CN101861156B - From the fatty or adherent cell of placenta tissue and purposes in the treatment thereof - Google Patents

Abstract

Disclose the method for ischemia in the experimenter needing it for the treatment of.The method include giving subject's effective dose selected from Placenta Hominis and the adherent cell of the tissue of fatty tissue, thereby treat the ischemia of experimenter.Also disclose that the method that treatment needs the medical condition of connective tissue regeneration and/or reparation.

Description

From the fatty or adherent cell of placenta tissue and purposes in the treatment thereof

Invention field and background

The method that the present invention relates to use the adherent cell treatment disease from fat or placenta tissue, more specifically, It is directed to use with adherent cell treatment ischemia and/or needs the medical condition of connective tissue regeneration and/or reparation.

In growing medical circle, have increasing need for substantial amounts of adult stem cell and implant for cellular transplant and tissue Engineering.Additionally, the positive sustainable development of adult stem cell therapy is in order to treat and to cure various situation, such as hematopoietic disorders, heart disease, Parkinson disease, Alzheimer's disease, apoplexy, burn, muscular dystrophy, autoimmune conditions, diabetes and arthritis.

In the last few years, substantial amounts of research activities concentrated on mesenchyma stromal cells (MSCs) and is used for including that damaged organ is such as Brain, heart, the tissue repair of skeleton regulating liver-QI and support bone marrow transplantation (BMT) are dived in the treatment of interior various medical applications Energy.MSCs is the heterogeneous cell population available from such as bone marrow, fatty tissue, Placenta Hominis and blood, can rely on from various biological activitys The impact of the factor is divided into different types of mesenchyme mature cell, and (such as, reticuloendothelial cell, fibroblast, fat are carefully Born of the same parents, bone precursor cells).Therefore, MSCs be widely studied in regenerative medicine as structure new organization such as bone, cartilage and The basis of fat for injury repairing or the displacement of pathological tissues and being used for treat heredity and acquired disease [Fibbe and Noort, Ann N Y Acad Sci (2003) 996:235-44；Horwitz etc., Cytotherapy (2005) 7 (5): 393-5； Zimmet and Hare, Basic Res Cardiol (2005) 100 (6): 471-81].And, the versatility of MSCs, they easy In separating and cultivate and their ex vivo (ex vivo) high proliferation potential making them become attractive treatment Instrument [Fibbe and Noort, above；Exp Biol Med (Maywood) (2001) 226 (6): 507-20 such as Minguell].

The MSCs being derived from Placenta Hominis demonstrates many and is isolatable from the labelling that the MSCs of other tissue has, such as CD105, CD73, CD90 and CD29, and lack hematopoietic cell, endotheliocyte and the expression of trophoderm-specific cell labelling.Suitably Under the conditions of cultivate the MSCs being derived from Placenta Hominis after complete into lipid, osteogenic and neurogenicity differentiation [Yen etc., Stem Cells (2005) 23 (1): 3-9].And, the MSCs being isolatable from Placenta Hominis In vitro culture has been demonstrated in the way of similar to MSCs to exempt from Epidemic disease is absolved.Therefore, Placenta Hominis provides MSCs source that is the most uncontested and that be easily obtained for experiment and clinical practice [Zhang etc., Exp Hematol (2004) 32 (7): 657-64].

Three-dimensional (3D) condition of culture (PCT application of the MSCs amplification being suitable to be derived from Placenta Hominis has been devised before the present inventor No. IL2007/000380), its complete content is by quoting and in herein.

The primary clinical use of MSCs is summarized in hereafter.

