CD34 that suppresses the TNF-alpha active +Or CD133 +Progenitor cell, wherein said progenitor cell was cultivated in substratum six days under the culture condition that allows propagation, and wherein said progenitor cell was cultivated in substratum six days under the culture condition that allows described progenitor cell proliferation and differentiation; After cultivating six days, collect described cell; And with pharmaceutically useful carrier and the combination of described cell.In a specific embodiments of this method, described contact was carried out in cultivation in first day.In another specific embodiments of this method, described contact is carried out twice in described six days cultivate at least.In another specific embodiments of this method, described compound is a small molecules immunomodulator compounds of the present invention.In another embodiment, the compound with cells contacting is Actimid TMOr Revimid TMIn being still another specific embodiments of this method, described progenitor cell separates from other hemocyte before described cultivation.In another specific embodiments of this method, described substratum also comprises GM-CSF and TNF-α in addition.At another of this method more specifically in the embodiment, described Actimid TMOr Revimid TMConcentration with 0.1 μ M to 10.0 μ M exists.At another of this method more specifically in the embodiment, described Actimid TMOr Revimid TMConcentration with 1.0 μ M exists.In another specific embodiments of this method, described cell is carried out cryopreservation after collection.
The present invention further provides the method for propagation progenitor cell populations in mammalian subject, comprise that administration is to treat the CD34 of significant quantity +Or CD133 +Progenitor cell and Actimid TMOr Revimid TMIn described acceptor mammalian subject.In the specific embodiments of this method, described progenitor cell breaks up in the acceptor mammalian subject.In another specific embodiments of this method, described progenitor cell with the cellular preparations form administration of essentially no red blood cell in described curee.In another specific embodiments of this method, described progenitor cell with the cellular preparations form administration that comprises medullary cell, placenta cells, cord blood cell or PBMC in the acceptor mammalian subject.In another specific embodiments of this method, described progenitor cell with the form administration of carrier associating in the acceptor mammalian subject.In another specific embodiments of this method, described progenitor cell is CD34 +CD133 +Progenitor cell.In another specific embodiments of this method, described progenitor cell is expressed the purpose genetic stocks that mixes.
The present invention also provides the cell as pharmaceutical composition by method for preparing.
Still in another embodiment, present invention resides in for example CD34 of cryopreservation and thawing back adjusting stem cell or progenitor cell +Progenitor cell is to eliminate cryopreservation and to be exposed to the method for freezing agent for the harmful effect of stem cell.In certain embodiments, the invention provides at cryopreservation and melt the back and regulate stem cell, be exposed to the freezing agent (for example, DMSO) and to the method for the harmful effect of stem cells hyperplasia and transfer ability with elimination.
3.1 definition
As used herein, term " bio-reactor " is meant and is used for propagated cell, production or expresses the vitro system of biomaterial and growth or culturing cell tissue, organoid, virus, albumen, polynucleotide and microorganism.
As used herein, " DC cell " is meant dendritic cell.
As used herein, " early progenitor cell " is meant CD34 +Progenitor cell, CD133 +Arbitrary equivalent in progenitor cell or Mammals, bird or the reptiles.
As used herein, term " embryonic stem cell " is meant the cell that comes from living cell mass in the blastocyst, and has versatility, and wherein said embryonic stem cell does not comprise human embryo stem cell.
As used herein, term " embryonic-like stem cell " is meant the non-cell of giving birth to cell mass in the blastocyst that comes from.As used herein, " embryonic-like stem cell " also may be meant " placenta stem-cell ".Preferred embryo sample stem cell is a versatility.Yet, can comprise embryonic-like stem cell, multipotency cell and committed progenitor from the stem cell that placenta obtains.According to method of the present invention, the embryonic-like stem cell that comes from placenta can be collected from isolating placenta, and bloodletting and perfusion that it has passed through for some time are enough to remove residual cell.Preferably, embryonic-like stem cell is the people source, though can derive from any Mammals.
As used herein, term " bloodletting " or " bloodletting " when use relevant with placenta, are meant from placenta and remove and/or discharge all basically Cord bloods.According to the present invention, the placenta bloodletting can be passed through, for example, but qualification thus, discharge, gravity inductive flow out, massage, push, aspirate or the like and finish.In a preferred embodiment, the bloodletting of placenta can be further by perfusion, rinsing or with containing or do not contain reagent, as the liquid wash placenta of antithrombotics, to help the bloodletting of placenta and finish.
As used herein, term " perfusion " or " perfusion " are meant that seeing through organ or tissue flows out or expel liquid, and preferably, the discharge that sees through the liquid of organ or tissue has enough strength or pressure to remove any residual cell, that is, come from the non-adherent cell of organ or tissue.As used herein, term " perfusion liquid " is meant the liquid of collecting along with discharging from organ or tissue.In a preferred embodiment, perfusion liquid comprises one or more antithrombotics.
As used herein, term " endogenous cell " refers to a kind of " non-external source " cell, that is, a kind of self or autologous cell, it comes from placenta.
As used herein, term " foreign cell " is meant " external " cell, that is, heterogenous cell (promptly, come from " non-oneself " cell of non-placenta donor source) or come from the autologous cell (that is, coming from " the self-cell " of placenta donor) of the organ or tissue of non-placenta.
As used herein, term " immunomodulator compounds " is meant the hereinafter disclosed compound of 5.3 joints.
As used herein, term " organoid " is meant with appearance form or as any organ or mammalian body, the preferably aggregate of one or more cell types of the practical structures form of the body of gland of human body assembling.
As used herein, term " multipotency cell " is meant the cell with the ability that is grown to the about 260 kinds of arbitrary subclass of cell type of mammalian body.Be different from multipotential cell, the multipotency cell does not have the ability that forms all cells type.
As used herein, term " multipotential cell " is meant to have and breaks up multifarious cell fully, that is, is grown to the ability of the about 260 kinds of cell types of mammalian body.But the multipotential cell self, and can in tissue, keep dormancy or static.Be different from totipotent cell (for example, the diploid ovum of fertilization), embryonic-like stem cell can not form new blastocyst usually.
As used herein, term " progenitor cell " is meant that directed differentiation is particular cell types or the cell that forms particular organization.
As used herein, term " stem cell " is meant and can regenerates indefinitely and the standard cell lines of the qualification cell of formative tissue and organ.Stem cell is the cell that has versatility or multipotency on growing.Stem cell can be divided and produced two filial generation stem cells, or filial generation stem cell and an ancestral (transformations) cell, and it breeds the cell for sophisticated, the Gestalt of organizing subsequently.
As used herein, term " totipotent cell " is meant and can forms a complete embryo (for example, a blastocyst) cell does not comprise human embryo stem cell.
4. accompanying drawing summary
Fig. 1. histogram has shown at Thalidomide (THD), Actimid TMAnd Revimid TMThere is cultivation cord blood CD 34 down with concentration 1 μ g/ml and 10 μ g/ml +The result of cell.Isopyknic DMSO is as negative control.Red corpuscle hematopoiesis burst forming unit (BFU-E), CFU-E (CFU-E), huge the biting of grain are colony forming unit (CFU-GM) and colony sum (CFU-Total) record under opticmicroscope.Y-axle: colony number.See 6.1 joints for details.
Fig. 2 (A-F). fluidic cell figure.Human cord blood CD45 +Cellular exposure is in Actimid TMAnd Revimid TMErythrocytic generation and CD34 have been suppressed +CD38 -The propagation of cell.Cord blood CD45 +Cell was cultivated 14 days having under cytokine IL3, IL6, G-CSF, Epo and the KL.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(5μg/ml)。D. Thalidomide (" Thal ") (5 μ g/ml).E.Actimid TM(5μg/ml)。F.Revimid TM(5μg/ml)。The painted relative intensity of X-axle: Gly-A (FL1-H).The painted relative intensity of Y-axle: CD34 (FL2-H).See 6.1 joints for details.
Fig. 3 (A-F). fluidic cell figure.Human cord blood CD45 +Cellular exposure is in Actimid TMAnd Revimid TMHaving suppressed is having cytokine IL3, KL and G -CSFCultivate 14 days human cord blood CD 34 down +The expression of CXCR4 in the cell.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast, CD45 +Cell.C.DMSO(0.3μg/ml)。D.Thal(0.3μg/ml)。E.Actimid TM(0.3μg/ml)。F.Revimid TM(0.3μg/ml)。The painted relative intensity of X-axle: CD34 (FL1-H).The painted relative intensity of Y-axle: CXCR4 (FL2-H).See 6.2 joints for details.
Fig. 4 (A-F). fluidic cell figure.Human cord blood CD45 +Cellular exposure is in Actimid TMAnd Revimid TMStimulated CD34 +And/or CD34 +CD38 -The propagation of cell colony.Cord blood CD45 +Cell was cultivated 14 days having under cytokine IL3, IL6, G-CSF, Epo and the KL.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(0.3μg/ml)。D.Thal(0.3μg/ml)。E.Actimid TM(0.3μg/ml)。F.Revimid TM(0.3μg/ml)。The painted relative intensity of X-axle: CD38 (FL1-H).The painted relative intensity of Y-axle: CD34 (FL2-H).See 6.2 joints for details.
Fig. 5 (A-F). fluidic cell figure.Human cord blood CD45 +Cellular exposure is in Actimid TMAnd Revimid TMProduced the important preservation of human cord blood progenitor cell.Cord blood CD45 +Cell was cultivated 14 days having under cytokine IL3, IL6, G-CSF, Epo and the KL.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(5μg/ml)。D.Thal(5μg/ml)。E.Actimid TM(5μg/ml)。F.Revimid TM(5μg/ml)。The painted relative intensity of X-axle: CD38 (FL1-H).The painted relative intensity of Y-axle: CD34 (FL2-H).See 6.2 joints for details.
Fig. 6 (A-F). fluidic cell figure.Human cord blood CD45 +Cellular exposure is in Actimid TMAnd Revimid TMSuppressed CD45 +The expression of the CXCR4 of cell and bred CD45 +Cell colony.Cord blood CD45 +Cell was cultivated 14 days having under cytokine IL3, IL6, G-CSF, Epo and the KL.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(5μg/ml)。D.Thal(5μg/ml)。E.Actimid TM(5μg/ml)。F.Revimid TM(5μg/ml)。The painted relative intensity of X-axle: CD45 (FL1-H).The painted relative intensity of Y-axle: CXCR4 (FL2-H).See 6.2 joints for details.
Fig. 7 (A-F). fluidic cell figure.Human cord blood CD45 +Cellular exposure is in Actimid TMAnd Revimid TMProduced CD34 +The quantitative propagation of progenitor cell and improved granulocyte and monocytic output.Cord blood CD45 +Cell was cultivated 14 days having under cytokine IL3, IL6, G-CSF, Epo and the KL.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(5μg/ml)。D.Thal(5μg/ml)。E.Actimid TM(5μg/ml)。F.Revimid TM(5μg/ml)。The painted relative intensity of X-axle: CD11b (FL1-H).The painted relative intensity of Y-axle: CD34 (FL2-H).See 6.2 joints for details.
Fig. 8 (A-F). fluidic cell figure.Human cord blood CD45 +Cellular exposure is in Actimid TMAnd Revimid TMBred CD34 +Progenitor cell and the inhibition of having eliminated the monocyte output of DMSO mediation.Cord blood CD45 +Cell was cultivated 14 days having under cytokine IL3, IL6, G-CSF, Epo and the KL.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(5μg/ml)。D.Thal(5μg/ml)。E.Actimid TM(5μg/ml)。F.Revimid TM(5μg/ml)。X-axle: CD14 expresses (FL1-H) painted relative intensity.The painted relative intensity of Y-axle: CD34 (FL2-H).See 6.3 joints for details.
Fig. 9 (A-F). fluidic cell figure.Human cord blood CD45 +Cellular exposure is in Actimid TMAnd Revimid TMShown in the less retarding effect that is divided on the B cell.Cord blood CD45 +Cell was cultivated 14 days having under cytokine IL3, IL6, G-CSF, Epo and the KL.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(5μg/ml)。D.Thal(5μg/ml)。E.Actimid TM(5μg/ml)。F.Revimid TM(5μg/ml)。The painted relative intensity of X-axle: CD38 (FL1-H).The painted relative intensity of Y-axle: CD19 (FL2-H).See 6.3 joints for details.
Figure 10 (A-F).Fluidic cell figure.Human cord blood CD45 +Cellular exposure is in Actimid TMAnd Revimid TMOvercome the inhibition of the CXCR5 that produces by being exposed to DMSO.Cord blood CD45 +Cell was cultivated 14 days having under cytokine IL3, IL6, G-CSF, Epo and the KL.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(5μg/ml)。D.Thal(5μg/ml)。E.Actimid TM(5μg/ml)。F.Revimid TM(5μg/ml)。The painted relative intensity of X-axle: CXCR5 (FL1-H).The painted relative intensity of Y-axle: CD34 (FL2-H).See 6.3 joints for details.
Figure 11 (A-E). fluidic cell figure.Human cord blood monocyte (MNCs) is exposed to Actimid TMAnd Revimid TMImproved CD34 +CD38 +The cell colony number.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(5μg/ml)。D.Actimid TM(5μg/ml)。E.Revimid TM(5μg/ml)。The painted relative intensity of X-axle: CD38 (FL1-H).The painted relative intensity of Y-axle: CD34 (FL2-H).See 6.3 joints for details.
Figure 12 (A-E). fluidic cell figure.MNCs is exposed to Actimid TMAnd Revimid TMDownward modulation CXCR4 +CD45 +Cell colony number, but high CXCR4 +CD45 -The cell colony number.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.A. contrast, immunoglobulin (Ig) (Ig).B. contrast does not add compound.C.DMSO(5μg/ml)。D.Actimid TM(5μg/ml)。E.Revimid TM(5μg/ml)。The painted relative intensity of X-axle: CD45 (FL1-H).The painted relative intensity of Y-axle: CXCR4 (FL2-H).See 6.3 joints for details.
Figure 13 (A-E). fluidic cell figure.Thalidomide, Actimid TMAnd Revimid TMIn the influence of hemopoietic progenitor cell on the pedigree directional trend of the karyocyte differentiation of Cord blood composition.Fluidic cell figure shows Actimid TMAnd Revimid TMImproved the per-cent of monokaryon pedigree cell compared with the control, shown the adjusting that has differentiation, it turns to the pedigree that causes lymph sample and medullary cell.A. contrast, immunoglobulin (Ig) (Ig).B.DMSO(5μg/ml)。C.Thal(5μg/ml)。D.Actimid TM(5μg/ml)。E.Revimid TM(5μg/ml)。See 6.4 joints for details.
Figure 14 .Actimid TMTo CD34 +Cytodifferentiation and maturation are the research sketch map of the influence of DC cell.At propagation and ripening stage (the 1st day to the 12nd day) or ripening stage (the 6th day to the 12nd day), CD34 +Cell is at 1.0 μ MActimid TMAnd cultivate under the condition of DMSO (dimethyl sulfoxide (DMSO)) existence or shortage.The 6th day or the monoclonal antibody of puting together with FITC and PE in the 12nd day the immunohistochemistry mark is tested.See 6.6 joints for details.
Figure 15 (A-D). fluidic cell figure has shown 6 days CD34 +The phenotypic characteristic of cell was exposed to Actimid from the 1st day TMActimid TMAlmost completely suppressed CD86 +CD1 +Cells whose development.Cell CD1aFITC and CD14PE or CD1aFITC and CD86PE double-tagging.A: with the CD34 of DMSO (contrast) processing +The CD86 that cell produces +CD1 +Cell.B: with the CD34 of DMSO (contrast) processing +The CD14 that cell produces +The CD1+ cell.C: use Actimid TMThe CD34 that handles +The CD86 that cell produces +The CD1+ cell.D: use Actimid TMThe CD34 that handles +The CD14 that cell produces +CD1 +Cell.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of the specific mark of recombinating surely.See 6.6 joints for details.
Figure 16 (A-D). fluidic cell figure has shown at CD34 +Cellular proliferative stage (the 1st day to the 6th day), be exposed to Actimid TMInfluence at the 6th day.Be exposed to Actimid TMCD34 +The CD38 cell was divided into CD34 after 6 days +CD38 -CD33 +Cell.Cell CD33FITC and CD83PE or CD38PE and CD34PE double-tagging.A: cell is handled with DMSO, and to CD34 and CD38 mark.B: cell is handled with DMSO, and to CD83 and CD33 mark.C: cell Actimid TMHandle, and to CD34 and CD38 mark.D: cell is with handling.The per-cent that shows on every cytological map represents to express the per-cent of the cell colony of certain heavy group echo.See 6.6 joints for details.
Figure 17. from the 0th day to the 6th day Actimid that cultivates TMInduce CD34 +The transformation of the cell colony in cell source.Cell was at different concns Actimid from the 0th day to the 6th day TMCD34 and CD38 or CD1a and CD14 double-tagging are used in following cultivation then.Actimid TMAt CD34 +CD38 -Cause significant raising in the cell, at CD1a +CD14 -Cause reduction in the cell.See 6.6 joints for details.
Figure 18. at different time Actimid TMThe CD34 that handles +Cell was at the 6th day phenotypic alternation.Cell inoculation was cultivated 6 days in culture dish.Use Actimid TMOnly from the 0th day to the 1st day, only the 0th day to the 2nd day, only the 0th day to the 3rd day, only the 0th day to the 4th day, only cell is handled in the 0th day to the 5th day or the 0th day to the 6th day.For the incubation that is less than 6 days, Actimid TMAt flush away that day of indication, and cell is resuspended among the DMSO.At the 6th day, cell CD34 and CD38 or CD1a and CD14 mark.See 6.6 joints for details.
Figure 19 (A-B). with the Actimid of single dose or multiple doses TMThe CD34 that handles +Progenitor cell was at the 6th day phenotypic alternation.Figure 19 A: condition 1: at the 0th day single dose Actimid TMCondition 2: at the 0th day and the 4th day administration Actimid TMCondition 3: at the 0th day, the 2nd day and the 4th day administration Actimid TMY-axis represents to express the per-cent of the cell of specific markers or reorganization mark.Figure 19 B: condition 1: at the 0th day single dose Actimid TMCondition 2: at the 0th day, the 4th day, the 6th day, the 8th day administration Actimid TMCondition 3: at the 0th day, the 2nd day, the 4th day, the 6th day, the 8th day administration Actimid TMCell assessment by the expression of CD11c and CD15, it is the granulocyte mark.Y-axis is represented and Actimid TMOr the CD11c of DMSO (contrast) contact +CD15 -And CD11c -CD15 +The per-cent of cell.See 6.6 joints for details.
Figure 20 (A-F). be exposed to Actimid from the 1st day to the 12nd day TMThe CD34 of (1 μ M) +The dendritic cell that cell produces were at the 12nd day fluidic cell figure.Actimid compared with the control TMCaused the reduction that CD86 and CD80 molecule stimulate altogether at the 12nd day.Cell CD1aFITC and CD86PE antibody, CD1aFITC and CD80PE antibody or CD1aFITC and CD14PE antibody double-tagging.A: cell is handled with DMSO, and at the 12nd day CD86 and CD1a is dyeed.B: cell is handled with DMSO, and at the 12nd day CD80 and CD1a is dyeed.C: cell is handled with DMSO, and at the 12nd day CD14 and CD1a is dyeed.D: cell Actimid TMHandle, and CD86 and CD1a are dyeed at the 12nd day.E: cell Actimid TMHandle, and CD80 and CD1a are dyeed at the 12nd day.F: cell Actimid TMHandle, and CD14 and CD1a are dyeed at the 12nd day.The per-cent that shows on every cytological map represents to express the per-cent of the cell of certain heavy group echo.See 6.6 joints for details.
Figure 21 (A-D). be exposed to Actimid from the 1st day to the 12nd day TMThe CD34 of (1 μ M) +The dendritic cell that cell produces were at the 12nd day fluidic cell figure.Be exposed to Actimid TMCause the change of CD54 adhesion molecule expression,, cause CD54 with respect to contrast (comparing) with subpanel E and F among Figure 21 D BrightThe reduction and the CD54 that express DimThe raising of expressing.A: cell is handled with DMSO, and with IgG1 to HLA -DR dyeing.B: cell is handled with DMSO, and to CD54 and CD40 dyeing.C: cell Actimid TMHandle, and HLA-DR is dyeed with IgG1.D: cell Actimid TMHandle, and to CD54 and CD40 dyeing.The per-cent that shows on every cytological map represents to express the per-cent of the cell of certain heavy group echo.See 6.6 joints for details.
Figure 22 (A-B) .Actimid TMPromotion is from CD34 +The granulocyte differentiation of progenitor cell.At DMSO (Figure 22 A) or Actimid TM(Figure 22 B) grew 12 days down, used the CD34 of the antibody labeling of granulocyte molecule marker CD15 then +The fluidic cell figure of cell.The per-cent that shows on every cytological map represents to express the per-cent of the cell of certain heavy group echo.See 6.6 joints for details.
Figure 23 (A-D) .Actimid TMDo not induce CD1a +Or CD14 +The apoptosis of precursor cell.CD1a +CD14 -And CD1a -CD14 +The fluidic cell figure of isolated cell (DC progenitor cell).CD34 +Progenitor cell is at SCF, Flt -3L, GM -Under CSF and the TNF-α through 6 days cultivation.At the 6th day, collect CD1a by magnetic cell sorter (Miltenyi) +CD14 -And CD1a -CD14 +There is GM in cell -CSF and TNF -α and existence or do not have Actimid TMCultivate CD1a under the condition of (1 μ M) +CD14 -And CD1a -CD14 +Extra 2 days of cell mass.Apoptotic cell is used annexin V-FITC subsequently, a kind of mark of apoptosis, and dyeing, with iodate third ingot (PI), a kind of active probe is united use.Per-cent on every cytological map is represented the per-cent to the cell of annexin V-FITC and/or the iodate third ingot stained positive.To annexin V-FITC positive cells quantity and Actimid TMThat handle and cell contrast (especially with every cytological map in B3 and B4 relatively) make comparisons.A: with the CD1a of DMSO processing +CD14 -Cell.B: use Actimid TMThe CD1a that handles +CD14 -Cell.C: with the CD1a of DMSO processing -CD14 +Cell.D: use Actimid TMThe CD1a that handles -CD14 +Cell.See 6.6 joints for details.
Figure 24 (A-D). by Actimid TMThere is or lacks 12 days CD34 of growth down +The fluidic cell figure of cell.Actimid TMCause the pinocytotic reduction of mannose receptor mediation, its and DMSO are compared, and the reduction of the dextran picked-up by the FITC mark obtains proving.A:DMSO and 4 ℃.B:ActimidTM and 4 ℃.C:DMSO and 37 ℃.D:ActimidTM and 37 ℃.Per-cent on every cytological map represents to present pinocytotic cell fraction.See 6.6 joints for details.