Ischemia

Peripheral arterial disease (PAD)

Peripheral arterial disease (PAD) is a kind of chronic disease, and it can limit the blood flow of limbs thus may result in serious by Progressive symmetric erythrokeratodermia Medical complication.This disease is the most relevant to other clinical conditions, including hypertension, cardiovascular diseases, hyperlipidemia, glycosuria Disease, obesity and apoplexy.Critical limb ischemia (CLI) suffers from the pain of chronic ischemia induction, ulcer, tissue defect for describing Or the patient of extremity gangrene.CLI represents the latter stage of PAD patient, and they need by vascular surgery or the Synthetic of blood vessel expert Treat.Sick with coronary artery and arteriae cerebri contrary, peripheral arterial disease (PAD) remains the situation not being completely understood, although sick Feelings are serious and very popular but be seldom diagnosed and be the most less treated.As a result, CLI frequently result in amputation or dead and The mortality rate of PAD patient exceedes the mortality rate of myocardial infarction and paralytic.

Multiple adult stem cell has been employed in order to treat ischemic conditions.Therefore, the stromal cell of fatty tissue it is derived from And co-culturing of endotheliocyte (EC) mainly causes EC survival rate, migration and tube chamber shape by the secretion of VEGE and HGF (ADSC) Become dramatically increases.Angiogenesis scoring after acellular matrix graft to ischemia mouse hind leg surrounding is improved [Nakagami Deng, JAtheroscler Thromb (2006) 13 (2): 77-81].[Cell Physiol Biochem. (2006) such as Moon 17:279-90] detect the ability of CFU-GM (ADSC) the treatment immunodeficient mouse limb ischemia being derived from fatty tissue and demonstrate,proved In clear ADSC-transplantation group, laser Doppler perfusion index dramatically increases.

Additionally, be transplanted to four when the mescenchymal stem cell being derived from Cord blood (UCB) accepted Drug therapy and operation Time in the Buerger's disease patient of therapy, the Ischemic rest pain of their suffering limb suddenly disappears [Kim etc., Stem Cells (2006) 24 (6): 1620-6].And, the human mesenchymal stem cell (FMhMSC) being isolatable from mature afterbirth is implanted into the big of infraction Rat heart is relevant to substantially reducing of capillary density increase, left ventricular function normalization and scar tissue, these situations It is enhanced [Ventura etc., (2007) when the mixed ester pretreatment formed with butanoic acid and tretinoin with hyaluronan by stem cell J.Biol.Chem., 282:14243-52].

Apoplexy

Apoplexy is one of main inducing of whole world death, causes all death of about 9% and consumes total doctor of about 2-4% Treatment expense.Although probably because improve the control to risk of stroke (especially hypertension, diabetes and smoking), send out The apoplexy mortality rate reaching country the most persistently reduces, but apoplexy still may result in permanent damage (such as, tissue injury, nerve Damage).

New curing apoplexy scheme includes stem cell therapy.Imagine stem cell or CFU-GM local or passed through intravenous Approach is implanted into damaged site to substitute nonfunctional cell, strengthen endogenous stem cells or the propagation of CFU-GM and/or differentiation and carry For necessary immunomodulator, and as the main strategy based on cell.The potential source of stem/progenitor cells includes Fetal nerve stem cell, embryonic stem cell, neural teratocarcinoma cells, it is derived from the non-hematopoietic stem cell of Cord blood, is derived from bone marrow Stem cell and be derived from the mescenchymal stem cell [Andres etc., NeurosurgFocus (2008) 24 (3-4): E16] of Placenta Hominis.

In a nearest research, what Koh etc. [Koh etc., Brain Res. (2008)] have detected transplanting is derived from people's umbilicus The mescenchymal stem cell (hUC-MSCs) with blood neuroprotective in Ischemic Apoplexy rat model and mechanism.External evoked After neuron differentiation 20 days, hUC-MSCs demonstrates the morphological feature of neuron and expresses neuronal cell labelling and nerve Unit's factor (such as, be derived from the neurotrophic factor of glial cell-line, be derived from the neurotrophic factor of brain).And, will The impaired brain hemisphere being transplanted to immunosuppressant Ischemic Apoplexy rat in hUC-MSCs body can improve nerve relative to control rats Behavioral function and minimizing infarct volume.After transplanting three weeks, hUC-MSCs occurred in impaired hemisphere and expresses neuron-special mark Note, but these cells do not become functional activity neuronal cell.

Orthopedic applications

Multiple situation and pathology need regeneration and/or the reparation of connective tissue (such as, bone, tendon and ligament).These include, Such as, fracture, burn, burn wound, depth wound, degeneration bone, connective tissue loss that various cancer is relevant (such as, osteocarcinoma, Osteosarcoma, Bone tumour) and articular cartilage defect.

Use autologous BM-MSCs to strengthen knitting be described for veterinary and the orthopedic applications of people and included percutaneous Injection of bone marrow passes through the autograft or of the same race of bone marrow in Ligament healing (Carstanjen etc., 2006), orthopedic clinic Allotransplant treatment Cranial defect (Horwitz etc., 1999, Horwitz etc., 2002), use are carried on by hydroxyapatite-phosphorus On the ceramic cylinder of acid DFP composition allochthonous [Arinzeh TL, etc., J Bone Joint Surg Am.2003, Or autologous [Bruder SP etc., J Bone Joint Surg Am.1998 Jul 85-A (10): 1927-35]；80 (7): 985-96] bone marrow-MSCs is in Canis familiaris L., or use the allochthonous MSCs (Chao etc., 2006.) of peripheral blood that is derived from rabbit Carry out the regeneration of critical size Cranial defect, and use MSCs is implanted in baboon and carries out substantial amounts of bone formation (Livingston Deng, 2003).

In horse art of orthopedic surgery, the mescenchymal stem cell of BM and adipose tissue-derived is the most experimentally for operative treatment Subchondral bone cyst, fracture repair [Kraus and Kirker-Head, VetSurg (2006) 35 (3): 232-42] and repair of cartilage [Brehm etc., OsteoarthritisCartilage (2006) 14 (12): 1214-26；Wilke etc., J Orthop Res (2007) 25 (7): 913-25] and be clinically used for treat the overworked tendon injury caused of horse.And, use different controlling Treatment method promotes Suspensory ligament healing (Herthel, 2001) of horse.Herthel (2001) has been proved a kind of auxiliary Suspensory ligament more The neontology method closed, it relates to damaging interior injection autologous stem cells and relevant bone marrow fraction to stimulate natural ligament again Raw.

The impaired rabbit models show of tendon organizes shape more higher than the tissue of natural reparation and hard through what MSC-processed (Gordon etc., 2005).The reparation biomechanics significantly improved is caused additionally, be inoculated at tendon crack by the MSCs of cultivation (Young etc., 1998, Osiris Therapeutics, www.osiris.com).

Detection Osiris chondrogen (adult mesenchymal stem cells) is to evaluate its safety and to have the most in the patient Effect property.In the animal that MSC processes, the meniscal tissue regeneration that operation removes, cartilage surface is protected and dynamic with compareing Thing is compared and is observed that joint injury reduces.These benefits continue at least one year (Osiris in animal model Therapeutics, www.osiris.com).

Summary of the invention

One side according to some embodiments of the invention, it is provided that treat the method for ischemia in the experimenter needing it, The method include giving subject's effective dose selected from Placenta Hominis and the adherent cell of the tissue of fatty tissue, thereby treatment is subject to The ischemia of examination person.

One side according to some embodiments of the invention, it is provided that treatment needs to need connective group in its experimenter Knit regeneration and/or the method for medical condition repaired, the method include giving subject's effective dose selected from Placenta Hominis and fat The adherent cell of the tissue of fat tissue, thereby needs the medical condition of connective tissue regeneration and/or reparation in treatment experimenter.

One side according to some embodiments of the invention, it is provided that thin selected from the adhesion of Placenta Hominis and the tissue of fatty tissue Born of the same parents' purposes in preparing the identified medicament for treating ischemia.

One side according to some embodiments of the invention, it is provided that thin selected from the adhesion of Placenta Hominis and the tissue of fatty tissue Born of the same parents' purposes in the medicament preparing the identified medical condition needing connective tissue regeneration and/or reparation for treatment.

One side according to some embodiments of the invention, it is provided that one prepares article, it includes comprising for treating The packaging material of the label of ischemia, these packaging material pack medicinal effective dose selected from Placenta Hominis and the adhesion of the tissue of fatty tissue Cell.