Figure 25 (A-D). cultivate 12 days CD34 +The fluidic cell figure of cell, and had or lacked Actimid from the 6th day to the 12nd day TMThe following cultivation.At the 1st to 5 day Actimid TMAfter lacking cultivation down, at the 6th to 12 day Actimid TMExist down and cultivate, compare, cause the pinosome of mannose receptor mediation with the DMSO contrast.Per-cent on every cytological map represents to present pinocytotic cell fraction.A:DMSO and 4 ℃.B:ActimidTM and 4 ℃.C:DMSO and 37 ℃.D:ActimidTM and 37 ℃.See 6.6 joints for details.
Figure 26 .Actimid TMReduce the CD34 that cultivated 12 days +The antigenic ability of presented by cells.CD34 +There is or is lacking Actimid in cell TMUnder cultivated 12 days; Actimid TMThe cell of handling at the 12nd day, is compared the hormesis index that demonstration reduces basically with DMSO.
Figure 27. to from the 6th day to the 12nd day at Actimid TMThere is the CD34 that cultivates down +Cell, Actimid TMHas the activity that antigen presenting cell (APC) reduces.CD34 +There is or is lacking Actimid in cell TMUnder cultivated 5 days.The antigen presentation ability and the contrast of the cell of handling, the cell that DMSO handles is made comparisons.See 6.6 joints for details.
Figure 28. the CD34 that in the presence of GM-CSF and TNF-α, cultivates +The differentiation pathway of hemopoietic progenitor cell.
Figure 29. show with Actimid TMFor the CD34 that in the presence of GM-CSF and TNF-α, cultivates +The summary chart of the effect of the differentiation pathway of hemopoietic progenitor cell.See 6.6 joints for details.
Figure 30. show mouse Sca +Lin -The chart of hemopoietic progenitor cell maturation condition.STEM CELL FACTOR (SCF), Flt-3L, grain are huge to be bitten G CFS (GM-CSF) and hugely bites G CFS (MCSF) and grew 10 μ MActimid 9 days 0.1%DMSO (contrast) time cell existing TMOr 10 μ M all-trans retinoic acid (ATRA) in order to drive cell to DC precursor cell phenotypic differentiation.Cell was cultivated in the presence of GM-CSF and TNF-α from the 9th day to the 12nd day subsequently, added DMSO, Actimid TMOr ATRA, to drive of the differentiation of mouse cell to immature dendritic cell.See 6.8 joints for details.
Figure 31 (A-E). the mouse cell is appearance (seeing embodiment 1, material and method, the description of Figure 30) in the 9th day under common culture condition.Cell antibody (Figure 31 A), the antibody (Figure 31 B) of CD11, the antibody (Figure 31 C) of CD32/16, the MHCII (I-A of CD80 b) antibody (Figure 31 D), the antibody (Figure 31 E) of CD14 or antibody (Figure 31 F) mark of Gr-1.Cell is on presenting by the antibody labeling of CD88, CD11 or CD32/16 molecule marker on the 9th day, by I-A bOn a little mark of the antibody of molecule marker, not by on the antibody labeling of CD14 or Gr-1 molecule marker.See 6.8 joints for details.
Figure 32 (A-I). with DMSO, Actimid TMOr the 12nd day the fluidic cell figure of mouse cell of ATRA processing.The cell antibody labeling of CD86 and CD11b.Figure 32 A-32C (top line): cytological map shows with DMSO (contrast), Actimid TMOr all-trans retinoic acid (ATRA) is expressed the percentage of cells of CD86 (Y-axle) when handling.Figure 32 D-32F (middle row): when handling, express the percentage of cells of the main II of histocompatibility complex (MHC-II) as top line.Figure 32 G-32I (end row): when handling, express the percentage of cells of CD11b as top line.See 6.8 joints for details.
5. detailed Description Of The Invention
The present invention's part is based on unexpected discovery, be stem cell or progenitor cell be exposed to compound of the present invention cause produce a kind of control stem cell or progenitor cell to specific progenitor cell populations differentiation or progenitor cell to particular cell types, the regulatable method of breaking up as dendritic cell, granulocyte, endotheliocyte or neurocyte.Especially, stem cell or progenitor cell are exposed to compound of the present invention causes generation hematopoietic cell special group, comprises CD34 +, CD38 +And CD133 +The regulatable differentiation and the propagation of cell.The regulation and control of this differentiation are not because of necrocytosis or be divided into the unwanted cells type or cell lineage causes under the situation of remarkable loss of output and finishes; In other words, compound of the present invention does not cause the apoptosis of one or more cell colonys.Further, hemopoietic progenitor cell is exposed to regulatable differentiation and the propagation that compound of the present invention causes particular cell types.
Therefore, the invention provides the method for mediator's differentiation of stem cells, particularly CD34 +Hemopoietic progenitor cell and CD133 +The differentiation of progenitor cell.Especially, the invention provides use and suppress TNF -The little organic molecule of alpha active is regulated progenitor cell along specific cells and the method for organizing the pedigree differentiation.Further, the present invention includes the propagation early progenitor cell, as people CD133 +Or CD34 +Cell, especially CD34 +CD38 -Cell is exposed to TNF to be implanted into the method in Mammals, birds or the Reptilia, to comprise with hemopoietic progenitor cell -Alpha inhibitor or antagonist, wherein inhibitor or antagonist are small-molecule substances.The present invention also provides the method that produces the other types cell from these early progenitor cells, includes but not limited to brain cell, nephrocyte, intestinal cell and muscle cell.Compound of the present invention is also to suppressing from early progenitor cell, as people CD34 +The dendritic cell differentiation of progenitor cell and promote granulocytic differentiation to work.
The example of the micromolecular compound that uses includes but not limited to related to the present inventionly, suppresses TNF -The compound of alpha active.The compound of Shi Yonging is described in detail at 5.3 joints in the method for the invention.In one embodiment, though also use Thalidomide hydrolysate, metabolite and precursor, preferred compound is a thalidomide analogs.In particularly preferred embodiments, compound is ImiDs TM(Celgene), as Actimid TMAnd Revimid TM
Method of the present invention comprises the regulation and control that stem cell or progenitor cell break up to the specific cells pedigree, include but not limited to mesochymal, hematopoiesis, living fat, living liver, living neural, collagen, living cartilage, angiogenic, myogenic, living pedigree cartilage or skeletonization, it comprises preferably external causes the cytophyletic cytodifferentiation of cell to expectation with stem cell or progenitor cell and one enough period of compound incubation of the present invention.In a specific embodiment, stem cell or progenitor cell are regulated to the differentiation of hematopoietic lineage cell.Specifically, method of the present invention can be used for regulating by CD34 in the relevant mode of a kind of dosage +, CD133 +And CD45 +Hemopoietic progenitor cell generates the generation of hemocyte colony.
Method of the present invention also comprises CD34 +Progenitor cell is to the adjusting of dendritic cell differentiation, and it comprises preferably external causes the cytophyletic cytodifferentiation of cell to expectation with progenitor cell and compound incubation of the present invention sufficiently long one period.In a specific embodiment, this progenitor cell passes through described cells contacting Actimid to the differentiation of dendritic cell pedigree cell TMOr Revimid TMOr its analogue or prodrug and regulate.In another specific embodiment, CD34 +The differentiation of progenitor cell is adjusted to suppress along the differentiation of marrow sample pedigree and to support differentiation along the granulocyte pedigree.In a more particular embodiment, CD34 +Progenitor cell to the differentiation of granulocyte pedigree cell by comprising with CD34 +Progenitor cell is regulated in the method that contacted in the 1st day that progenitor cell is cultivated with compound of the present invention.
Any mammalian stem cell or progenitor cell can use according to method of the present invention, include but not limited to, from Cord blood (" CB " cell), placenta and other isolating stem cells of originating.Stem cell can comprise multipotential cell, that is, have and break up diversity, energy self completely and keep dormancy or the immobilized cell in tissue.Stem cell also can comprise multipotency cell and committed progenitor.In a preferred embodiment, that the present invention utilizes is alive, the stem cell of immobilized, versatility, it is present in the placenta of foot phase, can be along with the childbirth and the placenta of the success of the recovery that causes multipotency and multipotent stem cells are given birth to, bloodletting and placenta perfusion and reclaimed.
In another preferred embodiment, progenitor cell is early progenitor cell, especially CD34 +Or CD133 +Cell.Preferably, CD34 +Or CD133 +Progenitor cell derives from people's marrow, placenta or Cord blood.Also can use from the equivalent of these cells in other Mammalss source.For example, in mouse, Sca +Progenitor cell can use in the method for the invention.Also can use from the early progenitor cell of the equivalence in birds or Reptilia source.
In a specific embodiment of the present invention, placenta is endogenous or by pouring into the cell that placenta produces postpartum, include but not limited to, embryonic-like stem cell, progenitor cell, multipotential cell and multipotency cell, be exposed to compound of the present invention when in isolating and dabbling placenta, cultivating, and induce differentiation.The endogenous cell of breeding in the dabbling placenta in postpartum can reclaim, and/or bioactive molecules can reclaim from perfusion liquid, substratum or from placenta cells self.
In another embodiment of the invention, be exposed to compound of the present invention and induce differentiation when the vitro culture coming from except the stem cell in other source of back placenta or progenitor cell.Therefore, the present invention includes and make mammalian stem cell be divided into the method for specific progenitor cell, it comprises makes stem cell under the situation of the condition of the differentiation that is suitable for expecting and/or substratum and under the situation that compound of the present invention exists stem cell is broken up.
Further, the present invention includes and regulate or regulate and control the method for specific progenitor cell populations to the particular cell types differentiation, it comprises makes described progenitor cell under the condition that is fit to described differentiation and break up in the presence of one or more compounds of the present invention.Selectively, stem cell or progenitor cell can be exposed to compound of the present invention, break up with appropriate condition subsequently.The example of conditions suitable comprises the nutritional medium preparation that replenishes with human serum and cell culture substrate, as replenishing with somatomedin
Method of the present invention also relates to different cell colonys, its can by with compound of the present invention at the different times of cultivating, no matter be propagation or differentiation phase to contact progenitor cell and produce.Referring to 5.4 joints, be specially following 5.4.2 joint.
In a specific embodiment, the invention provides the method that the hemopoietic in before hemopoietic progenitor cell is transplanted adjusting period was regulated and regulated and control to the amino binding substances that uses acid amides and imide small molecules, especially Thalidomide.
The present invention also provides and has used small-molecule substance of the present invention to regulate and regulate and control the method that hemopoietic progenitor cell is regulated the hemopoietic in period in the body of formerly external back.Method of the present invention comprises the regulation and control of stem cell or progenitor cell vitro differentiation, comprises external incubation compound and stem cell or progenitor cell, subsequently the cell that breaks up to curee's directly transplanting.
The present invention also comprise by with stem cell or progenitor cell and compound administration of the present invention in its have demand the patient and in vivo to the control and the regulation and control of stem cell or progenitor cell.
The present invention comprises that further the transplanting of pretreated stem cell or progenitor cell is with treatment or preventing disease.In one embodiment, the patient that needs are transplanted before transplanting, transplant in and/or transplant the back administration with compound of the present invention.In another embodiment, also undressed stem cell of patient's administration or the progenitor cell that needs are transplanted, for example cord blood cell, adult blood cell, peripheral blood cells or medullary cell.In another embodiment, method of the present invention comprises that to curee's administration with compound, described curee is the acceptor of unadjusted stem cell or progenitor cell, to induce the adjusting effect to the stem cell of having transplanted.
In certain embodiments, the present invention includes bone marrow transplantation, it comprises transplants Cord blood (or the stem cell that obtains from Cord blood), periphery (that is, adult) blood (or the stem cell that obtains from peripheral blood), and wherein said Cord blood or stem cell have used compound of the present invention pretreated.In addition, the present invention includes the leukocytic purposes that makes by the hemopoietic progenitor cell of in the presence of compound of the present invention, having broken up.For example, the white corpuscle that produces by the differentiation hemopoietic progenitor cell can be used for transplanting or can mixing with Cord blood or cord blood stem cell before transplanting.
In other embodiment, the present invention includes bone marrow transplantation, it comprises the early progenitor cell that obtains according to method of the present invention, as CD34 +Or CD133 +The transplanting of progenitor cell, wherein said progenitor cell have used compound of the present invention pretreated.In one embodiment of the invention, the precursor cell of described dendritic cell is CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Precursor cell.Further, the present invention includes the CD34 that in the presence of compound of the present invention, has broken up by +The purposes of the cell that progenitor cell makes.For example, pass through CD34 +The differentiation of progenitor cell and the CD34 that produces with compound of the present invention +CD38 -CD33 +Precursor cell, CD34 +CD38 -CD33 -Precursor cell, granulocyte etc. are in can be used for transplanting.With compound of the present invention from CD133 +The cell that cytodifferentiation is come is also included for the present invention.
The present invention further is included in cryopreservation and melts the back and regulates stem cell, to eliminate cryopreservation and to be exposed to the method for freezing agent to the harmful effect of stem cell.In certain embodiments, the invention provides the adjusting stem cell of following cryopreservation and thawing, be exposed to the freezing agent (for example, DMSO) to the method for the harmful effect of the propagation of stem cell and transfer ability with elimination.
5.1 stem cell and CD34 +Or CD133 +The adjusting of the differentiation of progenitor cell
5.1.1 stem cell
The invention provides the method for mediator's differentiation of stem cells.In certain embodiments, method of the present invention is included in the regulation and control of external stem cell or progenitor cell differentiation, comprise external with stem cell with the compound incubation, subsequently to the cell of curee's directly transplanting differentiation.In another embodiment, method of the present invention comprises the regulation and control of stem cell in the body or progenitor cell differentiation, comprises that the curee to unadjusted stem cell acceptor sends compound, subsequently to the direct administration of curee with compound.
The embryonic-like stem cell that obtains by method of the present invention can be induced the differentiation to the specific cells pedigree, and that these cell lineages include but not limited to is mesochymal, hematopoiesis, it is fat, livings liver to give birth to, it is neural, collagen to give birth to, give birth to cartilage, angiogenic, myogenic, living pedigree cartilage or skeletonization.
In certain embodiments, will induce differentiation, to be used for transplanting and the scheme of earlier external back interior therapeutic according to the embryonic-like stem cell that method of the present invention obtains.In certain embodiments, induce differentiation by the embryonic-like stem cell that method of the present invention obtains to special cell type, and be subjected to genetic manipulation so that the product of gene therapy to be provided.In a specific embodiment, the embryonic-like stem cell that obtains by method of the present invention external with compound of inducing its differentiation such as little organic molecule incubation, subsequently to the cell of curee's directly transplanting differentiation.In a preferred embodiment, be used to control and the compound of regulating and control differentiation of stem cells is not polypeptide, peptide, albumen, hormone, cytokine, oligonucleotide or nucleic acid.
The stem cell of using according to the present invention includes but not limited to that Cord blood (CB) cell, placenta cells, embryo do (ES) cell, embryonic-like stem cell, trophoderm stem cell, progenitor cell, bone marrow stem cell and multipotency, versatility and totipotent cell.
Especially, method of the present invention comprises that stem cell colony except that mescenchymal stem cell is to the regulation and control of particular organization's pedigree differentiation.For example, the generation and the specific muscle tissue of regeneration by promoting the specific muscle bone and reparation, neovascularity, as cardiac muscle and skeletal muscle, and the revascularization of various organs and tissue, described organ and tissue include but not limited to brain, spinal cord, liver, lung, kidney and pancreas, and method of the present invention can be used for regulating and control the multipotency stem cell to giving birth to cartilage, angiogenic, myogenic meat and differentiation skeletonization pedigree cell.Method of the present invention can be used for regulating and control the differentiation of multipotency stem cell to the pedigree of giving birth to fat, living cartilage, skeletonization, living nerve or living liver.
The reagent that is used for regulating differentiation can import postpartum dabbling placenta to induce the differentiation in the placenta cultured cells.Optionally, this reagent can be used for from placenta collecting or removes behind the cell in external adjusting differentiation.
Method of the present invention comprises the regulation and control of progenitor cell to hematopoietic lineage cell differentiation, is included in externally with progenitor cell and one enough period of compound incubation, causes the differentiation of cell to hematopoietic lineage.Especially, method of the present invention can be used for regulating by CD34 in the relevant mode of dosage +, CD133 +And CD45 +Hemopoietic progenitor cell generates the generation (about the discussion of dosage, seeing 5.7 joints) of hemocyte colony.
Preferably, method of the present invention can be used for suppressing specifically the generation of red blood cell or red corpuscle colony (BFU-E and CFU-E), and leukocyte increasing and thrombocyte colony form the generation of (CFU-GM) and the output that improves total colony forming unit simultaneously.Method of the present invention not only can be used for regulating and control the differentiation of stem cell, also can stimulate colony forming efficiency, the speed by improving the marrow implant and the recovery of white corpuscle and/or platelet production and provide significant benefits to hematopoietic stem cell transplantation.
In other embodiment, method of the present invention can be used for regulating and control the differentiation to specific neural cell type such as Sensory neurone (for example, visual cell, olfactory receptor cell, mechanical sense are answered neurone, chemical co-ordination neurone etc.), motor neuron, cortical neuron or relay cell of neuronal precursor for example or neuroblast.In other embodiment, method of the present invention can be used for the differentiation that regulation and control include but not limited to cholinergic neuron, dopaminergic neuron, GABAergic neuron, neurogliocyte (comprise oligodendrocyte, it produces myelin) and ependymocyte (it is arranged in ventricular system) cell type.Still in other embodiment, method of the present invention can be used for being regulated to the differentiation of the cell of organ component, include but not limited to the pul base alunite cell of heart, the bile duct epithelial cell of liver, β islet cells, renal cortex or the myelocyte of pancreas and the visual impression photo-cell of eyes.
The evaluation of the differentiation of stem cells state that obtains according to method of the present invention can be identified by the existence of cell surface marker.For example, embryonic-like stem cell of the present invention can pass through following cell surface marker: OCT-4 +Distinguish with ABC-pt.Further, the present invention includes the embryonic-like stem cell that has following mark: CD10, CD29, CD44, CD54, CD90, SH2, SH3, SH4, OCT-4 and ABC-p or lack following cell surface marker: CD34, CD38, CD45, SSEA3 and SSEA4 as described.Such cell surface marker can be according to method conventional determining well known in the art, for example, by the flow cytometry method, follows washing and with the antibody staining of anti-cell surface markers.For example, for measuring the existence of CD34 or CD38, cell can wash in PBS, uses the Phycocyanins, C-of anti-CD34 and the fluorescein isothiocyanate of anti-CD38 (Becton Dickinson, Mountain View, CA) two dyeing then.
5.1.2CD34 +And CD133 +Early progenitor cell
The present invention also provides mediator CD34 +Or CD133 +The method of cytodifferentiation.In certain embodiments, method of the present invention is included in the regulation and control of external stem cell or progenitor cell, comprises external incubation stem cell and compound, subsequently the cell directly transplanting of differentiation is gone into the curee.
Can induce differentiation along the specific cells pedigree by the progenitor cell that method of the present invention obtains, include but not limited to, for CD34 +Progenitor cell is along marrow sample or granulocyte pedigree, for CD133 +Cell is along endothelium or neurocyte pedigree.In certain embodiments, progenitor cell is induced differentiation to be used for transplanting with elder generation external back interior therapeutic scheme.In certain embodiments, progenitor cell is induced differentiation to special cell type, and is subjected to genetic manipulation so that the product of gene therapy to be provided.In specific embodiment, with progenitor cell external with induce the compound of its differentiation, as little organic molecule incubation together, subsequently to the cell of curee's directly transplanting differentiation.In a preferred embodiment, be used to control and the compound of regulating and control differentiation of stem cells is not polypeptide, peptide class, albumen, hormone, cytokine, oligonucleotide or nucleic acid.In another preferred embodiment, progenitor cell is facilitated to CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -The progenitor cell differentiation.
Preferably, method of the present invention can be used for suppressing specifically the generation of red blood cell or red corpuscle colony (BFU-E and CFU-E), and leukocyte increasing and thrombocyte colony form the generation of (CFU-GM) and the output that improves total colony forming unit simultaneously.Method of the present invention not only can be used for regulating and control the differentiation of stem cell, also can stimulate colony forming efficiency, the speed by improving the marrow implant and the recovery of white corpuscle and/or platelet production and provide significant benefits to hematopoietic stem cell transplantation.
In other embodiment, method of the present invention can be used for reducing CD34 +Progenitor cell is to CD1a +Cell, especially CD86 +CD1a +The differentiation of cell.In another embodiment, method of the present invention can be used for reducing or preventing CD34 +Progenitor cell is to CD14 +CD1a -The differentiation of cell.CD14 +CD1a -Cell is epidermis dendritic cell or monokaryon/huge progenitor cell of biting.In another embodiment, method of the present invention can be used for reducing the CD34 in propagation +The expression of CD80 and CD86 costimulatory molecules on the progenitor cell.In another embodiment, method of the present invention can be used for reducing the CD34 of propagation +Progenitor cell is to CD54 BrightThe differentiation of cell, and excitation is to CD54 DimThe differentiation of cell.In another embodiment, method of the present invention can be used for increasing CD133 +The quantity of cell, it is the endotheliocyte progenitor cell.Still in another embodiment, method of the present invention can be used for reducing the CD34 of propagation +Cell is to CD11c -CD15 +The differentiation of cell, and increase to CD11c +CD15 -The differentiation of cell is broken up thus from marrow sample dendritic cell pedigree and is changed the granulocyte pedigree into.
The evaluation of the differentiation of stem cells state that obtains according to method of the present invention can be identified by the existence of cell surface marker.For example, progenitor cell of the present invention can pass through CD34 +Or CD133 +Cell surface marker and distinguishing.Further, the present invention includes and have or show with respect to contrast high expression level one or more following mark: CD15, CD34, CD33, CD133 or CD54 as described DimThe progenitor cell of propagation.The present invention further comprises shortage or shows with respect to low one or more following mark: the HLA of expression of contrast -DR, CD1a, CD11c, CD38, CD80, CD86, CD54 BrightOr the progenitor cell of the propagation of CD14.In a preferred embodiment, the progenitor cell of propagation of the present invention presents CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Such cell surface marker can be according to method conventional determining well known in the art, for example, and by washing with the antibody staining of anti-cell surface markers, subsequently through flow cytometry method mensuration.For example, for measuring the existence of CD34 or CD38, cell can wash in PBS, uses the Phycocyanins, C-of anti-CD34 and the fluorescein isothiocyanate of anti-CD38 (Becton Dickinson, MountainView, CA) two dyeing then.