One side according to some embodiments of the invention, it is provided that one prepares article, it includes comprising for treating Needing the packaging material of the label of the medical condition of connective tissue regeneration and/or reparation, medicinal effective dose packed by these packaging material Selected from Placenta Hominis and the adherent cell of the tissue of fatty tissue.

According to some embodiments of the present invention, this adherent cell can suppress the immunoreation in experimenter.

According to some embodiments of the present invention, the adherent cell of at least 10% is in proliferation period.

According to some embodiments of the present invention, this ischemia is peripheral arterial disease (PAD).

According to some embodiments of the present invention, this peripheral arterial disease (PAD) is critical limb ischemia (CLI).

According to some embodiments of the present invention, this ischemia includes the ischemia of central nervous system (CNS).

According to some embodiments of the present invention, this ischemia is selected from peripheral arterial disease, ischemic vascular disease, ischemic heart Disease, ischemic encephalopathy, ischemic nephropathy and ischemia Placenta Hominis.

According to some embodiments of the present invention, this adherent cell is cultivated available from three-dimensional (3D).

According to some embodiments of the present invention, this three-dimensional (3D) is cultivated and is included 3D bioreactor.

According to some embodiments of the present invention, the cell in 3D cultivates is cultivated and is realized under perfusion.

According to some embodiments of the present invention, the condition of culture of dimensional culture includes selected from polyester and polyacrylic adhesion Material.

According to some embodiments of the present invention, cell is cultivated and is at least carried out 3 days.

According to some embodiments of the present invention, cell is cultivated and is carried out to the cell of at least 10% breeding.

According to some embodiments of the present invention, this adherent cell comprises the sun selected from CD73, CD90, CD29 and CD105 Property marker expression.

According to some embodiments of the present invention, this adherent cell comprise selected from CD3, CD4, CD45, CD80, HLA-DR, The negative marker of CD11b, CD14, CD19, CD34 and CD79 is expressed.

According to some embodiments of the present invention, this adherent cell comprises expresses overview the most as described herein.

According to some embodiments of the present invention, this adherent cell includes the cell comprising stromal stem cell phenotype.

According to some embodiments of the present invention, this stromal stem cell phenotype includes T cell inhibitory activity.

According to some embodiments of the present invention, this connective tissue includes tendon, bone and/or ligament.

According to some embodiments of the present invention, need the medical condition of connective tissue regeneration and/or reparation selected from fracture, Osteocarcinoma, burn wound, articular cartilage defect and deep wound.

According to some embodiments of the present invention, this medical condition selected from subchondral bone cyst, fracture, osteoporosis, Osteoarthritis, degeneration bone, osteocarcinoma, cartilage injury, articular cartilage defect, DDD, osteogenesis imperfecta (OI), burn, Burn wound, deep wound, wound healing delay, tendon is impaired and ligament is impaired.

Except as otherwise noted, all technology used herein and scientific terminology have and common skill of the art The identical implication that art personnel are generally understood that.Although can be used for implementing with those similar or equivalent methods described herein and material Or the inspection present invention, but the most still describe suitable method and material.If any repugnance, it is fixed to include with patent specification Justice is as the criterion.Additionally, material, method and embodiment are merely to illustrate and the most restrictive.

Accompanying drawing is sketched

The present invention it is described herein as the most by way of example with reference to accompanying drawing.Now with specific reference to the detailed content of accompanying drawing, emphasize , show that particular content and being merely to illustrate property discuss embodiment of the present invention by way of example, and be to carry What is considered as the most useful and the most intelligible explanation in terms of the principle of the invention and design for and proposes.In this, It is not intended to show the ratio more detailed present configuration details needed for the basic comprehension present invention, accompanying drawing description is made It will be appreciated that the several forms of the present invention embodies the most in practice.