In certain embodiments, the cell of differentiation can be tested and appraised the cytophagy ability of differentiation and characterize.For example, can be by measuring the phagolysis that intake is estimated the cell that has broken up or breaking up with FITC mark dextran and with known method.The ability of cytositimulation T cell differentiation or that just breaking up can be measured in mixed lymphocyte reacion (MLR), and wherein antigenic cell of Jia Ding load and T cytomixis are measured T cell activity level then.
5.1.3 the evaluation of cell and sign
In certain embodiments, the cell that has broken up can be identified (difference that for example, shows gene pool with the gene pool of the cell that has broken up that comes from progenitor cell in undifferentiated progenitor cell source) by the gene of performance differential expression.For example, nucleic acid amplification method such as polymerase chain reaction (PCR) or can be used for being presented in the genetic expression in the different cell colonys based on the amplification method of transcribing (for example, in-vitro transcription (IVT)), for example, by using the polynucleotide microarray.This method that is used to represent differential gene expression be in the art know (see, for example, Wieland etc., Proc.Natl.Acad.Sci.USA87:2720-2724 (1990); Lisitsyn etc., Science259:946-951 (1993); Meth.Enzymology254:291-304 such as Lisitsyn (1995); U.S. Patent number 5,436,142; U.S. Patent number 5,501,964; Lisitsyn etc., Nature Genetics6:57-63 (1994); Hubank and Schatz, 1994, Nucleic Acids Research22:5640-5648; Zeng etc., 1994, Nucleic Acids Research 22:4381-4385; U.S. Patent number 5,525,471; Linsley etc., U.S. Patent number 6,271,002 is entitled as " RNA amplification method ", is disclosed in 2001-08-07; Van Gelder etc., U.S. Patent number 5,716,785 is entitled as " process of using promotor to carry out genetic manipulation ", is disclosed in 1998-02-10; Stoflet etc., Science 239:491-494,1988; Sarkarand Sommer, 1989, Science244:331-334; Mullis etc., U.S. Patent number 4,683,195; Malek etc., U.S. Patent number 5,130,238; Kacian and Fultz, U.S. Patent number 5,399,491; Burg etc., U.S. Patent number 5,437,990; R.N Van Gelder etc., (1990), Proc.Natl.Acad.Sci.USA 87,1663; D.J.Lockhart etc., 1996, NatureBiotechnol.14,1675; Shannon U.S. Patent number 6,132,997; Lindemann etc., U.S. Patent number 6,235,503 is entitled as " method of cutting down hybridization and variance analysis ", is disclosed in 2001-05-22).
It is useful that commercial existing test kit represents for gene, for example, shows PROFILE TM(CA), it uses the treatment process based on gel to represent genetic expression to the reagents series box for Qbiogene, Carlsbad.This test kit utilizes restriction fragment difference to show that PCR (RFDD-PCR) compares gene expression pattern in eukaryotic cell.The PCR-selectivity is cut down test kit (Clontech) and PCR-selectivity differential screening reagent box (Clontech) also can use, and it allows to cut down the evaluation of differential expression clone in the library.Using PCR -After selectivity is cut down test kit generation difference expression gene storehouse, PCR -Selectivity differential screening reagent box obtains using.With directly by detect and driven element cluster synthetic probe with cut down library and hybridization, a kind ofly originate from the probe of cutting down cDNA, and a kind of system self-reversal is cut down the probe of cDNA (reduction second time that is reversed).Obtain differential expression with the clone who detects son rather than driven element probe hybridization; Yet the probe of Xiao Jianing is not sensitive inadequately for detecting micro-information.The probe of cutting down is that differential expression cDNA obtains a large amount of enrichments, but may provide false-positive result.(Clontech) according to manufacturers instructs probe that use is cut down and that do not cut down to identify difference expression gene.
In another embodiment, the stem cell of differentiation or progenitor cell obtain identifying and characterize by the colony forming unit assay method, and it is known to usually in the art, for example MesenCuIt TMSubstratum (stem cells technology, Inc., Vancouver British Columbia).
Stem cell or progenitor cell have been divided into the mensuration of special cell type and can have finished by method well known in the art, for example, use as flow cytometry method or immunocytochemistry (as, with the dyeing of tissue specificity or cell marking specific antibody pair cell) variation of measuring morphologic and cell surface marker, by with optics or the morphological examination of Laser Scanning Confocal Microscope pair cell, or use technology well known in the art, and show as PCR and genetic expression, measure the variation of genetic expression.
5.2 stem cell and progenitor cell populations
The invention provides the method for mediator's differentiation of stem cells.Any mammalian stem cell except that human embryo stem cell can use in the method for the invention, includes but not limited to, separates the stem cell from Cord blood (CB cell), peripheral blood, adult blood, marrow, placenta, mescenchymal stem cell and other sources.In not preferred embodiment, stem cell is an embryonic stem cell or from the source isolated cells of non-placenta.
The source of mescenchymal stem cell comprises marrow, placenta, umbilical cord and blood.For example, medullary cell can obtain from the cavity of iliac crest, leg section, tibia, rib or other marrows.
The stem cell of using according to method of the present invention can comprise multipotential cell, that is, have and break up diversity, energy self completely and keep dormancy or the immobilized cell in tissue.Stem cell also can comprise multipotency cell, committed progenitor and inoblast.In a preferred embodiment, the stem cell that utilizes of the present invention be from the stem cell dabbling mazolytic work of bloodletting of sufficient phase, immobilized, versatility.
Stem cell colony can be made up of the placenta stem-cell that obtains by commerce services, the LifeBank U.S. (Cedar Knolls for example, NJ), ViaCord (Boston MA), CordBlood Registry (San Bruno, CA) and Cryocell (Clearwater, FL).
Stem cell colony also comprises according to U. S. application, publication number US20020123141, be published in 2002-09-05, be entitled as " method of collecting placenta stem-cell " and U. S. application, publication number US20030032179 is published in 2003-02-13, is entitled as the placenta stem-cell that " postpartum Mammals placenta; it is used and placenta stem-cell thus " (at this, the full content of quoting each piece as a reference) disclosed method is collected.
In one embodiment, can use stem cell from Cord blood.Collection first from the blood of placenta is called Cord blood, and it mainly comprises CD34 +And CD38 +Hemopoietic progenitor cell.In dabbling first 24 hours of postpartum, the CD34 of high density +CD38 -Hemopoietic progenitor cell can be separated from placenta.After about 24 hours of perfusion, the CD34 of high density -CD38 -Cell can be along with aforesaid cell together separates from placenta.The placenta of the isolating perfusion of the present invention provides a large amount of enrichment CD34 +CD38 -And CD34 -CD38 +The source of human stem cell of stem cell; Poured into 24 hours or the longer time isolating placenta a large amount of enrichment CD34 are provided -And CD38 -The source of human stem cell of stem cell.
The preferred cell that uses according to method of the present invention is the embryonic-like stem cell that comes from the dabbling placenta of bloodletting, or the cell that obtains from embryo's sample placenta stem-cell.Embryonic-like stem cell of the present invention can obtain characterizing by using the variation of measuring genetic expression as round pcr as the variation and the use of flow cytometry method and immunocytochemical technique measurement morphology and cell surface marker.In one embodiment of the invention, this embryonic-like stem cell can obtain characterizing by the existence of following cell surface marker: CD10, CD29, CD44, CD54, CD90, SH2, SH3, SH4, OCT-4 and APC-p or the shortage of following cell surface marker: CD34, CD38, CD45, SSEA3 and SSEA4.In a preferred embodiment, such embryonic-like stem cell can obtain by the existence of cell surface marker OCT-4 and ABC-p characterizing.Such cell surface marker can for example, by the flow cytometry method, wash and use the antibody staining of anti-cell surface markers afterwards according to method conventional determining well known in the art.For example, for measuring the existence of CD34 or CD38, cell can wash in PBS, uses the Phycocyanins, C-of anti-CD34 and the fluorescein isothiocyanate of anti-CD38 (Becton Dickinson, Mountain View, CA) two dyeing then.
The embryonic-like stem cell that is derived from placenta has the characteristic of embryonic stem cell but is not to obtain from the embryo.In other words, the present invention includes OCT-4 +And ABC-p +The purposes of cell, it is from dabbling mazolytic undifferentiated stem cell in postpartum.This cell is the same with human embryo stem cell to be omnipotent (for example, versatility).As mentioned above, many different versatilities or multipotency stem cell can separate from dabbling placenta at different time points, for example, and CD34 +CD38 +, CD34 +CD38 -And CD34 -CD38 -Hematopoietic cell.According to method of the present invention, people's placenta is in the source of postpartum as embryonic-like stem cell.
For example, after giving birth to from the uterus, placenta by bloodletting as quickly as possible with prevention or reduce apoptosis.Subsequently, placenta is just perfusion as soon as possible after a bloodletting, to remove any other material that exists in blood, residual cell, albumen, the factor and the organ.The material fragment is also removed from placenta.Perfusion continues at least 2 hours extremely above 24 hours usually with suitable perfusion liquid.In some other embodiments, placenta is subjected at least 4,6,8,10,12,14,16,18,20,22 hours perfusion.In other words, the present invention to small part can be based on the cell in the postpartum placenta and activatory is found by bloodletting and perfusion time enough.Therefore, placenta can be easy to the rich and abundant source as embryonic-like stem cell, and its cell can be used for research, comprises drug discovery, treatment of diseases and prevention, and especially surgery is transplanted and therapy, and the generation that is used for committed cell, tissue or organoid.See series number 10/004,942 co-pending application, submit in 2001-12-05, be entitled as " method of collecting placenta stem-cell " and series number 10/076,180 application is submitted in 2002-02-13, is entitled as " postpartum Mammals placenta; it is used and placenta stem-cell thus ", quote herein every piece full content as a reference.
Embryonic-like stem cell can be from the placenta of drainage extracts by the method for the perfusion technique that utilizes Umbilical artery and umbilical vein.Placenta is preferably by bloodletting drainage (for example residual Cord blood) with collecting residual blood.Then, the placenta of drainage is handled under the state of setting up intravital, the natural bio-reactor environment in the external back of elder generation, and the embryonic-like stem cell that wherein resides at parenchyma and blood vessel external cavity obtains recruiting.This embryonic-like stem cell migrates in drainage, the empty microenvironment, according to method of the present invention it is collected, and preferably washes in the collection tube by perfusion.
That especially pay close attention to as part of the present invention is CD34 +And CD133 +Progenitor cell is to medullary cell, especially dendron or granulocytic adjusting.Nearest report shows that this cell is a versatility; Therefore, the present invention also pays close attention to the adjusting that these progenitor cells are grown to brain cell, nephrocyte, intestinal cell, liver cell or myocyte.
Any Mammals, birds or reptiles CD34 +Or CD133 +Stem cell or progenitor cell can use in company with method of the present invention, include but not limited to, comprise that from Cord blood (CB cell), peripheral blood, adult blood, marrow, placenta dabbling placenta (sees U. S. application publication number US20030032179, be published in 2003-02-13, be entitled as " postpartum Mammals placenta; it is used and placenta stem-cell thus ", quote its full content herein as a reference), mescenchymal stem cell and other isolating stem cells of originating.In a preferred embodiment, stem cell is hemopoietic stem cell or isolated cells from marrow.This cell can obtain from other organ or tissues, but these sources are less preferred.
In one embodiment, can use from Cord blood or the next progenitor cell of placenta in postpartum.As above put down in writing, Cord blood will comprise CD34 +And CD38 +Hemopoietic progenitor cell.In dabbling first 24 hours of postpartum, the CD34 of high density +CD38 -Hemopoietic progenitor cell can be separated from isolating, dabbling placenta.After about 24 hours of perfusion, the CD34 of high density -CD38 -Cell can be along with aforesaid cell together separates from placenta.In another embodiment, progenitor cell populations can obtain by commerce services, for example, the LifeBank U.S. (Cedar Knolls, NJ), ViaCord (Boston MA), Cord Blood Registry (San Bruno, CA) and Cryocell (Clearwater, FL).
5.3 compound of the present invention
The compound that the present invention uses is mentioned as " immunomodulator compounds " at this, and is comprised racemic, the steric isomer enrichment or pure immunomodulator compounds and drug acceptable salt, solvate, hydrate, steric isomer, inclusion compound and its prodrug.The preferred compound that uses in the present invention is the little organic molecule of molecular weight less than about 1000g/mol, and is not albumen, polypeptide, oligonucleotide, oligosaccharides or other macromole.
Unless show as used herein and in addition, a kind of compound represented in term " steric isomer is pure ", and it comprises a kind of isomer of this compound, and other isomer of essentially no this compound.For example, a kind of enantiomorph of opposition of essentially no this compound of steric isomer pure component of the compound with a chiral centre.Other diastereoisomer of a kind of essentially no this compound of steric isomer pure component of the compound with two chiral centres.Unless show a kind of steric isomer pure component of term " enantiomer-pure " expression as used herein and in addition with compound of a chiral centre.Unless show as used herein and in addition, a kind of a kind of steric isomer that comprises this compound that surpasses about 60% weight of term " the steric isomer enrichment " expression, preferably surpass about 70% weight, more preferably surpass a kind of steric isomer of this compound of about 80% weight.As used herein, steric isomer with compound of a chiral centre of term " enantiomer-pure " the expression component of isozygotying.Similarly, term " the enantiomorph enrichment " expression has the steric isomer enriched composition of the compound of a chiral centre.
Unless show term " immunomodulator compounds " or " IMiDs as used herein as used herein and in addition TM" (Celgene company) comprise little organic molecule, it significantly suppresses TNF-α, the monocytic IL1 β of LPS inductive and IL12, and part suppresses the generation of IL6.The concrete immunomodulator compounds of the present invention describes in detail below.This compound can obtain from for example Celgene is commercial, perhaps according to the method preparation of describing in patent of listing at this or the application.
TNF-α is a kind of inflammatory cytokine that is produced when the acute inflammation by scavenger cell and monocyte.In cell, TNF-α is the reason that forms the signal event different range.TNF-α may play pathology in cancer.Be not subjected to the restriction of particular theory, a kind of biological effect of being brought into play by immunomodulator compounds of the present invention is that TNF-α synthetic reduces.The degraded of immunomodulator compounds enhance TNF of the present invention-α mRNA.
Further, be not subjected to the restriction of particular theory, the immunomodulator compounds that the present invention uses is the effective stimulator altogether of T cell still, and strengthens the propagation of cell significantly in a kind of dose-dependent mode.Immunomodulator compounds of the present invention is also to CD8 +The T cell subtype compares CD4 +The T cell subtype is by stronger common stimulatory effect.
Immunomodulator compounds specific embodiment of the present invention includes but not limited to, as those at United States Patent (USP) 5,929, the cinnamic cyano group and the carboxy derivatives of disclosed replacement in 117; As at United States Patent (USP) 5,874,1-oxo-2-(2,6-dioxo-3-fluorine piperidines-3-yl) isoindoline compounds and 1 of describing in 448,3-dioxo-2-(2,6-dioxo-3-fluorine piperidines-3-yl) isoindoline compounds; As at United States Patent (USP) 5,798, quaternary 2-(2,6-dioxopiperidine-3-yl)-1-oxo isoindole quinoline compound of describing in 368; 1-oxo and 1,3-dioxo-2-(2,6-dioxopiperidine-3-yl) isoindoline compounds (for example the 4-methyl-derivatives and the EM-12 of Thalidomide) includes but not limited at United States Patent (USP) 5,635, those disclosed in 517; With at United States Patent (USP) 5,698, the non-polypeptide cyclic amide compounds of a disclosed class in 579 and 5,877,200.The full content that this paper quotes every piece of patent referred in this as a reference.Immunomodulator compounds of the present invention does not comprise Thalidomide.
Other concrete immunomodulator compounds of the present invention include but not limited to, as United States Patent (USP) 5,635, the 517 described 1-oxos of quoting in this combination and 1 that in the benzo ring, replaced by amino amino or that replace, 3-dioxo-2-(2,6-dioxopiperidine-3-yl) isoindoline compounds.These compounds have structure I:
Wherein having one in the middle of X and the Y is C=O, and another is C=O or CH in the middle of X and the Y 2, R 2Be hydrogen or low alkyl group, particularly methyl.Concrete immunomodulator compounds includes but not limited to:
1-oxo-2-(2,6-dioxopiperidine-3-yl)-4-aminoisoindoline;
1-oxo-2-(2,6-dioxopiperidine-3-yl)-5-aminoisoindoline;
1-oxo-2-(2,6-dioxopiperidine-3-yl)-6-aminoisoindoline;
1-oxo-2-(2,6-dioxopiperidine-3-yl)-7-aminoisoindoline;
1,3-dioxo-2-(2,6-dioxopiperidine-3-yl)-4-aminoisoindoline; With
1,3-dioxo-2-(2,6-dioxopiperidine-3-yl)-5-aminoisoindoline.
Other concrete immunomodulator compounds of the present invention belong to the 2-(2 that a class replaces, 6-dioxopiperidine-3-yl) 2-of phthalimide compound and replacement (2,6-dioxopiperidine-3-yl)-1-oxo isoindole compound is for example at United States Patent (USP) 6,281,230; 6,316,471; 6,335,349; With 6,476,052, and describe among International Patent Application PCT/US97/13375 (international publication WO98/03502) those, quote at this for every piece.The compound of this quasi-representative has following general formula:
R wherein 1Be hydrogen or methyl.In an independent embodiment, the present invention includes the purposes of the form (for example optically pure (R) or (S) enantiomorph) of the enantiomer-pure of these compounds.
Although other concrete immunomodulator compounds of invention belong at U.S. Patent application 10/032,286 and 09/972,487, and a disclosed class isoindole imide compound among International Patent Application PCT/US01/50401 (international publication WO02/059106), this paper quotes each piece of writing as a reference.Exemplary compounds has following general formula I I:
Figure S03813627919950430D000322
And pharmacologically acceptable salt, hydrate, solvate, inclusion compound, enantiomorph, diastereomer, racemic modification and stereoisomer mixture, wherein:
Having one in the middle of X and the Y is C=O, and another is CH 2Or C=O;
R 1Be H, (C 1-C 8) alkyl, (C 3-C 7) cycloalkyl, (C 2-C 8) thiazolinyl, (C 2-C 8) alkynyl, benzyl, aryl, (C 0-C 4) alkyl-(C 1-C 6) Heterocyclylalkyl, (C 0-C 4) alkyl-(C 2-C 5) heteroaryl, C (O) R 3, C (S) R 3, C (O) OR 4, (C 1-C 8) alkyl-N (R 6) 2, (C 1-C 8) alkyl-OR 5, (C 1-C 8) alkyl-C (O) OR 5, C (O) NHR 3, C (S) NHR 3, C (O) NR 3R 3', C (S) NR 3R 3' or (C 1-C 8) alkyl-O (CO) R 5
R 2Be H, F, benzyl, (C 1-C 8) alkyl, (C 2-C 8) thiazolinyl or (C 2-C 8) alkynyl;
R 3And R 3' be (C independently 1-C 8) alkyl, (C 3-C 7) cycloalkyl, (C 2-C 8) thiazolinyl, (C 2-C 8) alkynyl, benzyl, aryl, (C 0-C 4) alkyl-(C 1-C 6) Heterocyclylalkyl, (C 0-C 4) alkyl-(C 2-C 5) heteroaryl, (C 0-C 8) alkyl-N (R 6) 2, (C 1-C 8) alkyl-OR 5, (C 1-C 8) alkyl-C (O) OR 5, (C 1-C 8) alkyl-O (CO) R 5Or C (O) OR 5
R 4Be (C 1-C 8) alkyl, (C 2-C 8) thiazolinyl, (C 2-C 8) alkynyl, (C 1-C 4) alkyl-OR 5, benzyl, aryl, (C 0-C 4) alkyl-(C 1-C 6) Heterocyclylalkyl or (C 0-C 4) alkyl-(C 2-C 5) heteroaryl;
R 5Be (C 1-C 8) alkyl, (C 2-C 8) thiazolinyl, (C 2-C 8) alkynyl, benzyl, aryl or (C 2-C 5) heteroaryl;
R 6When occurring, be H, (C independently at every turn 1-C 8) alkyl, (C 2-C 8) thiazolinyl, (C 2-C 8) alkynyl, benzyl, aryl, (C 2-C 5) heteroaryl or (C 0-C 8) alkyl-C (O) O-R 5, perhaps R 6Can connect to form Heterocyclylalkyl;
N is 0 or 1; And
* represent the chiral carbon center.
In concrete formula II compound, when n is 0, R then 1Be (C 3-C 7) cycloalkyl, (C 2-C 8) thiazolinyl, (C 2-C 8) alkynyl, benzyl, aryl, (C 0-C 4) alkyl-(C 1-C 6) Heterocyclylalkyl, (C 0-C 4) alkyl-(C 2-C 5) heteroaryl, C (O) R 3, C (O) OR 4, (C 1-C 8) alkyl-N (R 6) 2, (C 1-C 8) alkyl-OR 5, (C 1-C 8) alkyl-C (O) OR 5, C (S) NHR 3Or (C 1-C 8) alkyl-O (CO) R 5
R 2Be H or (C 1-C 8) alkyl; And
R 3Be (C 1-C 8) alkyl, (C 3-C 7) cycloalkyl, (C 2-C 8) thiazolinyl, (C 2-C 8) alkynyl, benzyl, aryl, (C 0-C 4) alkyl-(C 1-C 6) Heterocyclylalkyl, (C 0-C 4) alkyl-(C 2-C 5) heteroaryl, (C 5-C 8) alkyl-N (R 6) 2(C 0-C 8) alkyl-NH-C (O) O-R 5(C 1-C 8) alkyl-OR 5, (C 1-C 8) alkyl-C (O) OR 5, (C 1-C 8) alkyl-O (CO) R 5Or C (O) OR 5And its dependent variable has and last identical definition.
In other concrete formula II compound, R 2Be H or (C 1-C 4) alkyl.
In other concrete formula II compound, R 1Be (C 1-C 8) alkyl or benzyl.
In other concrete formula II compound, R 1Be H, (C 1-C 8) alkyl, benzyl, CH 2OCH 3, CH 2CH 2OCH 3Or
In another embodiment of formula II compound, R 1Be
Or
Figure S03813627919950430D000343
Wherein Q is O or S, and R 7When occurring, be H, (C independently at every turn 1-C 8) alkyl, benzyl, CH 2OCH 3Or CH 2CH 2OCH 3
In other concrete formula II compound, R 1Be C (O) R 3
In other concrete formula II compound, R 3Be (C 0-C 4) alkyl-(C 2-C 5) heteroaryl, (C 1-C 8) alkyl, aryl or (C 0-C 4) alkyl-OR 5
In other concrete formula II compound, heteroaryl is pyridyl, furyl or thienyl.
In other concrete formula II compound, R 1Be C (O) OR 4
In other concrete formula II compound, available (C 1-C 4) alkyl, aryl or benzyl replace the H of C (O) NHC (O).
Other concrete immunomodulator compounds of the present invention belong to a disclosed class isoindole imide compound in U.S. Patent application 09/781,179, international publication WO98/54170 and United States Patent (USP) 6,395,754, and this paper quotes each piece of writing as a reference.Exemplary compounds has following general formula III:
And pharmacologically acceptable salt, hydrate, solvate, inclusion compound, enantiomorph, diastereomer, racemic modification and stereoisomer mixture, wherein:
Having one in the middle of X and the Y is C=O, and another is CH 2Or C=O;
R is H or CH 2OCOR ';
(i) each R 1, R 2, R 3Or R 4Independently of one another for halogen, have the alkyl of 1-4 carbon atom or have the alkoxyl group of 1-4 carbon atom; Or (ii) R 1, R 2, R 3Or R 4In the middle of have one be nitro or-NHR 5, all the other R 1, R 2, R 3Or R 4Be hydrogen;
R 5Be hydrogen or alkyl with 1-8 carbon;
R 6Be hydrogen, have alkyl, benzyl (benzo), chlorine or a fluorine of 1-8 carbon atom;
R ' is R 7-CHR 10-N (R 8R 9);
R 7Be metaphenylene or to phenylene or-(C nH 2n)-, wherein n has the value of 0-4;
Each R 8And R 9Be hydrogen or alkyl, perhaps R independently of one another with 1-8 carbon atom 8And R 9Be together tetramethylene, pentamethylene, hexa-methylene or-CH 2CH 2[X] X 1CH 2CH 2-, [X] X wherein 1Be-O-,-S-or-NH-;
R 10Be hydrogen, have an alkyl or phenyl of 1-8 carbon atom; And
* represent the chiral carbon center.
Most preferred immunomodulator compounds of the present invention is 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone and 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-piperidines-2,6-diketone.These compounds can make (for example referring to United States Patent (USP) 5,635,517, being incorporated herein by reference) by the standard synthetic method.These compounds can be from Celgene Corporation, Warren, and NJ obtains.4-(amino)-2-(2,6-dioxo-(3-piperidyl))-isoindoline-1,3-diketone (ACTIMID TM) have a following chemical structure:
Figure S03813627919950430D000361
3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-piperidines-2,6-diketone (REVIMID TM) following chemical structure arranged:
5.4 stem cell culture method
In certain embodiments of the invention, with stem cell or progenitor cell, include but not limited to pour in embryonic stem cell, embryonic-like stem cell, progenitor cell, multipotential cell, totipotent cell, multipotential cell, postpartum placenta interior living cell, cord blood cell, derive from the stem cell or the progenitor cell of peripheral blood or adult blood, or medullary cell is exposed to compound of the present invention and induces differentiation.These cells can be bred in external use method known in the field, or optionally, also can breed in the dabbling placenta in postpartum.
In certain embodiments, the interior living cell that pours into placenta postpartum can be by collecting in placenta or the substratum, and under appropriate condition external cultivation time enough, to induce differentiation to the cell type or the pedigree of expectation.Referring to U. S. application publication number US20030032179, be published in 2003-02-13, be entitled as " postpartum the Mammals placenta use and later placenta stem-cell ", quote its full content here.
In another embodiment of the invention, stem cell or progenitor cell do not derive from and pour into placenta postpartum, but on the contrary, separate from other sources, for example Cord blood, marrow, peripheral blood or adult blood are exposed to compound of the present invention and induce differentiation.In a preferred embodiment, differentiation is carried out external under appropriate condition, through adequate time, to induce differentiation to the cell lineage or the cell type of expectation.Compound of the present invention is by adding, produce in position, or makes stem cell or progenitor cell can touch any alternate manner of compound of the present invention and use in differentiation medium/substratum.
In another embodiment, the stem cell of cultivating, for example, the stem cell of vitro culture or in the process that the stem cell that cultivate in the dabbling placenta postpartum is all being cultivated irriate and breeding, for example, be subjected to the stimulation of following material: erythropoietin, cytokine, lymphokine, Interferon, rabbit, G CFS (CSF), Interferon, rabbit, chemokines, interleukin, the recombinant human hematopoietic factor comprises part, STEM CELL FACTOR, thrombopoietin (Tpo), interleukin, granulocyte colony-stimulating factor (G-CSF) or other somatomedin etc.
Cultured cells can detect test by colony forming unit and obtain identifying that it is known in this area, as Mesen CuIt after collecting and/or separating TMSubstratum (stem cells technology, Inc., Vancouver British Columbia).
Vitro culture stem cell or the method for progenitor cell is well known in the art, for example, referring to Thomson etc., 1998, Blood, 93 (4): 1253-63, with Hatzopoulos etc., 1998, Development125:1457-1468 (endotheliocyte progenitor cell); Slager etc., 1993, Dev.Genet.14 (3): 212-24 (neurocyte or muscle cell progenitor cell); Genbachev etc., 1995, Reprod.Toxicol.9 (3): 245-55 (cytotrophoblast such as placenta epithelial cell progenitor cell); .1984 such as Nadkarni, Tumori70:503-505, Melchner etc., 1985, Blood66 (6): 1469-1472, International PCT announces that WO00/27999 published Himori etc., 1984 on May 18th, 2000, Intl.J.Cell Cloning2:254-262, with Douay etc., 1995, Bone MarrowTransplantation 15:769-775 (hemopoietic progenitor cell); Shamblott etc., 1998, Proc.Natl.Acad.Sci.USA95:13726-31 (spermatogonium); Yan etc., 2001, Devel.Biol.235:422-432 (embryo's trophoderm stem cell).These methods can be used in the method for the invention through simple improvement, as long as the cultivation of progenitor cell is to be cultivated in the effect of compound of the present invention next step or multistep by culturing cell, at preset time, just can produce the colony of required noble cells.
5.4.1 external stem cell is cultivated
The inventive method comprises the adjusting in vitro differentiation of stem cell or progenitor cell, comprise cell and compound, at external incubation together, induce them to be divided into the cell lineage of specific expectation as little organic compound of the present invention, then the cell directly transplanting of differentiation is gone among the curee.In a preferred embodiment, cell induction is divided into hematopoietic cell lineage.
In certain embodiments, with the purpose stem cell cultivated in the external compound of the present invention that is exposed to 0.1 μ g/ml, 0.2 μ g/ml, 0.3 μ g/ml, 0.4 μ g/ml, 0.5 μ g/ml, 1 μ g/ml, 5 μ g/ml or 10 μ g/ml concentration.Preferably, the purpose cellular exposure is approximately the Thalidomide of 0.005pg/ml to 5mg/ml in concentration, and 0.005 μ g/ml is to the Actimid of 5mg/ml TM(NJ), (NJ), 0.005mg/ml is to the Actimid of 5mg/ml for CelgeneCorp., Warren to about 5mg/mlRevimid for 0.005 μ g/ml for CelgeneCorp., Warren TM(CelgeneCorp., Warren, NJ) or 0.005 μ g/ml to the Revimid of 5mg/ml TM(CelgeneCorp., Warren, NJ) in (also referring to 5.7 the joint, " pharmaceutical composition ").
In certain embodiments, by some nutritive substances, hormone, VITAMIN, somatomedin or its combination are incorporated in the primer solution embryonic-like stem cell is induced propagation in the placenta bio-reactor.Serum and other somatomedin also can be added in propagation primer solution or the substratum.Somatomedin is protein normally, includes but not limited to: cytokine, lymphokine, Interferon, rabbit, G CFS (CSF), Interferon, rabbit, chemokines and interleukin.Also can use other somatomedins, comprise the recombinant human hemopoieticgrowth factor, the somatomedin and the Urogastron in part, STEM CELL FACTOR, thrombopoietin (Tpo), granulocyte colony-stimulating factor (G-CSF), leukaemia inhibitory factor, trofermin, placenta source wherein arranged.
Add that somatomedin in the primer solution can stimulate undifferentiated embryonic-like stem cell, committed progenitor or the propagation of the cell (for example Fen Hua hematopoietic cell) that broken up.But the generation of the molecule of somatomedin stimulating organism material and biologic activity includes but not limited to some somatomedins of having described in immunoglobulin (Ig), hormone, enzyme or front.
The placenta of cultivating regularly " supply " reduces the cell that discharges to remove the substratum that has consumed, replenishes fresh culture.Cultivate placenta and should deposit under aseptic condition with the minimizing contamination of heavy, and keep growth to create a such environment under supercharging condition intermittently, regular, it keeps the sufficient nutrition supply to placenta cells.Should be realized that the perfusion of placenta and cultivation can realize that all automatization and computerize are to obtain high efficient and capacity.
5.4.2 external progenitor cell is cultivated
The inventive method also comprises progenitor cell, especially CD34 +And CD133 +Adjusting and regulation and control that progenitor cell is grown.In one embodiment of the invention, progenitor cell is induced to differentiate into a kind of hematopoietic cell lineage.In a specific embodiment, this cell lineage is the granulocyte pedigree.In another embodiment, CD133 +Cell induction is divided into endotheliocyte, brain cell, nephrocyte, liver cell or intestinal cell.
Progenitor cell can be cultivated by standard method above-mentioned.In addition, the cultivation of progenitor cell is handled cell in a plurality of times of cultivating or the time of setting possibly, so that progenitor cell is divided into different cell lineage.
Therefore, cultivating CD34 +Or CD133 +In the method for progenitor cell, cell was implanted in the time of 0 day and is contained in the substratum of STEM CELL FACTOR (SCF), Flt-3L, GM-CSF and TINT-α, cultivated 6 days.In the 6th day, cell is implanted in the substratum that contains GM-CSF and TNF-α again, continues to continue to cultivate six days.This method can produce dendritic cell.In the changing method of this method, cell is implanted in the substratum that contains GM-CSF and IL-4 at the beginning, changes the substratum (referring to Steinman etc., international publication number WO97/29182) of monocyte culture condition then in the 6th day into.In order to produce CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Progenitor cell contacted The compounds of this invention with progenitor cell with compound of the present invention at the 0th day, and collected CD34 at the 6th day +CD38 -CD33 +Or CD34 +CD38 -CD33 -Progenitor cell.
The inventor has been found that and adds especially Actimid of compound of the present invention TMOr Revimid TMTime for CD34 +Cell is to the differentiation pathway of specific cells pedigree, and CD133 +The differentiation of cell has crucial influence.CD34 +Progenitor cell is cultivated under standard conditions, is along the approach of development of bone marrow or pedigree, just, becomes dendritic cell in beginning to implant 12 days of back (just beginning to cultivate the back).Yet, beginning to cultivate in first six days, the time in several specific times is added compound of the present invention, has changed this approach basically.For example, if CD34 +The especially myeloid CD34 of cell +Cell was at first day that cultivates and compound of the present invention, especially Actimid TMOr Revimid TMContact, the path along the differentiation of medullary cell pedigree will be suppressed so, is not cultivating the 6th day CD34 as be exposed to compound of the present invention (just being exposed to DMSO) with respect to contrast +CD38 -The increase of cell quantity and CD1a +CD14 -Cell quantity minimizing proved.And, contacting the cells whose development that can cause expressing surface markers with compound of the present invention and be suppressed, surface markers is by dendritic cell system, expresses as CD80 and CD86.At first day that cultivates or cultivate any moment in back three days of the beginning, allow cell contact with compound of the present invention, all can cause CD34 +This growth of progenitor cell is regulated.If between the 0th day and the 6th day, giving repeatedly taking medicine of compound of the present invention, for example, administration in the 0th and the 2nd day, administration in the 0th and the 4th day, administration or the 2nd, the 4th in the 3rd and the 6th day, administration in the 6th day can aggravate CD34 +The increase of cell quantity.
One of the present invention be particularly useful aspect, at CD34 +Added compound of the present invention in first day of the progenitor cell cultivation, continued to expose in 12 days, can cause the growth of unique progenitor cell, this progenitor cell is expressed the reconstitution cell surface markers of uniqueness: CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Cell mass has been represented an intermediateness of differentiation.This cell mass is very useful as the bred group of progenitor cell, and they can be implanted on one's body the patient of the hematopoietic cell lineage that needs fast breeding very easily, for example granulocyte.In another embodiment, CD34 +Can propagation in the phase (about 6 days) (just do not add Actimid at standard medium TM, Revimid TMOr other analogues) implant in and cultivate, it is forwarded to comprise Actimid then TMOr in the same or analogous substratum of analogue, continued to cultivate the 12nd day always.In this embodiment, the cell that is breaking up generally shows the minimizing of CD80, CD86 and CD14 expression amount, but causes CD1a +The cell comparison is according to but continuing to increase.Cell in this differentiation can be divided into dendritic cell by obstruction.In another embodiment, CD34 +Cell continues three days with ActimidTM, RevimidTM or other compound treatment of the present invention at least in the propagation phase (implant back 1-6 days).Still in another embodiment, CD34 +Or CD133 +In progenitor cell first six days after implantation with the Actimid of twice or more multiples TM, Revimid TMOr other compound treatment of the present invention.This multiple treatment process can increase CD34 +And CD133 +The propagation of cell mass.Use Actimid TMOr other compound several different methods processing of the present invention, can cause CD34 +The transformation of progenitor cell differentiation is not divided into CD1c +CD15 -Cell lineage but be divided into CD1c -CD15 +Cell lineage just is not divided into marrow dendritic cell pedigree, and becomes granulocyte pedigree (Fig. 6 B).
The 0th day processing progenitor cell from cultivating especially adopted multidose in the 0th day and the 6th day, also can cause CD133 +The increase of progenitor cell quantity, especially CD34 +CD133 +The increase of progenitor cell.CD133 is the mark of a hematopoiesis, can be used as the alternative of CD34 isolate, because CD133 +Cell can be with CD34 +The same mode of subclass increases, and has kept the ability (referring to Kobariv etc., J.Hematother.StemCell Res.10 (2): 273-81 (2001)) of many cells pedigrees simultaneously.Reported CD133 +CD34 in the human fetal brain tissue source -Occur in the cell, and when it is injected in the newborn mice, demonstrate the potential transplanting, propagation, migration and Neural Differentiation (referring to Proc.Natl.Acad.Sci.U.S.A.19:97 (26): 14720-5 (2000)).CD133 +Hemopoietic stem cell shown and has been imbued with the progenitor cell activity, has by the enhanced colony to form ability and at NOD-SCID mouse higher transfer ability on one's body.
Although more than, if the CD34 in the propagation +Progenitor cell (any times in just beginning to cultivate 3-6 days) after cultivating three days contact with compound of the present invention, and the progenitor cell that has begun in the propagation of express cell surface markers CD1a can continue to increase this mark of expression with the contrast of DMSO processing relatively.What deserves to be mentioned is that this lasting increase can't cause cytotoxicity.In other words, use Actimid TMHandle the apoptosis that can not cause other cell masses.Pure effect is exactly the maintenance of existing immunological competence and the development of new immunological competence.
Therefore, in an embodiment of the inventive method, CD34 +It is modulated (just being suppressed) that cytodifferentiation becomes dendritic cell, and this is to pass through CD34 +Progenitor cell is realized with compound treatment of the present invention the 0th day that cultivates (just first day of cultivation).In another embodiment, CD34 +The cytodifferentiation myeloblast passes through CD34 +Progenitor cell contacts with compound of the present invention and strengthens the 0th day that cultivates (just cultivate first day).In another embodiment, CD34 +Cytodifferentiation becomes CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Progenitor cell contacts with compound of the present invention in first three days of cultivating by progenitor cell and strengthens.In another embodiment, CD34 +CD133 +The group contacts with the compound of the present invention of multidose by progenitor cell in the 0th day to the 6th day process of cultivation and strengthens or increase.In another embodiment, CD1a +Cell mass is by CD34 when cultivating the 6th day +Progenitor cell contacts with compound of the present invention and strengthens or increase, wherein said CD34 +Cytodifferentiation becomes to present the cell of CD1a surface markers, and wherein said cultivation comprises handling without above-claimed cpd and reaches six days most.
In the superincumbent embodiment, this Actimid TM, Revimid TMOr related compound is understandable to not being both of the administration of progenitor cell in vivo, for example, one transplanted or the patient of this cell of grafting in, as being applied to progenitor cell external.
Method of the present invention comprises the adjusting in vitro differentiation of stem cell or progenitor cell, be included in cell in vitro and inducing cell and be divided into specific cytophyletic compound, as little organic molecule of the present invention incubation together, then the cell directly transplanting of differentiation is gone into the curee.In a preferred embodiment, cell is induced and is divided into a kind of hematopoietic cell lineage.In a selectable embodiment, with CD133 +Cell induction is divided into endotheliocyte, brain cell, nephrocyte, liver cell or intestinal cell.
What deserves to be mentioned is, method described herein with from Mammals people's CD133 preferably +Progenitor cell uses together and receives publicity, but receives publicity using also with birds or reptiles progenitor cell.But also there is different effects in compound of the present invention, and this depends on the species in progenitor cell source.Aspect cultural method, exist some differences, especially, also receive publicity about used compound concentrations.For example, the progenitor cell in mouse source is to compound of the present invention, for example Actimid TMLess responsive, require higher concentration just can reach the effect of people source progenitor cell under 1 μ M.It will be appreciated by those skilled in the art that this optimization is routinely.
5.5 the genetic engineering of stem cell and progenitor cell
In another embodiment of the invention, use, for example, virus vector such as adenovirus or retrovirus vector, or by using the DNA picked-up of mechanical means such as liposome or chemistry mediation, will according to the stem cell of method differentiation of the present invention or progenitor cell before or after compound of the present invention contacts, carried out genetically engineered.In specific embodiment, CD34 +Progenitor cell is used compound treatment of the present invention then through genetically engineered; In more specific embodiments, described compound is Actimid TM, Revimid TMOr its analogue.In another embodiment, described cell compound treatment of the present invention is carried out genetically engineered then.
By certain methods well known in the art, for example transfection, conversion, electroporation, infection, microinjection, cytogamy, DEAE-dextran, calcium phosphate precipitation, liposome, LIPOFECTIN TM, lysosome fusion, synthetic cation lipid, particle gun use or dna vector transhipment, can import in the purpose cell containing genetically modified carrier, like this transgenosis is delivered in the daughter cell, as by embryonic-like stem cell splitted filial generation embryonic-like stem cell or progenitor cell.Be used to transform or the different methods of transfection mammalian cell, referring to Keown etc., 1990, MethodsEnzymol.185:527-37; Sambrook etc., 2001, Molecular Cloning, A Laboratory Manual, the third edition, cold spring harbor laboratory publishes, New York.
Preferably, utilize any technology that transgenosis is imported, only otherwise destroy nuclear membrane or other the already present cell or the genetic construction of cell.In certain embodiments, can transgenosis be inserted in the interior genetic material of nuclear by microinjection.Cell and cyto-architectural microinjection are known in this field and skilled.
Mammalian cell for stable transfection is cultivated as cultivating the cell in placenta, has only the sub-fraction cell foreign DNA can be incorporated on its genome.The efficient of integrating depends on carrier and used rotaring dyeing technology.For identifying and filter out integron, generally can change the gene that has a selection markers (for example, antibiotics resistance) over to embryonic-like stem cell together with the sequence of goal gene.Preferred selection markers comprises that those have the antiradiation drug resistance, as G418, Totomycin and methotrexate.Stable transfection the cell of exogenous nucleic acid can identify (cell that just is integrated with the selected marker can be survived, and other cells are all dead) by drug screening.Particularly useful during the homologous recombination of this method before recombinant mammalian cells (for example embryonic-like stem cell) imports or be transplanted to main body or patient.
Many screening systems can be used for screening the host transformed stem cell, as embryonic-like stem cell or progenitor cell, resemble CD34 +Or CD133 +Progenitor cell.Especially, this carrier can comprise some observable or selectable mark.Other system of selection includes but not limited to screen another mark as herpes simplex virus thymidine kinase (Wigler etc., 1977, Cell, 11:223), xanthoglobulin-guanine phosphoribosyltransferase (Szybalska and Szybalski, 1962, Proc.Natl.Acad.Sci USA 48:2026), adenine phosphoribosyl transferase (Lowy etc., 1980, Cell 22:817), these genes can use in tk-, hgprt-or aprt-cell respectively.Equally, the metabolic antagonist resistance also can be given methotrexate resistance (Wigler etc., 1980, Proc.Natl.Acad.Sci.USA 77:3567 as the basis of the following gene of screening: dhfr; O ' Hare etc., 1981, Proc.Natl.Acad.Sci.USA78:1527); Gpt, give mould fen acid resistance (Mulligan and Berg, 1981, Proc.Natl.Acad.Sci.USA78:2072); Neo, give aminoglycoside G-418 resistance (Colberre-Garapin etc., 1981, J.Mol.Biol.150:1); And hygro, give hygromycin resistance (Santerre etc., 1984, Gene30:147).
Transgenosis can be incorporated on the genome of purpose cell, preferably passes through random integration.In other embodiment, transgenosis can be integrated by direct method, as by direct homologous recombination (just in the purpose cell, " knock in " or " knock out " goal gene), Chappel, the U.S.. the patent No. 5,272,071; PCT publication number WO91/06667 is published in 1991-05-16; United States Patent (USP) 5,464,764, Capecchi etc. are disclosed in 1995-11-07; United States Patent (USP) 5,627,059, Capecchi etc. are disclosed in 1997-05-06; United States Patent (USP) 5,487,992, Capecchiet etc. are disclosed in 1996-01-30).
By the homologous recombination modified target dna method that it changes cell over to is well known in the art.This construct will comprise the part of the goal gene that passes through genetic modification at least, and will comprise and target site homologous zone, just the copy of endogenic goal gene on host genome.The DNA construct of random integration needn't comprise that homologous region is with the mediation reorganization with respect to those for homologous recombination.Mark can be included in the target construct or at random in the construct, to form just screening or the negative screening that transgenosis is inserted.
For producing the cell of homologous recombination, the for example embryonic-like stem cell of homologous recombination, endogenous placenta cells or the foreign cell in placenta, cultivated, the carrier for preparing a kind of homologous recombination, wherein goal gene is positioned at 5 of the endogenous gene order of target cell genome ' and 3 ' end, so that between goal gene that is carried by carrier and the target cell genome native gene homologous recombination takes place.Remaining nucleic acid of both sides is wanted sufficiently long so that with the target cell genome on the homologous recombination of native gene success.Usually, the DNA of both sides on the carrier (5 ' and 3 ' end) has several thousand base pairs.Make up homologous recombination vector and be known in the art by the method that the reorganization stem cell obtains the homologous recombination animal.(referring to, for example Thomas and Capecchi, 1987, Cell 51:03; Bradley, 1991, Curr.Opin.Bio/Technol.2:823-29; PCT publication No. WO90/11354, WO91/01140, and WO93/04169).