In accompanying drawing:

Figure 1A-G is described in the bioreactor system comprising 3D carrier the class bone microenvironment produced.Figure 1A-B is for describing Nature bone (Figure 1A) and inoculation adherent cell, 7 days PluriX after analog bone microenvironment^TMThe comparison of 3D carrier structure (Figure 1B) Electron micrograph.Fig. 1 C-F is the PluriX of the adherent cell inoculation that description bone marrow produces^TM3D substrate 20 days after inoculation (Fig. 1 C-D amplifies X 150 and 250 respectively) and the electron micrograph of 40 days (Fig. 1 E-F amplifies X 350 and 500 respectively).Figure 1G is to have by the figure of the Plurix 3D plug-flow bioreactor of the separate part of number definition: culture medium storehouse (1), mixed Close gas supply (2), filter (3), injection point (4), wherein the placement post (5) of 3D carrier, flow monitor (6), flow Valve (6a), separation container (7), cell growth analysis instrument (8), peristaltic pump (9), sample point (10), molten O₂Measure electrode (11), pH Measure electrode (12), control system (13), fresh growth medium (14), used growth medium (15).

Fig. 2 is that to be described in bioreactor system the adherent cell deriving from Placenta Hominis of growth under 3D growth conditions different The figure (batch 5-8) of production batch.By adherent cell (2X10⁶) it is inoculated into life with the density of 10000-15000 cell/carrier Thing reactor.After cultivating 12 days, the density of 3D-adherent cell reaches 150,000-250,000 cell/carrier or containing 150 22.5-37.5X10 in the bioreactor of individual carrier⁶。

Fig. 3 A-B is to describe to compare the membrane marker (mulberry) expressed in the 3D-adherent cell be derived from Placenta Hominis and in routine The bar diagram of the expression difference of the membrane marker (lavender) in the placenta cells cultivated under 2D condition of culture.Adherent cell exists Growth 4-6 week in culture bottle (2D), or in polystyrene support (3D) upper growth 2-3 week in bioreactor system.From training After supporting bottle or carrier harvesting, incubated cell and with identify adherent cell (Fig. 3 A) or hematopoietic cell (Fig. 3 B) distinctive film mark One group of monoclonal antibody (MAb) of note combines.Notice and in the adherent cell that 3D cultivates express MSC membrane marker compared with MSC membrane marker expression (as shown in CD90, CD 105, CD73 and CD29 membrane marker) that 2D cultivates in cell is considerably higher, Especially CD 105, it is display 56% expression in 3D cultivates cell, and comparing in 2D cultivates cell is 87% (Fig. 3 A).2D Any hemopoietic membrane marker (Fig. 3 B) is not the most expressed with the adherent cell in both 3D culture.

Fig. 4 A-D be describe compare 2D and 3D condition or under 2D and 3D conditioned medium cultivate from Placenta Hominis produce The bar diagram of the protein level of adherent cell.Fig. 4 A-C describes and (is standardized as 1X10 by elisa assay with pg/ml⁶Individual carefully Born of the same parents/ml) be unit 2D and 3D cultivate adherent cell conditioned medium in Flt-3 part (Fig. 4 A), IL-6 (Fig. 4 B) Level with SCF (Fig. 4 C).Result represents one of three independent experiments.The expression of the different cell protein of Fig. 4 D display, as By the analytical reagent composition of the protein example that iTRAQ reagent labelling compares therebetween.Protein sample picks up from 2D (informal voucher) and 3D (ash Bar) under the conditions of growth adherent cell.This figure represents two and repeats one of experiment.Notice cell and 2D and 3D condition of culture The difference of the expression of some albumen in conditioned medium.

Fig. 5 A-D is the micro-photograph that the 3D-adherent cell describing and being derived from Placenta Hominis is divided into osteoblastic ability in vitro Sheet.The adherent cell being derived from Human plactnta (is resisted containing 10%FCS, 100nM dexamethasone, 0.05mM at Osteogenic Induction Medium Bad hematic acid 2-phosphate, the DMEM of 10mMB-glycerophosphate) middle cultivation 3 time-of-weeks.Fig. 5 A-B Explicit Expression calcified matrix Cell, as shown in Alizzarin Red S dyes.Fig. 5 C-D shows the compared with control cells processed without Osteogenic Induction Medium, its It is kept into fibrocyte sample phenotype and proof does not has mineralising.