In a specific embodiment, (U.S. Patent number 5,942,496 is entitled as method and compound that polygene changes osteocyte over to, is disclosed in 1999-08-24 to utilize the method for Bonadio etc.; PCT WO95/22611 is entitled as the method and the compound that stimulate osteocyte, is disclosed in 1995-08-24) nucleic acid is imported in the purpose cell, as stem cell, progenitor cell or the foreign cell of in placenta, cultivating, for example osteoprogenitor cells.
5.6. the stem cell of being adjusted that is used to break up and the purposes of progenitor cell
5.6.1. general use
Stem cell of the present invention, CD34 +And CD133 +Progenitor cell can induce differentiation to be used for transplanting with elder generation external back interior therapeutic scheme.In one embodiment, population of stem cells is divided into specific cell type, and through the genetically engineered gene therapy product that provides.In another embodiment, progenitor cell propagation is early progenitor cell, and through genetic engineering modified so that the gene therapy product to be provided.In another embodiment, progenitor cell is divided into specific cell type such as granulocyte, and through genetic engineering modified so that the gene therapy product to be provided.
Compound of the present invention also is applied clinically, and transplanting has at present become and recovers the elementary object that the marrow white cell produces, as the neutropenia that caused by disease and/or the excision of clinical marrow and the counter-rotating of leukopenia.These compounds also are used in the recovery of early progenitor cell or granulocyte generation, and it is by disease, and various known treatment side effects or marrow excise and causes.Compound of the present invention also is applied in some cases, and the wherein inhibition that preferably red blood cell is generated does not produce and can not suppress marrow.
In certain embodiments, with the stem cell and the untreated cell of compound treatment of the present invention, as the stem cell co-administered that comes from Cord blood or peripheral blood is in its required patient.In other schemes, used the CD34 of compound treatment of the present invention +Or CD133 +Cell and untreated cell, as the stem cell co-administered that comes from Cord blood or peripheral blood is in its required patient.In a scheme, from first day the CD34 that cultivates with compound treatment of the present invention +Progenitor cell and untreated cell co-administered are in its required patient.At one more specifically in the scheme, the progenitor cell of transfer is CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Progenitor cell.
Stem cell, for example embryonic-like stem cell or hemopoietic stem cell, or progenitor cell, its differentiation is regulated according to method of the present invention, can be made into injectable preparation (referring to PCT WO96/39101, quoting its full content herein as a reference).In another possibility, cell and tissue, its differentiation is regulated according to method of the present invention, can be as U.S. Patent number 5,709,854; 5,516,532; Or 5,654,381 describe, and makes preparation with the hydrogel that polymeric is crosslinked, and the full content of quoting each piece here as a reference.
Embryonic-like stem cell can be used for substituting treatment or the specific progenitor cell monoid in the research method (for example chondrocyte, liver cell, hematopoietic cell, pancreas parenchyma, neuroblast, muscle progenitor cell etc.) that uses progenitor cell general.
5.6.2. tissue is replaced or is increased
Stem cell, embryonic-like stem cell especially of the present invention, and progenitor cell, its differentiation is regulated according to method of the present invention, can be used for a variety of methods of treatment, and described method is pointed to the transplanting or the fusion of required cell colony, as stem cell or progenitor cell.Stem cell or progenitor cell can be used to replace or increase existing tissue, import new or tissues replaced or connect biological tissue or structure.
In an embodiment preferred of the present invention, stem cell is as the embryonic-like stem cell that comes from placenta, or progenitor cell such as hemopoietic progenitor cell, its differentiation is regulated according to method of the present invention, can use from body homology or allos, comprise coupling or unmatched HLA type, hematopoiesis is transplanted.The service condition of transplanting as allos hematopoiesis with embryonic-like stem cell is consistent, and it can treat the host better, reduces the immunological rejection to donorcells, and as U.S. Patent number 5,800, on September 1st, 539,1998 announced; U.S. Patent number 5,806 described in announcing on September 15th, 529,1998, is quoted each piece of writing here as a reference.
For example, embryonic-like stem cell, its differentiation is regulated and control according to method of the present invention, in the transplanting scheme that can be used for treating, for example increase or replace the stem cell or the progenitor cell of liver, pancreas, kidney, lung, neural system, musculature, bone, marrow, thymus gland, spleen, mucosal tissue, sexual gland or hair.Similarly, hemopoietic progenitor cell, its differentiation is regulated and control according to method of the present invention, can be used for replacing marrow or endothelial progenitor cells.
Stem cell, embryonic-like stem cell for example, its differentiation is regulated and control according to method of the present invention, can be used for the increase of cartilage, tendon or ligament, repairs or replaces.For example, in certain embodiment, false organ (for example false stern) is the replacement cartilaginous tissue structure that grows up at the embryonic-like stem cell that surface coverage one deck is cultivated by the inventive method.In other embodiment, joint (for example knee) can be reconstituted by the cartilaginous tissue structure that embryonic-like stem cell grows up to.The cartilaginous tissue structure also can be applicable in the reconstruction operations in dissimilar joints (as for scheme, referring to as Resnick, D., and Niwayama, G., eels., 1988, bone and disorder of joint diagnosis, second edition, W.B.Saunders Co.).
Stem cell and the progenitor cell handled according to the inventive method can be used in tissue and the organ damage that reparation is caused by disease.In this embodiment, the patient can regenerate with embryonic-like stem cell or repairs tissue or the organ damage that is caused by disease by administration, for example strengthens functions of immune system after chemotherapy or the radiotherapy, repairs the heart tissue after the myocardial infarction.According to this method and with the stem cell or the progenitor cell of immunomodulatory compounds of the present invention processing, or with the stem cell or the progenitor cell of immunomodulatory compounds administration of the present invention, can implant in the individuality that needs, to repair or replacement liver, pancreas or heart tissue.
Stem cell and the progenitor cell handled according to the inventive method also can be used for increasing or replacing medullary cell in bone marrow transplantation.The people is in body homology and allos bone marrow transplantation just are being applied to treat illness disease as leukemia, lymphoma and some other life threatening at present.Yet the shortcoming of these methods need to be a large amount of donor bone marrows, to guarantee having enough cells to be used for transplanting.
The embryonic-like stem cell of collecting according to method of the present invention can provide stem cell and progenitor cell, can reduce necessity of a large amount of marrow donations.Certainly, also can obtain a spot of donations marrow, before injecting or being transplanted to acceptor, in placenta, cultivate propagation, obtain a large amount of stem cells and progenitor cell according to method of the present invention.
By a large amount of embryonic-like stem cells and/or the progenitor cell that present method obtained, in certain embodiments, can reduce the necessity of a large amount of marrow donations.In bone marrow transplantation, approximately need every kg of patient body weight 1 * 10 8To 2 * 10 8Myelomonocyte is used for transplanting (just, needing about 70 milliliters marrow for heavy 70 kilograms contributor).The marrow that obtains 70 milliliters needs highdensity beneficence, and a large amount of losses of blood are arranged in the donations process.In specific embodiment, the cell (for example 7-10 milliliter) that is obtained by a small amount of marrow donations can obtain enlarging by propagation, for example in the placenta bio-reactor before being injected into acceptor.Stem cell and progenitor cell be CD34 particularly +Or CD133 +Progenitor cell, its differentiation is regulated according to method of the present invention, also can provide stem cell and/or progenitor cell, to eliminate the necessity of a large amount of marrow donations.
Also can be used in the specific embodiment by mazolytic embryonic-like stem cell, replace some special disease of therapy for treating with homology or isodynamic enzyme, include but not limited to lysosomal storage disease, as Tay-Sach disease (Tay-Sachs), Niemann's disease (Niemarm-Pick), fabry disease (Fabry ' s), familial splenic anemia (Gaucher ' s), Heng Teshi (Hunter ' s) syndromes, what Le Shi (Hurler ' s) syndromes, and other ganglioside deposition disease, mucopolysaccharidosis and glycogenosis.
In other embodiment, cell can use in gene therapy, correct some inborn errors of metabolisms as homology or heterologous transgene carrier, as adrenoleukodystrophy, gallbladder cystic fibrosis, glycogenosis, thyroprivia, sickle cell anemia disease, pendant Ademilson (Pearson) syndromes, other Pa Shi (Pompe ' s) disease, phenylketonuria (PKU), Tay-Sach disease, porphyria, maple syrup urine disease, homocystinuria, mucopolysaccharidosis, granulomatous disease and tyrosinemia, or treatment cancer, tumour or other illness.
In other embodiment, cell can use in homology or allos tissue regeneration or replacement therapy, include but not limited to that corneal epithelium defective, repair of cartilage, face go scar, mucous membrane, eardrum, intestines internal layer, nervous tissue (for example olfactory nerve unit of the auditory neuron at retina, basilar membrane place, olfactory sensory epithelium), also can be used in the burn and trauma repair of skin injury, during scalp (hair) is transplanted, or other are impaired, in the reconstruction of the organ of disease, tissue.
And a spot of stem cell and progenitor cell are round-robin in blood flow normally.In other embodiments, ectogenic stem cell or progenitor cell are collected by the separation property blood transfusion, and in this program, blood is drawn out of, and one or more compositions are optionally separated, and remaining blood is still refilled in the donor body.The foreign cell that obtains by the separation property blood transfusion increases with the inventive method, can get rid of the necessity of donating marrow fully.
In another embodiment, be used for replacement therapy after the chemotherapy with the hemopoietic progenitor cell of the inventive method amplification.Most of chemotherapeutics is a target and tumoricidal, can kill the cell in all propagation, just is in the cell in the cell fission.Because marrow is one of most active tissue of proliferation in vivo, hemopoietic stem cell just often is subjected to the damage and the destruction of chemotherapeutics, and therefore the generation of hemocyte reduces or stop.Chemotherapy needs to stop interimly so that patient's hemopoietic system can the supply hemocyte before chemotherapy once more.This may need one month or the longer time make before the immobilized stem cells hyperplasia, make leukocyte count reach once more the acceptable level of chemotherapy (when chemotherapy once more, medullary cell wound in damaged condition).
When hemocyte when chemotherapy is regenerated interval, tumour is also grown up if having time certainly, and because natural selection and chemotherapeutics become have more resistance.Therefore, the time of chemotherapy is long more, and pitch time is short more, and the probability of kill tumor is just big more.For shortening the interval of chemotherapy, the embryonic-like stem cell or the progenitor cell that break up according to the inventive method can be injected in patient's body.This therapy can reduce the time that the patient is in low quantity of leucocyte, can be therefore from chemotherapy, recovering more early.
In another embodiment, people's placenta stem-cell can be used to treatment or prevention genetic diseases, as the chronic granuloma disease.
5.6.3. inflammation is improved
Stem cell and progenitor cell, its differentiation is regulated according to method of the present invention, can be used as common anti-inflammatory preparation.The inventor finds, when the stem cell in for example Cord blood source and progenitor cell are in being transplanted to patient's body, can reduce or the reaction that diminishes inflammation fully.Therefore, in one embodiment, method of the present invention comprises that it is broken up the stem cell or the progenitor cell that have been subjected to one or more compounds adjustings of the present invention delivers medicine to a patient who produces inflammatory reaction, or among the patient of the reaction that may be inflamed.In specific embodiment, stem cell is an embryonic-like stem cell, and progenitor cell is a hemopoietic stem cell, particularly CD34 +Or CD133 +Progenitor cell.
The inventor also finds to use compound of the present invention, and IMiDs treatment just is individual, has stimulated those to regulate, improve or reduce the cells whose development and the differentiation of inflammatory reaction.Therefore, another embodiment of the invention comprises the patient that treatment produces inflammatory reaction, or the patient's of the reaction that may be inflamed method, comprises that the effective dose with one or more compounds of the present invention delivers medicine to described individuality.In another embodiment, this method contacts stem cell or progenitor cell with compound of the present invention before being included in and delivering medicine to described individuality, and the dose therapeutically effective with described cell delivers medicine to described individuality then.Still in another embodiment, the cell of so handling can deliver medicine to described individuality simultaneously with the dose therapeutically effective of one or more compounds of the present invention.
In other embodiment, other compounds are combined administration with compound of the present invention and/or cell also can reduce inflammation.For example, these additional compounds comprise steroid, as prednisone, or other on-steroidal anti-inflammatory agent, suppress acetylsalicylic acid (acetylsalicylic acid), ibuprofen, acetaminophen, the special inhibitor of cox-1 as cox-1/cox-2, or the derivative of any of these compound.These additional anti-inflammatory agents can be by standard the method transmission, as vein, part, intracutaneous or by inhalation, also can be simultaneously and compound of the present invention and/or cell transmission, or in the different time.
Above method can be used for treating that those are relevant with inflammation, by its some diseases that causes or cause.For example, this method can be used to treat the inflammation that is caused by the outer damage of trauma, and this method also can be used for treating the especially blood vessel relevant with surgical operation relevant operation such as healthy tissues transplanting, artificial blood vessel's transplanting, heart valve or caused inflammation of angioplasty or damage.This method also can be used for preventing the narrow or restenosis behind the cardiac valve procedure.This method also can be used for treating the inflammation that any disease causes, and includes but are not limited to heart disease, arteriosclerosis, sensitivity or hypersensitization disease, immune disorders, autonomous immune disorders such as sacroiliitis, or by infecting caused inflammation.Except treating already present inflammation, cell of the present invention and/or compound also deliver medicine to preventability individuality, to reduce the generation of inflammation.Its form as the preceding treatment of operation is particularly useful, and can reduce postoperative inflammatory reaction thus, improves the chance that the patient becomes work recovery, shortens hospital stays and sick and wounded phase.
The monitoring of the effect of above anti-inflammatory therapy can be finished by a lot of known methods, as visual inspection MRI or cat scan, and system or local temperature mensuration etc.Because a kind of protein of c reactive protein by name is a mark of inflammation, the minimizing of the amount of the c reactive protein that the validity of above methods of treatment just can be by individuality detects, particularly the part before the inflammation generation.
5.6.4. the generation of dendritic cell and granulocyte colony
Compound of the present invention can carry out administration specifically, to regulate stem cell and/or progenitor cell with respect to the development pathway of dendritic cell and along the differentiation of granulocytic development pathway.Similarly, cell of the present invention also can be in vivo or external adjusting to produce the dendritic cell or the granulocyte group of propagation.
Dendritic cell can be used as the medicament based on the treatment of immunity.For example, can cause the in vitro activation of the T cell of antigen-specific with dendritic cell and T lymphocyte and antigen protein in external co-cultivation.Activated T cells is carried out immediately from the immunne response (WO97/24438) of body administration to cause antigen-specific in vivo.In another embodiment, the T cell can be activated external, by the T lymphocyte with contact from the proteic dendritic cell of the direct antigen expressed of recombinant precursor.Activated T cells can be used as from body infusion (WO97/29183).
Become anti-albumen, cell or biological immunizing agent with specific polypeptide or protein fragments activated T cells, and they are special peptide or proteic sources.For example, dendritic cell may have the peptide of tumour-specific.The concrete application of external t cell activation that comes from dendritic cell, is disclosed in 963 and has carried out claim at U.S. Patent number 5,788 with the treatment prostate cancer.The dendritic cell of demonstration derived from bone marrow such as Mayordomo carry out pulse with synthetic tumour peptide and bring out protectiveness and curative antineoplastic immune (Nature Medicine1:1297-1302 (1995); J.Exp.Med., 183:1357-1365 (1996)).U.S. Patent number 5,698,679 have described and in vivo antigenic peptide have been passed to the domain-immunoglobulin fusion proteins that the targeting antigen that comprises dendritic cell presents cell (APCs).This identical method can be used to produce virus, bacterium or parasitic vaccine to be derived from virus, cell or parasitic peptide or antigen.
Dendritic cell also are for the therapeutic interventional target in the treatment of various immunologically mediated diseases.For example, dendritic cell have been implied to be the pathogenesis of acquired immune deficiency syndrome (AIDS) and a kind of important factor in the physiopathology person of possessing of HIV virus (for example, as).Referring to Zoeteweij etc., J.Biomed.Sci.5 (4): 253-259 (1998); Grouard etc., Curr.Opin.Immunol.9 (4): 563-567 (1997); Weissman etc., Clin.Microbiol.Rev.10 (2): 358-367 (1997).At U.S. Patent number 5,627, the in vitro method that the material standed for that the HIV of dendritic cell infects is eliminated in screening has been described in 025.In another embodiment, can use the dendritic cell inducing T cell to donor tissue or organ the anergy (referring to U.S. Patent number 6,375,950) in acceptor.
Granulocyte can be used for the granulocyte blood transfusion in treatment of infecting or prevention, the infection that for example bacillary newborn septicemia, the neutrophil leucocyte in the cancer patients are relevant and move latent infection among the patient who grows accepting marrow.Granulocyte also can be used in hypersensitive prevention and treatment.For example, can carry out deactivation with the granulocyte (that is, with the granulocyte of IgE antibody sandwich, some of them have specificity for anaphylactogen) of the inflammation-related of IgE mediation and be used to relax set up at the immunoreactive symptom of anaphylactogen (referring to U.S. Patent number 6,383,489).
Thereby, in one embodiment of the invention, granulocyte population in individuality is bred by progenitor cell of the present invention by a kind of method, this method comprises that wherein said amount is enough to induce described individuality by endogenous CD34 to the compound of the present invention of significant quantity on the described individual drug treatment +Cell produces various granulocytes.In another embodiment, the granulocyte population is bred by a kind of method in individuality, this method comprises described individual administration CD34 +Or CD133 +Progenitor cell, wherein said cell contacts at least 3 days with compound of the present invention, and to the described cell population of described individual administration.In another embodiment, by the amplification in individual of a kind of method, this method comprises with CD34 with the granulocyte population +Or CD133 +Progenitor cell population and compound of the present invention deliver medicine to above-mentioned individuality together, and the dosage of wherein said compound of the present invention is enough to cause that various described cell populations are divided into granulocyte.In the concrete scheme in above embodiment, described CD34 +Progenitor cell is CD34 +CD38 -CD33 +Cell.
5.6.5. other diseases and treatment of conditions
Stem cell that the present invention has broken up and progenitor cell, or compound of the present invention, also can be separately or combined utilization to treatment or prevent in the various other diseases.In certain embodiments, for example, disease or illness include but not limited to blood vessel or cardiovascular disorder, arteriosclerosis, diabetes, aplastic anemia, myelodysplasia, myocardial infarction, epilepsy, multiple sclerosis, apoplexy, hypopiesia, heartbeat stops, amyotrophy, inflammation, the forfeiture of the cognitive function relevant with the age, radiation injury, middle cerebral artery aneurysm, nerve retrograde affection, Alzheimer, Parkinson's disease, Lay Fu Shi disease, the AIDS dementia, the loss of memory, amyotrophic lateral sclerosis (ALS), the kidney local asphyxia, brain or chorda dorsalis injury, cardiopulmonary bypass, glaucoma, retinal ischemia, retina injury, lysosomal storage disease such as Tay-Sach disease, niemann-Pick disease, fabry disease, familial splenic anemia, hunter syndrome, what reins in syndrome, resemble other ganglioside deposition disease, mucopolysaccharidosis, glycogenosis, inborn errors of metabolism such as adrenoleukodystrophy, gallbladder cystic fibrosis, glycogenosis, thyroprivia, sickle cell anemia disease, pendant Ademilson syndromes, other Pa Shi disease, phenylketonuria (PKU), porphyria, maple syrup urine disease, homocystinuria, mucopolysaccharidosis, chronic granuloma, tyrosinemia, Tay-Sach disease, cancer, situation tumour or other pathology or that become knurl.
In other embodiments, cell of the present invention (for example, being exposed to the cell of compound of the present invention) can be used to treat any by the wound caused wound of wound of inflammation particularly.These diseases relevant with wound comprise central nervous system (CNS) damage, comprise the damage, CNS surrounding tissue of brain, the notochord damage to peripheral nervous system (PNS); Or the damage of health rest part.This wound may be caused by accident, maybe may be normal and abnormal result of therapy such as surgical operation or angioplasty.This wound may with blood vessel break or inaccessible relevant, as apoplexy or phlebitis.In specific embodiment, cell can be used for from body or allos tissue regeneration or replaces in treatment or the scheme, include but not limited to that corneal epithelium defective, repair of cartilage, face remove scar, mucous membrane, eardrum, intestines internal layer, nervous tissue (retina for example, the auditory neuron at basilar membrane place, the olfactory nerve unit of olfactory sensory epithelium) treatment, also can be used in the burn and trauma repair of skin injury, or other are impaired, in the reconstruction of the organ of pathology, tissue.
In a specific embodiment, disease or illness are aplastic anemia, myelodysplasia, leukemia, marrow illness or hematopoietic disease or illness.In another embodiment, the curee is the people.
5.7. pharmaceutical composition
The present invention includes pharmaceutical composition, it comprises potion and/or multi-agent one or more compounds of the present invention, and wherein said potion or multi-agent are effectively for single or multiple administering modes, and it is with adjusted or unadjusted people CD34 +Or CD133 +Progenitor cell or stem cell transplantation in the individuality before or after, to suppressing, regulate and/or regulate and control these stem cells and/or progenitor cell is divided into specific cell type, for example, hematopoietic lineage cell, particularly marrow pedigree cell can play good effect.At this, just as the elsewhere of the context of the invention, " individuality " represents any individuality of medicine or cell administration, for example, and Mammals, birds or Reptilia.
Therefore, in specific embodiment, described a times or the many multiple doses that delivers medicine to individual compound of the present invention regulated endogenous CD34 +Progenitor cell is divided into dendritic cell.In scheme more specifically, one times or many multiple doses have improved granulocytic quantity in the described individuality, this body and function described one times or the administration of many multiple doses.At another more specifically in the scheme, one times or many multiple doses have improved CD34 in the Mammals +CD38 -CD33 +Or CD34 +CD38 -CD33 -The quantity of progenitor cell, this Mammals is with described one times or the administration of many multiple doses.
In other embodiment, purpose CD34 +Or CD133 +Progenitor cell or stem cell transplantation go into to have in this people curee or patient's body who needs.After the transplanting, compound administration of the present invention is in people curee or patient, to regulate the differentiation of the purpose cell of implanting in vivo.In specific embodiment, these cells are divided into granulocyte in vivo.The embodiment that also has, the differentiation of intravital purpose progenitor cell of people curee or patient or stem cell is regulated by the administration of compound of the present invention in position.
Still in other embodiment, the invention provides pharmaceutical composition, it comprises isolating cord blood stem cell or progenitor cell, and they obtain amplification the hemopoietic progenitor cell of having broken up be exposed to the compound that suppresses the TNF-alpha active according to method of the present invention.In another embodiment, the invention provides the pharmaceutical composition that comprises Cord blood, described Cord blood is replenished stem cell or the progenitor cell with compound treatment of the present invention; In a concrete scheme, described stem cell or progenitor cell are by described compound differentiation.