Fig. 6 uses chemotherapy (continuous 2 weeks peritoneal injection 25mg/kg busulfans in 3.5 weeks after the transfer for describing (busulfan) chart of people's CD45+ cell percentages of detection in the NOD-SCID mouse bone marrow cells (BM)) treated.Will be from being derived from The individually transplanting of the CD34+ cell (100,000) of the mononuclear cell purification of Cord blood (a), or train with under the conditions of 2D by 5 mices The 0.5X10 supported⁶Individual adherent cell co-transplantation (the 2D-adherent cell being derived from Placenta Hominis；2 mices, b), or with at pluriX^TM In bioreactor under the conditions of 3D cultivate be derived from Placenta Hominis adherent cell (3D-adherent cell) co-transplantation (5 mices, c). Then BM is collected from mice Thigh bone and tibia.By the people's cell in flow cytometry detection BM.By by cell with Anti-human CD45-FITC hatches the percentage ratio measuring the people's cell expressing CD45.Notice and process the people in mice with alone HSC The percentage ratio (a) of class cell is compared, with 2D-adherent cell (b) and with the mouse bone marrow cells of 3D-adherent cell (c) co-transplantation In the percentage ratio of people's cell (hCD45+) higher.Observe than warp in cultivating, through 3D-adherent cell, the mice that cell processes 2D-adherent cell cultivates the higher immigration of mice that cell processes, and indicates through higher specific to the adherent cell of 3D cultivation Treatment advantage.

Fig. 7 A-B is only with people's graft CD45+ cell (Fig. 7 A) in the mice of CD34+ cell transplantation and CD34+ cell Add and be derived from the facs analysis that the adherent cell (Fig. 7 B) of fatty tissue compares.Notice the mice processed with alone people CD34+ (7B-12%) compare, with the human hematopoietic cell group (hCD45+) in the mice of the adherent cell co-transplantation being derived from fatty tissue Percentage ratio (7A-29%) is significantly higher.

Fig. 8 A for be described in human cord blood mononuclear cell (CB) and equivalent (3000Rad) cord blood cell (iCB) via radiation, Be derived from the mononuclear cell (PBMC) of human peripheral, 2D cultivate (2D) or 3D cultivate (3D) be derived from Placenta Hominis adherent cell or Carry out mixing lymph between the combination (PBMC+2D and PBMC+3D) being derived from Placenta Hominis adherent cell that PBMC and 2D and 3D cultivates thin The bar diagram of born of the same parents' reaction.The size of CB cell mass by³H-thymidine absorbs (measuring with CPM) and represents, last 18 little cultivate Time interior measurement.The CB cell proliferation of irriate raises and shows higher immunne response level.Notice and hatch with adherent cell Cell demonstrates relatively low immunne response level, the CB immunne response particularly when jointly hatching with adherent cell, to PBMC Reduce.Three repetitions are made in each reaction.

Fig. 8 B is that Celligen is passed through in description^TMThe flow chart of 3D adherent cell (named PLX-C cell) is produced from Placenta Hominis.

Fig. 8 C is the Celligen from the debugging of New Brunswick Scientific website^TMBioreactor conduit and end The diagram of mouth.

Fig. 9 A-B is described and is carried out by Plurix (named PLX, Fig. 9 B) and Celligen (named PLX-C, Fig. 9 A) The cell cycle analysis that 3D adherent cell produces.Cell is fixed on 70%EtOH O.N, is centrifuged and is resuspended in propidium iodide (PI) solution, then passes through facs analysis.

Figure 10 A-C describes expression rather than the table of endothelium typical marks of the fibroblast-typical marks on PLX-C Reach.Figure 10 A describes the negative expression of endothelial marker CD31；Figure 10 B describes the negative expression of endothelial marker KDR；And Figure 10 C Describe the positive expression of human fibroblasts labelling (D7-FIB).Notice the red histogram table of isotype IgG1 (FITC) Show negative control and blue histogram represents positive stained cells.