In another embodiment, the invention provides pharmaceutical composition, it comprises one or more immunomodulator compounds of the present invention and stem cell of the present invention and/or progenitor cell.Said composition can be before administration preparation in 1,2,3,4,5,6,7,8,9,10,11 or 12 day, to regulate the differentiation of stem cell and/or progenitor cell along different growth/differentiation pathway.
Still in another embodiment, pharmaceutical composition of the present invention also comprises stem cell or progenitor cell self, and wherein said cell obtains differentiation according to method disclosed herein.Therefore, the invention provides a pharmaceutical composition that comprises a plurality of stem cells and/or progenitor cell, wherein said a plurality of stem cell or progenitor cell contact with one or more immunomodulator compounds of the present invention, and the differentiation that its concentration and time length are regulated and control described cell for described compound is sufficient.
Therefore, pharmaceutical composition of the present invention comprises the compound of the present invention that delivers medicine to individuality; With compound of the present invention or individually dosed in the cell of the present invention of individuality; And contact with compound of the present invention, deliver medicine to individual cell of the present invention.
The invention provides the method for treatment and preventing disease or illness, compound of the present invention by the drug treatment significant quantity or composition be to Mammals, preferred people, and the curee is can effectively regulate the CD34 that transplants or reside among the curee +Or CD133 +Propagation of progenitor cell or stem cell and/or differentiation.In one embodiment, the invention provides adjusting CD34 +And CD133 +Progenitor cell or differentiation of stem cells are to increase the method for the intravital granulocytic quantity of Mammals.In another embodiment, any CD34 that derives from +And/or CD133 +The cell lineage of progenitor cell or stem cell all can be by delivering medicine to Mammals, and especially people's compound of the present invention is regulated.Term used herein " Mammals " comprises any Mammals.Preferred Mammals needs this treatment and prevention.Mammiferous embodiment includes but not limited to, ox, horse, sheep, pig, cat, dog, mouse, rat, rabbit, cavy, monkey etc., more preferably people.
The administration of compound of the present invention can be system or partial.In most of the cases, will make system of compounds of the present invention ground discharge (that is, entering blood flow) to the Mammals administration.The method of administration comprises the enteron aisle approach, as per os, cheek, hypogloeeis and rectum; Topical is as through skin and intracutaneous; Also has the parenteral admin approach.The parenteral approach that is fit to comprises via hypodermic needle or tube injection, as the injection in intravenous, muscle, subcutaneous, intracutaneous, endoperitoneal, endarterial, intraventricular, the sheath, intraocular, the anterior chamber, and non-injecting pathway, as vagina, rectum or nasal administration approach.Preferably, compound of the present invention or composition are with oral administration.In specific embodiment, the zone that one or more compound administrations of the present invention need be treated in the part preferably.This can reach, for example, by the local infusion in the operation, overall applicability for example, is united use with wound dressing after operation, mode by injection, conduit, suppository, implantation, said implant is material porous, atresia or gel, comprises film, as ptyalectasis film or tunica fibrosa.
Compound of the present invention can for example be used parcels such as liposome, microparticle, microcapsule, capsule by general and off-gauge transfer system administration.For example, compound of the present invention and composition can place in the folliculus, particularly liposome and transmitted (referring to Langer, 1990, Science 249:1527-1533; Treat communicable disease and cancer with liposome, Lopez-Berestein and Fidler (editor) Liss, New York, pp.353-365 (1989); Lopez-Berestein, the same, the 317-327 page or leaf; Usually referring to as preceding).In another example, compound of the present invention and composition are transmitted in an in check release system.In one embodiment, can use pump (referring to Langer, supra; Sefton, 1987, CRCCrit.Ref Biomed.Eng.14:201; Buchwald etc., 1980, Surgery88:507 Saudek etc., 1989, N.Engl.J.Med.3:574).In another example, can use polymeric material (referring to, the medical use of in check release system, Langer and Wise (editor), CRC Press, Boca Raton, Florida (1974)); In check medicine biological effectiveness, the design of medicament production and characteristic, Smolen and Ball (editor), Wiley, New York (1984); Ranger and Peppas, 1983, J.Macromol.Sci.Rev.Macromol.Chem.23:61; Be also shown in Levy etc., 1985, Science228:190; During etc., 1989, Ann.Neurol.25:351; Howard etc., 1989, J.Neurosurg71:105).Still in another example, in check release system can place near the target region that needs treatment, for example, and liver, therefore also just only need part system's dosage (see, for example, Goodson, the application of in check release system in medical science, the same, volume 2,115-138 page or leaf (1984)).Also can use other the release system of in summary, discussing (Langer, 1990, Science249:1527-1533).When as the composition administration, compound of the present invention can be prepared with an amount of pharmaceutically useful vehicle or carrier, can provide for the suitable administering mode of Mammals.Term " pharmaceutically useful " is meant through federation management mechanism or state government to be checked and approved, or is listed in American Pharmacopeia or other suitable Mammalss especially in people's the generally acknowledged pharmacopeia.Term " vehicle " is meant thinner, auxiliary agent, vehicle or carrier, and compound of the present invention can be made them and deliver medicine to Mammals.These vehicle pharmaceutically can be liquid, and Ru Shui and oil comprise the oil that derives from oil, animal, plant or synthetics, as peanut oil, soybean oil, mineral oil, sesame wet goods analogue.Drug excipient can be analogues such as salt, Sudan Gum-arabic, gel, starch paste, talcum, Keratin sulfate, colloidal silica, urea.In addition, auxiliary, stablizer, thickening material, lubricant, tinting material etc. also can use.Preferably, when delivering medicine to Mammals, compound of the present invention and composition and pharmaceutically useful carrier, vehicle or thinner are aseptic.When compound intravenous administration of the present invention, water medium is preferred vehicle, as water, salt brine solution, D/W and glycerine solution.
Compound of the present invention can be made capsule, tablet, pill, pilule, lozenge, pulvis, granule, syrup, elixir, solution, suspension, emulsion, suppository or its extended release preparation, or any other suitable delivers medicine to mammiferous form.In a preferred embodiment, compound of the present invention and composition prepare with conventional procedure, just as the program of preparation human oral or intravenous (IV) drug.In one embodiment, pharmaceutically useful vehicle is a hard gelatin capsule.The suitable pharmaceutically useful vehicle and the embodiment of its preparation method are as describing in Remington: medicament science and practice, and Alfonso R.Gennaro edits, the .Easton of Mack publishing company, PA, the 19th edition, 1995,86,87,88,91 and 92 chapters are incorporated herein by reference.
The medicament forms of capsule, tablet, pill or any compression is preferably made in the oral medicine that compound of the present invention and composition are prepared.And when tablet or pill, but compound and composition need of coating continue the activated time to postpone them in GI degraded and absorption thereby can exceed or prolong.Osmotically active around the selectively permeable membrane drives the oral administration that compound also is fit to compound of the present invention and composition.In the platform of these back, absorbed by advancing compound at the liquid of capsule surrounding environment, described compound replaces the composition of promoting agent or promoting agent by the slit.These transmit platform can provide a kind of unordered basically transfer curve opposite with the description of snap-out release prepared product.Time lag material such as Zerol or Vinlub also can use.Oral compositions comprises standard excipients, vehicle and thinner, as Magnesium Stearate, sodium saccharinate, Mierocrystalline cellulose, magnesiumcarbonate, lactose, glucose, sucrose, sorbyl alcohol, N.F,USP MANNITOL, starch, Sudan Gum-arabic, Calucium Silicate powder, Microcrystalline Cellulose, polyvinylpyrrolidone, water, syrup and methylcellulose gum, preparation also can comprise lubricant, as talcum, Magnesium Stearate and mineral oil, wetting agent, emulsifying agent and suspension agent, sanitas such as methyl hydroxybenzoate and propyl ester.The preferred pharmacy level of these vehicle.Oral administration of compound of the present invention and composition can arbitrarily comprise one or more sweeting agents, as fructose, aspartame or asccharin; One or more seasoningss such as spearmint oil, wintergreen oil or cherry seasonings; Or one or more tinting materials, thereby provide pharmaceutically good to eat preparation.
Mainly depend on its characteristic and seriousness for treatment special illness or treatment of diseases effective dose, also can decide by the standard clinical techniques that the foundation practitioner has been done judgement.In addition, the interior detection of external or body can be used to help to identify optimal dosage.Certainly, can constitute the amount for the treatment of the compound of the present invention of going up effective dose and also depend on route of administration.In general, arrive about 20mg compound of the present invention about the about 0.001mg of per kilogram of body weight every day greatly for the oral administration suitable dosage ranges, preferably about 0.7mg is to about 6mg, and more preferably from about 1.5mg is to about 4.5mg.In a preferred embodiment, particularly human oral compound of the present invention about 0.01mg every day is to about 1000mg for Mammals, and more preferably from about 0.1mg is to about 300mg, and perhaps about 1mg takes separately or separately to about 250mg.Dosage as described herein refers to the total amount of taking medicine; That is to say that if more than a kind of compound of the present invention of taking, so preferred taking dose is suitable with the total amount that compound of the present invention is taken.Oral compositions preferably comprises the compound of the present invention of 10% to 95% weight.Preferred oral unit dosage form comprises pill, tablet and capsule, more preferably capsule.Usually these unit dosage comprise the about 0.01mg of compound of the present invention, 0.1mg, 1mg, 5mg, 10mg, 15mg, 20mg, 50mg, 100mg, 250mg or 500mg, preferably, each dose unit contains the about 5mg of compound of the present invention to about 200mg.
In another embodiment, compound of the present invention and composition can parenteral admins (for example, by in intramuscular, the sheath, intravenously and intra-arterial approach), preferred intravenously.Usually, the compound of the present invention and the composition that are used for intravenous injection are the aqueous solution that is dissolved in sterile isotonic, as water, salts solution, Ringer's solution, glucose solution.If need, composition also can comprise helping separates agent.The intravenous injection composition comprises that optionally local anesthetic such as lignocaine alleviate the pain of injection site.For intravenous injection, compound of the present invention and composition can the disinfectant lyophilized powder or encloses container in the enriching agent that dewaters and providing, as the injection or the anther sac of ampoule, reservoir shows the amount of promoting agent.Powder or enriching agent dissolve with the suitable aqueous solution earlier before intravenous injection.One ampoule sterilized water, salt brine solution or other suitable aqueous solution can dissolve before injection for powder or enriching agent.Perhaps composition can provide and administration with the pre-mixing form.Compound of the present invention or composition will be used as intravenous words, use the injection bottle packing that comprises pharmaceutical grade water, salt brine solution or other suitable media to it so long.
Rectal administration can be subjected to the influence of suppository, and suppository is prepared from by vegetables oil and other fatty alkali of traditional sucrose such as theobroma oil, modification.Suppository is to be prepared by known method with known form, for example referring to Remington: medicament science and practice, and Alfonso R.Gennaroed., the .Easton of Mack publishing company, PA, the 19th edition, 1995, the 1591-1597 page or leaf is quoted as a reference at this.
Prepare and deliver topical dosage forms, can use known through skin and intracutaneous transmission medium such as washing lotion, emulsifiable paste, ointment and transdermal delivery device such as patch.(Ghosh, T.K.; Pfister, W.R.; Yum, S.I. be through skin and local drug delivery system, InterpharmPress, Inc.p.249-297 quotes as a reference at this).For example, storage type patch design can be made up of the substrate film that is coated with layer of adhesive, includes the storage crack of compound of the present invention or composition, can separate (for example, U.S. Patent number 4,615,699 is quoted as a reference at this) by semi-permeable membranes and skin.The substrate layer that covers tackiness agent can extend providing one to seal with the concentric of skin along storage edge, and the maintenance storage is adjacent with skin.
The present invention also provides pharmacy packing material or medicine box, comprises that one or more is filled with the container of one or more compounds of the present invention.What preferably be associated with this container can be a kind of bulletin of the form of leaving with government organs, production, use or the sale of these bulletin regulation pharmacy or biology product, this bulletin has reflected the approval of production, use or marketing organization for human administration.In one embodiment, medicine box is made up of multiple compound of the present invention.In another scheme, medicine box comprises compound of the present invention and other biological is learned promoting agent.
Compound of the present invention was preferably detected before being used for human body in vitro and in vivo, to obtain required treatment or prophylactic activity.For example, vitro detection can be more effective with specific administration that determines whether compound of the present invention or compound Combined Preparation of the present invention.Also can be by using animal model system to measure compound of the present invention and whether medicine is effective.Other method is understood by some masterful technique persons, and is also contained in the scope of the invention.
5.8. use the inventive method to detect
Methodology described above is just checked IMiDs such as Actimid TMTo early progenitor cell such as CD34 +The influence of cytodifferentiation can be applied to any its purpose compound to the influence of differentiation of wanting to know.This can finish in several ways.
In one embodiment, compound can substitute Actimid simply TMOr any other compounds of the present invention.Here, CD34 +Progenitor cell and/or CD133 +Progenitor cell can allow progenitor cell to contact with various concentration with the purpose compound under the condition of cell proliferation orientation and/or differentiation fully and/or differentiation.Cultural method disclosed herein particularly at the disclosed cultural method of 5.4 joints, can use.Decide even if the effect of purpose compound is the variation of the cell mass that come by progenitor cell differentiation by assessment, this variation is by the change monitoring of any phenotype, but preferably by measuring having or not having and assess of cell surface marker.Resemble the inventive method, any time of having cultivated from the beginning all can adopt the purpose compound administration of single dose to obtain the cell of final differentiation.Alternatively, the purpose compound also can be in the propagation phase, differentiation phase or two administrations in period.The phenotype of progenitor cell variation cell preferred and contrast culture comes comparison in the proliferation/differentiation, as the cell of handling with DMSO.Specific purpose will influence propagation and differentiation, as, but be not limited to: the adjusting of growth fraction; The adjusting of differentiation ratio; Progenitor cell is to the adjusting of specific direction-sense precursor differentiation; Prevent differentiation to specific cell type; And promotion is to the differentiation of particular cell types.
In another embodiment, cultivate, breed and as above generation of differentiation, but purpose compound and progenitor cell are together with Actimid TMContact together.In this mode, the polyvoltine compound may be that synergitic effect can obtain measuring.It should be noted that especially any compound may not have or only have a bit the effect of propagation or differentiation separately, but and Actimid TMTime spent has but had obvious effects together.In another embodiment, any two kinds of purpose compounds all can contact with progenitor cell under culture condition, and as above, this condition allows progenitor cell proliferation and differentiation usually.Here, a preferred experiment, wherein precursor cell contacts with two kinds of purpose compounds, comprises a kind of a contact that contrasts in the wherein progenitor cell and described two kinds of compounds; One contrasts wherein cell and Actimid TMContact; One contrasts wherein that cell does not contact with compound, but contacts with DMSO.Once more, in dosage and the variation on action time all as described in 5.4 joints.
6. embodiment
6.1. embodiment 1: Thalidomide, Actimid TMAnd Revimid TMTo CD34 +The influence of progenitor cell differentiation
In following embodiment, Thalidomide (Thal), Actimid TM(" Actimid TM") and Revimid TMTo CD34 +The existing research of the influence of the generation of (hemopoietic progenitor cell) cytodifferentiation and colony forming unit.It should be noted that the result shows Actimid TMAnd Revimid TMCan be used for suppressing specifically the formation of red blood corpuscle colony (BFU-E and CFU-E), leukocyte increasing and thrombocyte form the generation of colony (CFU-GM) and strengthen the output of whole colony forming unit (CFU-Total) simultaneously.
Therefore the inventive method can be used for regulating the differentiation of stem cell, also can be used for stimulating the ratio of colony formation, and by the speed that improves the recovery of bone marrow transplantation and white cell and/or platelet production hematopoietic stem cell transplantation being produced provides important help.
Cord blood CD 34 +Progenitor cell is implanted in the 96 hole culture dish with the density of 1000 cells in every hole, replenishes 20% foetal calf serum and cytokine (IL-3, G-CSF and kit-ligand (R ﹠amp in IMDM; D Systems, Inc.).Cellular exposure is in Thalidomide (Thal), Actimid TMAnd Revimid TM, or DMSO (control compound), cultivated then 6 days.Cord blood CD 34 +Cell is implanted in the 96 hole culture dish with the density of 1000 cells in every hole, replenishes 20% foetal calf serum and cytokine (IL-3, G-CSF and kit-ligand (KL) (R ﹠amp in IMDM; D Systems, Inc.).After the cultivation, carry out cell dyeing, (FACS) carries out sorting with fluorescence-activated cell sorter.Gather in the crops the painted cell of 400 microlitres, be diluted to 1.0ml with the phosphate buffered saline (PBS) (PBS) that contains 1% foetal calf serum.Measure the impact effect that differentiation of stem cells is regulated by cell counting.The cytometer numerical value that obtains is presented among Fig. 1.
These results show that compound of the present invention is effective in the adjusting of hematopoiesis ancestral stem cell pedigree directional trend.So Actimid TMAnd Revimid TMCan be used for suppressing specifically the generation of red blood cell or red blood corpuscle colony (BFU-E and CFU-E), and increase the generation that white cell and thrombocyte form colony (CFU-GM), strengthen the output of total colony forming unit.Therefore the inventive method can be used for the differentiation of stem cell, also can be used to stimulate the ratio of special colony formation, also can produce hematopoietic stem cell transplantation by the speed that improves the recovery of bone marrow transplantation and white cell and/or platelet production important help is provided, they are by primordial stem cell coming towards the cell lineage directed differentiation of wanting to transplant.Pass through Actimid TMAnd Revimid TMTo CD34 +CD38-progenitor cell red blood corpuscle generates and being suppressed among Fig. 2 of amplification further describes.
6.2. embodiment 2: Thalidomide, Actimid TMAnd Revimid TMTo human cord blood CD 34 +The influence of cell proliferation and differentiation
In the following embodiments, Actimid TMAnd Revimid TMTo human cord blood (CB) monocyte propagation be divided into CD34 +The existing research of (hematopoiesis ancestral) cell.The Cord blood monocyte is that a blended cell mass comprises a small set of hematopoiesis ancestral (CD34 +) cell.The CD34 that this is little +The cell mass subclass comprises a group (about 1% total CB monocyte) CD34 +CD38 +Cell and even the CD34 of groupuscule (being less than total CB monocytic 1%) more +CD38 -Cell.Significantly, the result shows, compares Actimid with negative control with positive TMAnd Revimid TMCan significantly suppress or slow down the differentiation (Fig. 3-7) of hemopoietic stem cell or progenitor cell.
6.2.1. material and method
CB CD34 +Cell is with 4 * 10 4Cell is implanted in 24 orifice plates for every milliliter, adds cytokine (1L3, GCSF and Kit-ligand) (R ﹠amp in 20%FCSIMDM (calf serum/Iscove improvement Dulbecco substratum); D Systems, Inc.).Thalidomide (Thal), Actimid TMOr Revimid TMBe included in the substratum with three kinds of different concentration: 5 μ g/ml, 1 μ g/ml and 0.3 μ g/ml.The DMSO of same volume uses in contrast.Negative control does not add any compound (representing with " nothing ") in Fig. 3-7.Cell is at 37 ℃, 5%CO 2Cultivated 7 days in the incubator.Collect the cell in every hole then.
Every porocyte sum is measured (Fig. 3-7) by counting in CELL-DYN1700 (Margaret Abbott diagnosis) and with the expression of FACS (fluorescence-activated cell sorter) staining analysis CXCR4, CD45, CD34, CD38, CD11b and the expression of Gly-A.
CB cell (CB2276 and CB2417) single culture, mensuration and analysis (table 1) from two different donors.
6.2.2. result and discussion
Thalidomide, Actimid TMOr Revimid TMThe CD34 that the pair cell factor stimulates +The influence of cell amplification detects.As shown in Figure 4, Thalidomide, Actimid TMAnd Revimid TMTo lacking IL-3, the CD34 that Kit-ligand (KL) and G-CSF cultivate +The propagation of cell is compared with negative control (" nothing ") significantly not to be influenced.Yet, Thalidomide, Actimid TMAnd Revimid TMCompare as if the growth of inducing cell slightly with the DMSO contrast.The propagation of considering DMSO pair cell in the experiment generally has negative interaction, and these results show Thalidomide, Actimid TMAnd Revimid TMCompound is to having IL-3, the CD34 that Kit-ligand (KL) and G-CSF cultivate +The propagation of cell has hormesis, owing to be that DMSO with equivalent does carrier.Aspect this, Actimid TMAnd Revimid TMHas bigger effect than Thalidomide.
Thalidomide, Actimid TMAnd Revimid TMThe influence of pair cell expression of differentiation is determined by facs analysis surface protein CXCR4 and CD34 (Fig. 3 and 4).Actimid TMAnd Revimid TM, but be not Thalidomide, demonstrate the restraining effect that CXCR4 expresses.Actimid TMCompare Revimid TMBy stronger effect.
As for surface protein CD34 +, Actirnid TMCause the CD34 of cultivation at CB2276 and CB2417 +The rise of cell (increasing propagation).Yet, Thalidomide and Revimid TM, only in another donor, do not demonstrate similar effect at one.What is interesting is, use Actimid TMAnd Revimid TMTogether in the cell of Chu Liing, most of CD34 +And CD34 -Cell is CD38 -, the cell mass of handling with DMSO control treatment and Thalidomide then mainly is CD38 +This shows Actimid TMAnd Revimid TMCan be used for suppressing specifically the formation of red blood cell or red blood corpuscle colony (BFU-E and CFU-E), and increase the generation that white cell and thrombocyte form colony (CFU-GM), strengthen the output of total colony forming unit.Thereby method of the present invention can be used to regulate the differentiation of stem cell, and also can be used for stimulating colony forming efficiency, hematopoietic stem cell transplantation produced important help is provided by improving speed that bone marrow transplantation and white cell and/or platelet production recover.Referring to following table 4.
Thalidomide, Actimid TMAnd Revimid TMThe effect of pair cell expression of differentiation determines that by the facs analysis cell surface protein these surface proteins are CD34 +CD38 -To CD34 +CD38 +Or CD11b +The result places table 1.
Table 1: the embodiment of the influence that pair cell differentiation and CD34, CD38 and CD11b cell surface marker are expressed in the hemopoietic progenitor cell in Cord blood source
The CD34+CD38-/CD34+CD38+ cell colony
CB2276
　 None. DMSO Thal Actimid TM Revimi
5μg/ml 1.2/6.3 2.7/3.7 3.5/6.5 17.3/0.2 d TM
1μg/ml 1.5/8.5 2.6/5.3 1.0/3.8 15.0/0.2 10.3/1
0.3μg/ml ND 1.5/5.7 3.2/15.1 5.9/0.2 9.8/1.7
CB2417
None DMSO Thal Actimid TM Revimid TM
5μg/ml 0.5/5.7 0.8/5.2 1.1/3.0 14.7/0.1 4.2/0.3
1μg/ml 0.5/4.9 0.7/3.9 1.0/3.8 12.0/0.8 3.8/0.6
0.3μg/ml ND 0.5/4.4 0.8/5.0 5.9/0.2 3.4/0.9
CD11b +Cell colony
CB2276
None DMSO Thal Actimid TM Revimid TM
5μg/ml 11.7％ 5.0％ 8.1％ 1.6％ 4.8％
1μg/ml 9.1％ 7.3％ 6.2％ 3.8％ 8.6％
0.3μg/ml ND 7.0％ 7.6％ 6.9％ 13.3％
CB2417
None DMSO Thal Actimid TM Revimid TM
5μg/ml 12.0％ 7.2％ 6.5％ 2.5％ 5.5％
1μg/ml 7.2％ 5.3％ 5.2％ 3.9％ 8.2％
0.3μg/ml ND 5.1％ 7.8％ 8.4％ 11.2％
At CD11b +Not significant variation the on the size of cell mass, and for Revimid TM, under low concentration, observe bigger CD11b +Cell mass.
Yet the CD11b expression levels is at Actimid TMAnd Revimid TMDescend in the cell of two processing, measure demonstration CD11b expression by average immunofluorescence (MIF) and be suppressed.This just points out CD11b +Cell is at Actimid TMAnd Revimid TMExist and when cultivating, be in a low differentiation state.
6.3. embodiment 3:Actimid TMAnd Revimid TMInfluence to human cord blood MNC cell
Among the embodiment formerly, demonstrate at cord blood CD 34 +In the cell, Actimid TMAnd Revimid TMObviously the expression of downward modulation CXCR4 increases CD34 +CD38 -Cell mass.In this embodiment, demonstrate Actimid TMAnd Revimid TMIn Cord blood monocyte (MNC), has similar activity.
6.3.1. material and method
Through freezing preservation of standard method and the Cord blood MNCs that thaws, by standard Ficoll separation method it is separated, and with 0.5 * 10 6Cell/ml cultivates three parts in 24 orifice plates in the 20%FCS-IMDM that contains somatomedin (IL6, KL and G-CSF respectively are 10ng/ml).Experimental group is None (having only cytokine), DMSO (1.7 μ l), Actimid TM(5.0 μ g are in 1.7 μ lDMSO), Revimid TM(5 μ g are in 1.7 μ lDMSO).To cultivate week back cultured cells collection, by the FACS staining analysis.The result is summarised among table 2 and Fig. 8-12.Data with the mean value of three separate wells+/-SD represents.
Table 2
None DMSO Actimid TM Revimid TM
Total cell count (1 * 10 6) 0.50+/-0.10 0.30+/-0.17 0.223+/-0.06 0.30+/-0
CD34(％) 2.50+/-0.33 2.73+/-0.07 3.31+/-0.64 2.34+/-0.22
Total CD34+ cell count 7933+/-7310 8133+/-4623 7800+/-2600 7166+/-802
CD34+CD38-(％) 0.05+1-0.01 0.06+/-0.04 1.70+/-0.22 0.80+/-0.29
CXCR4+CD45+(％) 8.7+/-0.54 12.1+/-1.30 2.9+/-0.5 3.6+/-0.9
CXCR4+CD45-(％) 0.48+/-0.15 0.66+/-0.04 4.27+/-0.23 3.28+/-0.89
6.3.2. result and discussion
Shown in table 2 and Fig. 8-12, with DMSO, Actimid TMOr Revimid TMThe MNCs total cellular score of cultivating will be less than control group (" None, " have only somatomedin).This may be owing to the effect of DMSO.Demonstrate higher CD34 with the MID1 cultured cells than other groups +Cells ratio, and CD34 +Total cellular score is similar in all groups.CD34 +CD38 -Cell count is apparently higher than using IMJD1 and Revimid TMThe cell of handling is with the CD34 with the compound treatment purifying +The unanimity as a result of cell gained.Acceptablely be CD34 +CD38 -Cell is the hemopoietic progenitor cell of few differentiation, its after transplanting with than CD34 +CD38 +The higher efficient of cell transplant with propagation (.1998 such as Dao, Blood 91 (4): 1243-55; .1994 such as Huang, Blood 83 (6): 1515-26).
As if DMSO can be in the expression of Cord blood MNCs moderate stimulation CXCR4.When comparing with two control groups, Actimid TMAnd Reviniid TMAt CD45 +Obviously suppressed the expression of CXCR4 in the cell.
Using Actimid TMAnd Revimid TMHandle most CXCR4 in the cultured cells +Cell is the CD45 feminine gender.Using Actimid TMAnd Revimid TMIn the cell of handling, this cell mass is high significantly.
The result shows Actimid TMAnd Revimid TMIs useful to offset cryopreservation, to melt and/or be exposed to the freezing agent adjusting stem cell aspect the deleterious effect of stem cell.The result shows that further DMSO is to CD34 +And CD14 +The inhibition of cellular products can be by using Actimid TMOr Revimid TMHandle and offset, it has improved CD34 +And CD14 +The multiplication capacity of cell.
6.4. embodiment 4:: Thalidomide, Actimid TMAnd Revimid TMInfluence to monocyte output
6.4.1. materials and methods
The human cord blood CD 34 of purifying +Cell is (greater than 90%CD34 +) with 4 * 10 4Cell/ml cultivated 14 days in the moist incubator of the 20%FCSIMDM substratum that has added cytokine (IL3, IL6, G-CSF, KL and Epo), 37 ℃, 5%CO2.Experimental group is formed by following group, (i) does not add DMSO or compound (" None "), and DMSO is (ii) only arranged, and (iii) Thalidomide is dissolved among the DMSO, (iv) Actimid TMBe dissolved among the DMSO and (v) Revimid TMBe dissolved among the DMSO.Collecting the part cell uses in conjunction with the CD34-PE of monoclonal antibody with in conjunction with the CD14-FITC of monoclonal antibody and dyes.
6.4.2. result and discussion
The result is presented in " None " group, all only has 0.95% to be CD34 in the cell +In the DMSO treatment group, 0.17% cell is CD34 +, show that DMSO is to CD34 +Amplification has negative interaction with preservation.In the Thalidomide treatment group, 0.24% cell is CD34 +, it is not significantly different with the cell that DMSO handles.But at Actimid TMAnd Revimid TMTreatment group has higher CD34 +The per-cent of cell (being respectively 18.7% and 7.1%).(also referring to the experimental result of describing in Figure 13 and the appended legend).
CD14 is a monocytic mark.In " None " group, 11.5% cell is CD14 +, and in the DMSO treatment group, the 3.7%th, CD14 +, show that monocytic generation reduces.As the embodiment that top CD34 expresses, the Thalidomide treatment group is similar to the result of DMSO treatment group.Because Actimid TMAnd Revimid TMTreatment group also is exposed to DMSO, and can reason out the monocyte output that is suppressed by DMSO can be by using Actimid TMOr Revimid TMHandle and overcome.
6.5. embodiment 5: use Actimid TMThe effect of the karyocyte that derives from Cord blood and placenta that pre-treatment has been transplanted
Experimental results show that and use Actimid TMPre-treatment has increased the survival of placenta karyocyte (PLNC), Cord blood karyocyte (UCBNC) and the medullary cell (BMNC) transplanted.
6.5.1. materials and methods
Placenta karyocyte (PLNC), Cord blood karyocyte (UCBNC) and medullary cell (BMNC) obtain from people's donor.PLNC and UCBNC obtain from placenta and Cord blood with the described method of top 5.4 joints.
By being added with 2% people CB serum and 10g/mlActimid TMDMEM in incubation came pretreatment cell in 24 hours.Washed cell is suspended in it in autologous plasma then, and intravenous administration is in being subjected to body maturation SJL/L mouse (Jackson laboratory), and it suffers from the marrow excision that produces by the radiation (900cGy) that causes death according to standard method.This radiation is better than radiation back 50 days 90% cause death (Ende etc., 2001, LifeSciences69 (13): 1531-1539; Chen and Ende, 2000, J.Med.31:21-30; Ende etc., 2000, LifeSci.67 (1): 53-9; Ende and Chen, 2000, Am.J.Clin.Pathol.114:89).
6.5.2. result and discussion
Use Actimid TMThe effect of the PLNC that pre-treatment has been transplanted, UCBNC and BMNC is presented in the following table 3.Seen in table 3, Actimid TMPre-treatment has increased the survival of placenta karyocyte (PLNC), Cord blood karyocyte (UCBNC) and the medullary cell (BMNC) transplanted.
Table 3
The # of # raying at the 50th day at the 50th day
Experimental group is handled the initial body weight of animal % of animal dead
1 PLNC5 * 10 6Intravenously 31 84
2 PLNC50 * 10 6Intravenously 30 89
3 PLNC5×10 6+Actimid TM 4 0 102
The intravenously pre-treatment
4 UCBNC100 * 10 6Intravenously 30 81
5 UCBNC10×10 6+Actimid TM 3 0 84
The intravenously pre-treatment
6 UCBNC10 * 10 6Intravenously 1 78
7 BMNC0.5×10 6 3 3 79
8 BMNC5×10 6 3 3 74
9 BMNC50×10 6 3 2 83
10 contrasts, 12 11 N/A
Abbreviation:
PLNC: placenta karyocyte
UCBNC: Cord blood karyocyte BMNC: medullary cell
N/A: inapplicable
6.6.CD34 +The adjusting of progenitor cell differentiation
6.6.1. material and method
Marrow and cord blood CD 34 +Progenitor cell obtains from Clonetics, cultivates in Iscove ' sMDM that contains SCF, Flt-3L, GM-CSF and TNF-α and BIT95000 (stem cells technology) 6 days, cultivates 6 days having under GM-CSF and the TNF-α then again.
Cell surface phenotype analytical: united the mAbs pair cell at the 6th day and the 12nd day with FITC and PE and carry out two processing (30 minutes, 4 ℃) of dying.The antibody that uses is from BDPharmingen:CD34 (PE), CD38 (FITC), CD33 (FITC), CD1a (FITC), CD86 (PE), CD14 (PE), CD83 (PE), CD54 (PE), CD11b (PE), CD (PE), HLA-DR (PE), CD15 (FITC) with from the CD133 (PE) of Miltenyi.Fluorometric analysis is being carried out on FACS wandering cells counter in 10,000 results of acquisition (Coulter) back.Result displayed is the representative of four independent experiments.
Apoptosis detects: according to producing specification sheets, use annexin V-FITC associating iodate third ingot (BD Pharmingen apoptosis detection kit I) dyeing can measure the exposure of phosphatidylserine.
Phagolysis: the endocytosis activity of cell can be analyzed by measuring the picked-up of FITC-dextran.Cell 37 ℃ of incubations 1 hour in the perfect medium of 1mg/ml dextran-FITC (Sigma), 4 ℃ of incubations 1 hour are as negative control.Result displayed is the representative of two independent experiments.
T cell proliferation detects: cultivate after 13 days, collect to come from CD34 +The DC cell, with ametycin (50 μ g/ml, Sigma) handle after, with doing to the ripe CD3 of allos +The irritation cell of T cell, the former is from healthy volunteer's peripheral blood lymphocytes (PBMCs) purifying.CD3 +The T responsive cell is with 5 * 10 4The concentration of cells/well is used.Irritation cell joins in the T cell in black 96 hole plates with dose gradient, and this clearly bottom organizes culture plate to can be used for chemiluminescence detection.Cultivation is to carry out in the RPMI1640 substratum that has added 10% hot deactivation FBS, glutaminate and penicillin-Streptomycin sulphate.Cultivate after 6 days, measure the propagation of cell according to manufacturing instructions with BrdU chemiluminescence detecting method (Roche, Nutley NJ).The result shows to cultivate the mean value ± SD that obtains from three times.
6.6.2. result and discussion
Actimid TMThrough finding obviously to change the growth of the DC that derives from the CD34 progenitor cell.In order to study Actimid TMTo the effect that DC generates, CD34 +Progenitor cell is being with or without Actimid TMCultivate 12 days one-period in amplification and ripening stage (the 1st to the 12nd day) under (1 μ M), or cultivate 6 days one-period (Figure 14) in the ripening stage (the 6th to the 12nd day).Added Actimid from the 1st day to the 12nd day TMSuppress the acquisition (Figure 15) of DC phenotype, rolled up CD34 +CD38 -The group has changed CD34 +CD38 -Cell is to CD34 +CD38 +The normal differentiation of cell (Figure 16).Yet, Actimid TMThe CD34 that handles +Cell has obtained CD33 marrow sample mark really, and these cells presented CD34 in the time of the 6th day +CD38 -CD33 +Phenotype.Actimid TMAlmost completely hindered CD1a at the 6th day +The generation of cell, especially two positive CD86 +CD1a +The generation of cell.This two positive group is considered to the precursor of epidermis pancreas islet DC.Actimid TMAlso reduced CD14 +The generation of CD1a cell, it can make corium DC and monocyte/macrophage produce.Early progenitor cell group (CD34 +CD38 -Cell) increase and marrow DC progenitor cell (CD1a +CD14 -And CD1a -CD14 +Cell) retardance is a dose-dependently, at Actimid TMReach maximum value (Figure 17) when being 1 μ M.This effect is a reversible, if CD34 +Progenitor cell Actimid TMCultivated at least 3 days, and only observed and hindered CD34 differentiation path (Figure 18).
At the 0th day Actimid to the 6th day multiple doses TMStrengthened CD34 +Group's increase (Figure 19 A).
Actimid is being arranged TMThe time CD34 that cultivates +Progenitor cell is the same expression (Figure 20) that shows the reduction of costimulatory molecules (CD86, CD80) at the 12nd day.The CD54 adhesion molecule is along with CD54 BrightThe expression and the CD54 that reduce DimThe expression that the group increases and change (Figure 21).The HLA-DR molecule be expressed in Actimid TMThe CD34 that handles +Reduce in the progenitor cell.
When after not handling cultivation in 0-6 days, added Actimid at the 6th day TMAnd work as CD1a +The group produces Actimid TMStrengthened CD1a +Group's lasting (table 1).Used Actimid at the 12nd day TMThe culture of handling comprises more relatively CD1a than the DMSO contrast +Precursor cell.CD34 at 6 days +Add Actimid in the cell that has broken up TMReduced CD14 quite a lot ofly +The expression of the generation of precursor cell and costimulatory molecules (CD86, CD80).
Table 1. had Actimid at the 6th day to the 12nd day TMExist down, by CD34 +Progenitor cell produces DC at the 12nd day phenotypic characteristic
The 6th day to the 12nd day Actimid of the 12nd day DMSO TMThe 6th day to the 12nd day
CD1a + 8.5％ 11.5％
CD14 + 19.0％ 8.0％
CD86 + 28.8％ 18.5％
CD80 + 19.6％ 13.8％
Actimid TMWhether promote granulocytic differentiation: produce retardance and change with different marrow differentiation path and interrelate for measuring DC, we have detected the expression of granulocyte mark CD15.Surface molecular CD15 is having Actimid TMThe CD34 that cultivates +Expression in the progenitor cell is (Figure 22) that increases.Under the situation that the cytokine mixture that can promote the DC differentiation is arranged, add Actimid TMAmplification/the maturation that makes progenitor cell is to the more phenotype transfer of granulocyte sample.We have also studied departing from the marrow differentiation, by detecting the expression of two marks: CD11c, be that islet cells and mesenchyme DC express by marrow DC progenitor cell, and CD15 are expressed by the grain progenitor cell.CD11c +CD15 -Group minimizing with at CD11c -CD15 +Increase be associated (Figure 19 B) same period among the granulocyte group.Be Actimid interestingly TMMultiple dosing promoted transformation to the granulocyte pedigree.
It is not to be caused death and mediated by specific DC progenitor cell that DC generates retardance: mediate CD34 for determining that the DC progenitor cell reduces whether to be caused death by specificity +Progenitor cell is cultivated 6 days stage under the situation that SCF, Flt-3L, GM-CSF and TNF-α are arranged.At the 6th day, with magnetic cell sorter (Miltenyi) separation of C D1a +CD14 and CD1a -CD14 +Cell (DC progenitor cell).The purifying group is having GM -CSF and TNF -α is with or without Actimid TMCultivated in addition 2 days when (1 μ M).Actimid TMHandle, at annexin V +-PI -(early apoptosis) and annexin V +-PI +(apoptosis in late period) group level does not significantly increase (Figure 23).
Result from CD34 +The DC functionally active of progenitor cell changes: derive from CD34 +Progenitor cells is with cytokine and be with or without Actimid TMCultivate, its phagocytic activity is by the dextran mannose receptor mediation in the 12nd day -The FITC endocytosis detects.When added Actimid from the 1st day to the 12nd day TM, comparing phagocytic activity with the DMSO control group has very strong decline (Figure 24).When added Actimid from the 6th day to the 12nd day TM, compare phagocytic activity quite (Figure 25) with the DMSO control cells.
CD34 +Cell is with cytokine and be with or without Actimid TMCultivate, its antigen presentation ability (APC) is induced CD3 by measuring them at the 12nd day with mixing white cell reaction (MLR) +The ability of allos T cell proliferation and assessing.Added Actimid when the 1st day to the 12nd day TM, compare CD34 with the DMSO contrast +Cell shows the ability of the stimulation T cell proliferation of reduction.On the contrary, added Actimid when the 6th day to the 12nd day TM, stimulate the ability of T cell proliferation and suitable (Figure 27) of DMSO control cells.CD34 +The normal differentiation pathway of cell is described in Figure 28.
These results show, Actimid TMWeakened CD34 significantly +Progenitor cell is divided into dendritic cell.Thereby, Actimid TMThe cell of handling presents the APC ability of low phagocytic activity and reduction.The more important thing is Actimid TMIncreased early stage hemopoietic progenitor cell, CD34 +CD38 -Cell.These early stage hemopoietic progenitor cell have demonstrated in the NOD-SCID mouse models can better transplant and rebuild (Tamaki etc., J.Neurosci.Res.69 (6): 976-86 (2002)).And, Actimid TMMake CD34 by the marrow differentiation to the conversion of granulocyte pedigree +The differentiation of cell departs from, even when cytokine pressure helps dendritic cell differentiation.In addition, find Actimid TMTo CD34 +Cell does not have toxic action, can not weaken the ability of cell proliferation.The promotion of the adjusting of dendritic cell function and granulocyte differentiation can be to have significant therapeutic effect in various cancers, immune disorders, communicable disease, organ transplantation and reproducibility medical science.
Referring to the summary of Figure 29 for above figure.
6.7.Actimid TMRegulate CD133 +The progenitor cell differentiation
Multiple doses Actimid TM, except strengthening CD34 +The group increases, and also increases the expression of CD133, and it is usually by CD34 BrightHemopoietic progenitor cell and some primary CD34 -Expression of Subsets (Figure 19 A, 19B).Actimid TM, by enrichment CD34 +CD133 +Primitive hematopoietic cell recovers the tool clinical meaning to hematopoiesis after the stem cell transplantation.In addition, CD133 +Stem cell can produce the endotheliocyte pedigree, and wound healing is had contribution.Multiple doses Actimid TMCan not aggravate the retardance that pancreas islet DC precursor cell generates.
6.8. come from marrow (BM) Sca +The generation of the mouse dendritic cell of hemopoietic stem cell
6.8.1. materials and methods
The mouse marrow that is come by inbred C57BL/6 mouse obtains from Clonetics.Enrichment hematopoiesis Sca +Lin -Progenitor cell uses SpinSep mouse progenitor cell enrichment mixture (stem cells technology), BIT95000 (stem cells technology) being arranged and cultivation being arranged among Iscove ' sMDM of mouse somatomedin SCF, Flt3L, GM-CSF and M-CSF 9 days, promotes Sca +Cell amplification and DC precursor phenotype cultivate in addition in GM-CSF and TNF-α are arranged then that to impel cell over 3 days be immature DC phenotype.Referring to Figure 30.The Sca of enrichment +Lin -Cell had DMSO (0.1%), Actimid since the 0th day TMBe that 10 μ M or all-trans retinoic acid (ATRA) (ICN biomedicine) are to cultivate among the 10 μ M.Compound added in the cell at the 0th day and the 9th day.
Mouse cell surface phenotype analytical: the mouse cell was united mAbs pair at the 9th day and the 12nd day with FITC and PE and is dyed processing (14 minutes, room temperature).The antibody that uses is from BD Pharmingen; Sca (PE), CD11b (FITC), Gr-1 (FITC), CD86 (PE), CD14 (PE), CD80 (PE), I -Ab (PE), CD40 (PE) and from the CD32.1/16.1 (FITC) of Miltenyi.Fluorometric analysis is being carried out on FACS wandering cells counter in acquisition 10,000 results (Coulter) back.
6.8.2 result and discussion
Actimid TMDerive from Sca through finding to change +The growth of the mouse DC of progenitor cell.Present DC precursor cell phenotype at the 9th day cell, the high surface expression of dendron shape/marrow shape mark CD32/16 (Fc acceptor), CD11b, CD80, I-A bWith the low expression of CD86, pedigree mark such as CD14 and Gr-1 (Figure 31) do not express.Actimid TMBe shown to the 9th day pair cell surface markers and express not obviously influence, and ATRA demonstrates CD80, I-Ab and Sca +The obvious downward modulation of expressing (data do not show).Yet, by the 12nd day, Actimid TMDemonstrate the downward modulation that CD86 and brightI-Ab express, the rise (Figure 32) that CD11b expresses.ATRA demonstrates similar but compares Actimid TMRemarkable influence more.In addition, Actimid TMDemonstration does not influence and these molecules of the obvious downward modulation of ATRA demonstration (not demonstration) the expression of CD40 and CD80.
These results suggest Actimid TMExpression by downward modulation CD86 and MHCII suppresses the differentiation of DC progenitor cell to immature DC.The effect of compound does not resemble in the artificial blood progenitor cell viewed so noticeable, this and Actimid TMLow active similar in external and other model body of mouse.Actimid TMEffect more not obvious than ATRA, the latter is the teratogen of mouse.
6.9. the application that the differentiation of compound except that IMiD is detected
Methodology described above is just checked ImiD such as Actimid TMTo early progenitor cell such as CD34 +The influence of cytodifferentiation, any purpose compound of understanding is wanted in applicable influence to differentiation.Extend to the example of other compounds as this detection method, with respect to contrast (DMSO processing) cell, we have compared vitamin A acid (ATRA) and acetylsalicylic acid and Actimid TMTo CD34 +Cell is to the influence of the differentiation of DC pedigree.The research vitamin A acid is because effect, the use in some oncotherapies of its on cell proliferation and differentiation are known, and its known teratogenic effect.On the contrary, we study acetylsalicylic acid is because it is general anti-inflammatory medicaments, does not have the immunomodulator characteristic.CD34 +Progenitor cell is having SCF, Flt-3L, GM-CSF and TNF-α, is with or without in the compound period of cultivating 6 days, and the 6th day result is presented on that (arrow that makes progress is represented the increase of cell mass in the following table 2; The arrow that descends is represented to reduce).
Table 2. Actimid TM, vitamin A acid and acetylsalicylic acid be to CD34 +The comparison of cytodifferentiation influence.
Cell mass Actimid TMAlltrans RA acetylsalicylic acid
CD34 +CD38 -Not ↑ ↓ do not change
CD34 -CD38 +Not ↓ ↑ do not change
CD1a +CD14 -Not ↓ ↓ do not change
CD1a -CD14 +Not ↓ ↓ do not change
CD15 +Not ↑ ↑ do not change
In the literature, other medicine has shown the differentiation that can regulate cell, and for example, nearest bibliographical information reflunomide is to from CD34 +The adjusting of the DC that progenitor cell generates.Its description is different from Actimid TMBe accompanied by CD1a +Group's increase and CD14 +Group's minimizing.
6.10. embodiment 10: be divided into inducing of particular cell types
By being exposed to somatomedin, cord blood cell and/or embryonic-like stem cell can be induced and be divided into particular cell types.Being used for the inductive somatomedin includes but not limited to: GM-CSF, IL-4, Flt3L, CD40L, IFN-α, TNF-α, IFN-γ, IL-2, IL-6, vitamin A acid, basic fibroblast growth factor, TGF-β-1, TGF-β-3, pHGF, Urogastron, cardiotropin-1, angiotensinogen, angiotensin I (AI), angiotensin II (AII), AIIAT 2Type 2 receptor antagonists, or analogue or its fragment.
6.10.1. be divided into neuronic inducing
This embodiment has described and has induced cord blood cell and/or embryonic-like stem cell to be divided into neurone.Following scheme is applied to induce Neural Differentiation:
1. placenta stem-cell is grown in the substratum before inducing, is made up of DMEM/20%FBS and 1mM beta-mercaptoethanol.
2. discard the substratum before inducing, cell cleans with PBS.
3. add the nerve-inducing substratum that comprises DMEM and 1-10mM beta-mercaptoethanol.As selection, the inducing culture that comprises DMEM/2%DMSO/200 μ M butyric ester hydroxyl anisole can use to strengthen the efficient of Neural Differentiation.
4. in certain embodiments, form and change molecule can betide and be exposed to behind serum free medium and the beta-mercaptoethanol 60 minutes.(Woodbury etc., J.Neurosci.Res.61:364-370).RT/PCR also can be used to the expression of definite for example trk C and nerve fiber heavy chain gene.
6.10.2. be divided into inducing of adipocyte
This embodiment has described and has induced cord blood cell and/or embryonic-like stem cell to be divided into adipocyte.Scheme below using is induced living fat differentiation:
1. placenta stem-cell is grown in MSCGM (Bio Whittaker) or is supplemented with among the DMEM of 15% Cord blood serum.
2. use inducing/keeping of three cycles.Each cycle is made up of following: carry steatogenesis inducing culture (Bio Whittaker) to placenta stem-cell, culturing cell 3 days (at 37 ℃, 5%CO 2), then be that cultivation in 1-3 days keeps substratum (Bio Whittaker) at steatogenesis, used inducing culture comprises 1 μ M dexamethasone, 0.2mM indomethacin, 0.01mg/ml Regular Insulin, the high sugar of 0.5mM IBMX, DMEM-, FBS and microbiotic.
3. induce completely at 3/hold period after, cell keeps cultivating 7 days in the substratum at steatogenesis in addition, changes substratum in every 2-3 days.
4. steatogenesis can be determined by the generation of most endochylema inner lipid vesicles, and these vesicles can use lipotropy dyestuff oil red O to observe at an easy rate.Can use RT/PCR to detect the expression of checking lipase and fatty acid-binding protein gene.
6.10.3. inducing of differentiating cartilage-forming cell
This embodiment has described cord blood cell and/or embryonic-like stem cell is induced differentiating cartilage-forming cell.Scheme below using induces cartilage to form differentiation:
1. placenta stem-cell remains on MSCGM (Bio Whittaker) or is supplemented with among the DMEM of 15% Cord blood serum.
2. placenta stem-cell is distributed in the aseptic polypropylene test tube.Cell carries out centrifugal (150 * g, 5 minutes), and the full cartilage that toos many or too much for use forms substratum (Bio Whittaker) and cleans twice.
3. last is all over after washing, and cell suspension forms in the substratum (Bio Whittaker), with 5 * 10 (5) cells/ml at the complete cartilage that comprises 0.01 μ g/mlTGF-β-3.
4.0.5ml cell is distributed in the 15ml culture test tube.Cell precipitates 5 minutes with 150 * g.Precipitation is stayed in the substratum in good condition.
5. cover test tube loosely, 37 ℃, 5%CO 2Cultivated 24 hours.
6. forming substratum with freshly prepared complete cartilage in the every 2-3 of cell precipitation days supplies with.
7. precipitation is by keeping suspending in substratum with the low speed eddy current every day.
8. after 14-28 days that cultivate, collect the throw out of chondroblast.
Can by as the generation of observable endogenous fibers matrix, determine that cellular form and/or RT/PCR check that collagen 2 and collagen 9 genetic expressions identify the formation of cartilage.
6.10.4. break up osteoblastic inducing
This embodiment has described and has induced cord blood cell and/or embryonic-like stem cell to be divided into osteocyte.Scheme below using is induced the osteogenesis differentiation:
1. the adhesion of placenta stem-cell is cultivated and to be at MSCGM (Bio Whittaker) or to be supplemented with among the DMEM of 15% Cord blood serum and to carry out.
2. culture kept in tissue culture flasks 24 hours.
3. the osteogenesis differentiation is induced by replacing MSCGM with osteogenesis inducing culture (Bio Whittaker), and the osteogenesis inducing culture comprises 0.1 μ M dexamethasone, 0.05mM xitix-2-phosphoric acid ester, 10mM beta-glycerophosphate
4. supplied with freshly prepared osteogenesis inducing culture in the every 3-4 of cell days, continue 2-3 week.
5. use special dyestuff and the RT/PCR of calcium to come detection of alkaline phosphatase and osteogenin genetic expression.
6.10.5. be divided into hepatocellular inducing
This embodiment has described and has induced cord blood cell and/or embryonic-like stem cell to be divided into liver cell.Scheme below using is induced hepatogenic differentiation:
1. placenta stem-cell is cultivated in the DMEM/20%CBS that is supplemented with 20ng/ml pHGF, 100ng/ml Urogastron.The KnockOut serum substitute can be used to substitute FBS.
2.50ng/ml IL-6 add to and induce in the bottle.
6.10.6. be divided into inducing of pancreatic cell
This embodiment has described and has induced cord blood cell and/or placenta sample differentiation of stem cells to become pancreatic cell.Scheme below using is induced the differentiation of pancreas:
1. placenta stem-cell is cultivated in the DMEM/20%CBS that is supplemented with 10ng/ml Prostatropin and 2ng/ml transforming growth factor-beta-1.The KnockOut serum substitute can be used to substitute CBS.
2. the substratum from the next adjustment of nestin-positive neuron cell culture adds to the substratum with 50/50 concentration.
3. with cell cultures 14-28 days, reloaded again in every 3-4 days.
4. by detecting Regular Insulin albumen or detecting the insulin gene expression by RT/PCR and identify differentiation.
6.10.7. be divided into inducing of heart cell
This embodiment has described and has induced cord blood cell and/or embryonic-like stem cell to be divided into heart cell.Scheme below using is induced myogenic differentiation:
1. placenta stem-cell is cultivated in the DMEM/20%CBS that is supplemented with 1 μ M vitamin A acid, 10ng/ml Prostatropin, 2ng/ml transforming growth factor-beta-1 and 100ng/ml Urogastron.The KnockOut serum substitute can be used to substitute FBS.
2. optionally, placenta stem-cell was cultivated 24 hours in the DMEM/20%CBS that is supplemented with 50ng/ml heart Methylatropine Bromide-1.
4. optionally, placenta stem-cell kept in protein-free medium 5-7 days, and the myocardium extract of choosing then stimulates (the dosage analysis of rising step by step).The myocardium extract is by the generation that homogenizes in being supplemented with the 1%HEPES damping fluid of 1% cord serum of 1gm people's myocardium.Suspended substance was cultivated 60 minutes, and is centrifugal then, collects the upper strata stillness of night.
4. cell cultures 10-14 days, supply again in every 3-4 days.
5. detect by cardiac actin RT/PCR genetic expression and determine differentiation.
6.10.8. cord blood cell and/embryonic-like stem cell before differentiation/or after feature
Embryonic-like stem cell, cord blood cell and/or the cord blood cell group that suppresses with embryonic-like stem cell before differentiation/or after obtain identifying, the variation that it measures its form and cell surface marker by use as technology such as flow cytometry and immunocytochemistries, and by using the variation of measuring genetic expression as technology such as PCR.The cell that is exposed to the cell of somatomedin and/or has broken up can be identified by existing or lacking following cell surface marker: CD10 +, CD29 +, CD34 -, CD38 -, CD44 +, CD45 -, CD54 +, CD90 +, SH2 +, SH3 +, SH4 +, SSEA3 -, SSEA4 -, OCT -4 +And ABC -p +Preferably, embryonic-like stem cell can pass through cell surface marker OCT before differentiation -4 +, APC -p +, CD34 -And CD38 -Existence and identify.It to be omnipotent (for example versatility) that stem cell with these marks is the same with human stem cell.Cord blood cell can pass through cell surface marker CD34 before differentiation +And CD38 +Existence and identify.The cell source of differentiation is from embryonic-like stem cell, cord blood cell and/or with the cord blood cell group who does not preferably express the embryonic-like stem cell of these marks.
Scope of the present invention is not subjected to the restriction of specific embodiments described herein.In fact, except described herein, from the foregoing description, multiple improvement of the present invention is conspicuous to those skilled in the art.These improve also considers to fall in the scope of appended claim.
7. document
The full content degree as a reference of quoting each discrete publication, patent or patent application with this paper for all purposes is the same, and the full content that this paper quotes all reference for all purposes as a reference.
Quote any publication and all be since its open day early than the application's day and can not be interpreted as a kind of permission, i.e. the present invention can not do sth. in advance this open date owing to the effect of invention formerly.

Claims (81)

1. compound is used for when the Mammals non-embryonic stem cells purposes that differentiation phase is regulated the medicine of described cytodifferentiation in the presence of described compound in preparation, wherein said compound is selected from 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone and 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone.
2. the purposes of claim 1, wherein said differentiation of stem cells is a hemocyte.
3. the purposes of claim 1, wherein said stem cell is selected from placenta stem-cell, cord blood stem cell, peripheral hematopoietic stem cells and bone marrow stem cell.
4. the purposes of claim 1, wherein said cell breaks up in cell culture.
5. the purposes of claim 1, wherein said compound concentration is 0.005 μ g/ml to 5mg/ml.
6. the purposes of claim 1, wherein said stem cell is a human stem cell.
7. compound is used for as Mammals CD34 in preparation +Or CD133 +Progenitor cell is bred in the presence of described compound or differentiation phase is regulated purposes in the medicine of described cell proliferation or differentiation, wherein said compound is selected from 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone and 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone.
8. the purposes of claim 7, wherein said progenitor cell is divided into CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Cell.
9. the purposes of claim 7, wherein said cell breaks up in cell culture.
10. the purposes of claim 7, wherein said compound and contacting of described cell be enough to cause aspect differentiation or the propagation with respect to the detectable difference of contrast.
11. the purposes of claim 7, wherein said CD34 +Or CD133 +Progenitor cell has passed through cryopreservation and thawing before described differentiation.
12. external adjusting CD34 +Or CD133 +The method of progenitor cell differentiation, it comprises:
(a) breaking up the colony that described progenitor cell is provided under the condition that can take place;
(b) contact described progenitor cell with compound, wherein said compound is selected from 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone and 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-piperidines-2,6-diketone; With
(c) allow described progenitor cell break up under the condition that is fit to differentiation, wherein said compound contacts the part-time of described at least progenitor cell differentiation with described progenitor cell.
13. the method for claim 12, wherein in step (b), described contact was carried out in the 0th day to the 6th day any time of cultivating.
14. the method for claim 12, wherein in step (b), described contact described progenitor cell cultivate initial the time carry out.
15. the method for claim 12, wherein in step (b), described contact has been bred at least two days later at described progenitor cell and has been carried out.
16. the method for claim 12, wherein in step (b), described contact is carried out at least after described progenitor cell has been bred six days.
17. the method for claim 12, wherein said progenitor cell are CD34 +Progenitor cell.
18. the method for claim 12, wherein said progenitor cell is divided into the cell that represents the cell surface marker feature, and this cell surface marker feature is selected from:
Reduction with respect to contrast CD11c expression;
Reduction with respect to contrast CD38 expression;
Reduction with respect to contrast CD80 expression;
Reduction with respect to contrast CD86 expression;
Reduction with respect to contrast CD1a expression;
Reduction with respect to contrast CD14 expression;
With respect to contrast CD54 BrightThe reduction of expressing;
Reduction with respect to contrast HLA-DR expression;
Increase with respect to contrast CD15 expression;
Increase with respect to contrast CD33 expression;
With respect to contrast CD54 DimThe increase of expressing;
Increase with respect to contrast CD133 expression; With
More than the combination of any marker characteristic;
Wherein said contrast for do not have in the presence of the described compound with the same condition of described progenitor cell under the CD34 that cultivates +Progenitor cell.
19. the method for claim 12, wherein said progenitor cell is divided into CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Cell.
20.CD34 +Progenitor cell is united the purposes of compound in preparing the medicine for the treatment of the individuality that needs progenitor cell that suppresses the TNF-alpha active, wherein said compound is selected from 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone and 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone.
21. the purposes of claim 20, wherein said CD34 +Progenitor cell is a part that comprises the cellular preparations of medullary cell, placenta cells or cord blood cell.
22. the purposes of claim 20, wherein said CD34 +Progenitor cell contains carrier.
23. the purposes of claim 20, wherein said CD34 +Progenitor cell is CD34 +CD38 -CD33 +Or CD34 +CD38 -CD33 -Progenitor cell.
24. the purposes of claim 20, wherein said CD34 +Progenitor cell is CD34 +CD133 +Progenitor cell.
25. the purposes of claim 20, wherein this progenitor cell is expressed the purpose genetic material that inserts.
26. pharmaceutical composition, it comprises Mammals non-embryonic stem cells and drug acceptable carrier, wherein said stem cell has contacted time enough with the differentiation that causes described stem cell or the adjusting of propagation with the compound that suppresses the TNF-alpha active, and wherein said compound is selected from 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone and 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-piperidines-2, the 6-diketone.
27. the pharmaceutical composition of claim 26, wherein stem cell is selected from placenta stem-cell, cord blood stem cell, peripheral hematopoietic stem cells and bone marrow stem cell.
28. the pharmaceutical composition of claim 26, wherein said contact procedure is carried out in cell culture.
29. the pharmaceutical composition of claim 26, wherein said compound concentration are 0.005 μ g/ml to 5mg/ml.
30. the pharmaceutical composition of claim 26, wherein said stem cell are human stem cell.
31. the pharmaceutical composition of claim 26, wherein said being divided into is divided into CD14 +Cell, CD11b +Cell or CFU-GM.
32. pharmaceutical composition, it comprises the cell colony of isolating cord blood cell and isolating differentiation, wherein Fen Hua cell generates by a kind of method, this method is included under the appropriate condition and at 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone or 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-piperidines-2, the 6-diketone makes CD34 under existing +Or CD133 +The non-embryonic stem cells differentiation,
Separate the cell of differentiation thus then.
33. the pharmaceutical composition of claim 32, wherein differentiation step is carried out in cell culture.
34. the pharmaceutical composition of claim 32, wherein said compound concentration are 0.005 μ g/ml to 5mg/ml.
35. the pharmaceutical composition of claim 32, wherein stem cell is a human stem cell.
36. the pharmaceutical composition of claim 32, wherein stem cell is a progenitor cell.
37. the pharmaceutical composition of claim 36, wherein progenitor cell is orientated the specific cells pedigree.
38. the pharmaceutical composition of claim 37, wherein progenitor cell is a hemopoietic progenitor cell.
39. a pharmaceutical composition, it comprises the CD34 of cultivation +Or CD133 +Progenitor cell and drug acceptable carrier, wherein said progenitor cell contacts in the first six day of cultivating under the condition that promotes described progenitor cell proliferation and differentiation with compound, wherein said compound is selected from 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone and 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone.
40. the pharmaceutical composition of claim 39, wherein said progenitor cell is collected and cryopreservation after cultivating six days.
41. the pharmaceutical composition of claim 39, wherein said progenitor cell are CD34 +CD38 -CD34 -Or CD34 +CD38 -CD34 +Cell.
Suffer from the individual of disease or illness or be transplanted to purposes in the described intraindividual medicine 42. the Mammals non-embryonic stem cells is used for the treatment of in preparation, wherein said disease or illness are that myeloplast produces defectiveness or minimizing, tissue or organ damage, hematopoietic cell or blood cell proliferation defective or inflammation, and wherein said cell is at 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone or 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone was handled under existing.
43. the purposes of claim 42, wherein said medicine also comprises undressed cell.
44. the purposes of claim 43, wherein undressed cell is selected from placenta cells, cord blood cell, peripheral blood cells and medullary cell.
45. the purposes of claim 42, wherein said stem cell be cryopreservation and thawing before administration.
Suffer from the individual of disease or illness or be transplanted to purposes in the described intraindividual medicine 46. the non-embryo's progenitor cell of Mammals is used for the treatment of in preparation, wherein said disease or illness are that myeloplast produces defectiveness or minimizing, tissue or organ damage, hematopoietic cell or blood cell proliferation defective or inflammation, and wherein said cell is at 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone or 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone was handled under existing.
47. the purposes of claim 46, wherein said medicine also comprises undressed cell.
48. the purposes of claim 47, wherein undressed cell is selected from placenta cells, cord blood cell, peripheral blood cells and medullary cell.
49. the purposes of claim 46, wherein said progenitor cell be cryopreservation and thawing before administration.
Suffer from the individual of disease or illness or be transplanted to purposes in the described intraindividual medicine 50. promoting agent is used for the treatment of in preparation, wherein said disease or illness are that myeloplast produces defectiveness or minimizing, tissue or organ damage, hematopoietic cell or blood cell proliferation defective or inflammation, and described promoting agent is following promoting agent:
(a) suppress the compound of TNF-alpha active time enough with the adjusting that causes non-embryonic stem cells differentiation or propagation, wherein said compound is selected from 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone and 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone; With
(b) stem cell that in the presence of described compound, has broken up; Or
(c) progenitor cell that in the presence of described compound, has broken up.
51. noble cells suffers from the individual of disease or illness or is transplanted to purposes in the described intraindividual medicine in preparation treatment, wherein said disease or illness are that myeloplast produces defectiveness or minimizing, tissue or organ damage, hematopoietic cell or blood cell proliferation defective, or inflammation, and the cell of wherein said differentiation generates by a kind of method, this method is included under the appropriate condition and at 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone or 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone makes CD34 under existing +Or CD133 +Non-embryo's progenitor cell differentiation.
52. the purposes of claim 51, wherein progenitor cell is in vitro differentiation.
53. the purposes of claim 51, wherein progenitor cell broke up in the dabbling placenta in postpartum.
54. the purposes of claim 51, wherein Fen Hua cell is included in the cellular preparations that comprises cord blood cell.
55. the purposes of claim 51, wherein Fen Hua cell comprises carrier.
56. the purposes of claim 51, wherein hematopoietic cell or blood cell proliferation defective are neutrocytopenia or leukopenia.
57. the purposes of claim 51, its Chinese traditional medicine is through the general administration.
58. the purposes of claim 51, its Chinese traditional medicine is through intravenous administration.
59. the purposes of claim 51, wherein noble cells is expressed the purpose genetic material that inserts.
60. the purposes of claim 51, wherein noble cells is allogenic.
61. the purposes of claim 51, wherein said individuality are the people.
62. one kind from CD34 +The method of the cell of progenitor cell production differentiation, it is included in the substratum that allows propagation and differentiation and cultivates described cell, reach with 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone or 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone contacts described progenitor cell.
63. the method for claim 62, wherein said contact is carried out first day of described cultivation.
64. the method for claim 62, wherein said contact is carried out twice in the first six day of described cultivation at least.
65. the method for claim 62, wherein said contact was carried out at first day that is no earlier than described cultivation.
66. the method for claim 62, wherein said CD34 +Progenitor cell is CD34 +CD133 +Progenitor cell.
67. the method for claim 62, wherein said noble cells is being cultivated separation in the 6th day.
68. the method for claim 62, wherein said noble cells is being cultivated separation in the 12nd day.
69. the method for claim 62, wherein said CD34 +Cell separates from other hemocytes before described cultivation.
70. the method for claim 62, wherein said substratum also comprise GM-CSF and TNF-α.
71. the method for claim 62, wherein said 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1, the 3-diketone exists with the concentration of 0.1 μ M to 10.0 μ M.
72. the method for claim 62, wherein said 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1, the 3-diketone exists with the concentration of 1.0 μ M.
73. one kind prepares and is used for the treatment of the method for suffering from the individual of disease or illness or being transplanted to described intraindividual pharmaceutical composition, wherein said disease or illness are that myeloplast produces defectiveness or minimizing, tissue or organ damage, hematopoietic cell or blood cell proliferation defective or inflammation, and described method comprises:
(a) contact CD34 with compound +Or CD133 +Progenitor cell, wherein said progenitor cell was cultivated six days under the culture condition that allows described progenitor cell proliferation and differentiation, described compound is selected from 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone and 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone;
(b) cultivate the described cell of collection after six days; With
(c) cell is placed drug acceptable carrier.
74. the method for claim 73, wherein said contact was carried out at first day that cultivates.
75. the method for claim 73, wherein said contact is carried out twice in six days of described cultivation at least.
76. the method for claim 73, wherein said progenitor cell separates from other hemocytes before described cultivation.
77. the method for claim 73, wherein said culture condition are included in and cultivate described progenitor cell in the substratum that comprises GM-CSF and TNF-α.
78. the method for claim 73, wherein said 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone or 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-and piperidines-2, the 6-diketone exists with the concentration of 0.1 μ M to 10.0 μ M.
79. the method for claim 73, wherein said 4-(amino)-2-(2,6-dioxo-(3-piperidyl)) isoindoline-1,3-diketone or 3-(4-amino-1-oxo-1,3-dihydro-isoindole-2-yl)-piperidines-2, the 6-diketone exists with the concentration of 1.0 μ M.
80. the method for claim 73, wherein said cell is at described collection back cryopreservation.
81. pass through the pharmaceutical composition of the method preparation of claim 73.
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