Figure 11 A-D describes stimulation molecule and the expression of costimulatory molecules on PLX-C cell.Figure 11 A describes CD80's PLX-C expresses；Figure 11 B describes the PLX-C of CD86 and expresses；Figure 11 C describes the PLX-C of CD40 and expresses, and Figure 11 D describes The PLX-C of HLA-A/B/C expresses.Negative control is prepared with irrelevant isotype fluorescence molecule.Notice red histogram expression table Reaching the cell mass of PLX-C labelling, blue histogram represents the cell mass expressing bone marrow (BM) labelling, and green histogram represents table Reach the cell mass of mononuclear cell (MNC) labelling.

Figure 12 A-B describes PLX-C to lymphopoietic suppression.Figure 12 A describes utilization equivalent via radiation (3000Rad) be derived from PB MNC (donor B) stimulate 2x10⁵The individual MNC (donor A) of peripheral blood (PB) that is derived from is then to cultivation Thing adds the MLR detection that the PLX-C cell of increasing amounts is carried out.By three repeated inoculations of each group in 96 orifice plates.Pass through [³H] thymidine incorporation measurement multiplication rate；The MNC being derived from peripheral blood (PB) that Figure 12 B description ConA (1.5mg/ml) stimulates. The PLX-C cell of increasing amounts is added in culture.By three repeated inoculations of each group in 96 orifice plates.By [³H] thymidine mixes Enter to measure multiplication rate.

Figure 13 A-C describes and co-cultures rear proinflammatory and anti-inflammatory response cytokine secretion with peripheral blood cells PLX-C regulates.Figure 13 A-B describes after co-culturing with the MNC (being isolatable from peripheral blood) being derived from people and PLX-C of ConA stimulation IFN γ (Figure 13 A) and the secretion of TNF α (Figure 13 B)；Figure 13 C describes the MNC being derived from people stimulated with LPS and (is isolatable from periphery Blood) co-culture the secretion of rear IFN γ, TNF α and IL-10 with PLX-C.Collect supernatant and use ELISA to carry out cytokine to divide Analysis.

Figure 14 describes the luciferase expression vector for infecting PLX-C cell.Expression vector from OmicsLink Lv33 is for herein.This luciferin gene is cloned into ORF.

Figure 15 describes the high luciferase expression of the PLX-C cell of infection.Use luciferase expression vector infection cell And within 48 hours, make it visualize by IVIS system after infection.Notice that cell shows high-caliber luciferase expression.

Figure 16 A-D describes to SCID/Beige injected in mice 2x10⁶The PLX-C cell of individual expression luciferase.One Mice IM injects, an IV injection.IVIS system monitoring is used to be injected mice to evaluate the vivo biodistribution distribution of PLX-C.Aobvious Show the 1st day (Figure 16 A), the 4th day (Figure 16 B), the 6th day (Figure 16 C) and the IVIS result of the 22nd day (Figure 16 D).

Figure 17 is hip and the foot perfusion increase of the mice that description adherent cell of the present invention (named PLX-C) is treated Figure.This Figure illustrates mice hip and the intermediate value of foot perfusion percent.Use non-contact laser doppler instrument after surgery the 0th, 6, 9,14 and 21 days (display for the measured value of the 21st day) measures hip and the blood flow of foot from both sides.Result is expressed as ischemia in test The ratio of limb blood flow and normal limb blood flow.

Figure 18 is the figure of the interior evaluating describing limb function and ischemic injuries.Following marking system is used to be carried out continuously scarce The semi-quantitative assessment of the impaired purposes of blood limb: 3=drags foot, 2=without dragging foot but without plantar flexion, 1=plantar flexion, and 0=bending toe with The opposing mild traction to tail.

Figure 19 A-C describes the capillary density increased after PLX-C processes.Figure 19 A describes the mice processed with PBS Capillary density；Figure 19 B describes the capillary density of the mice processed with PLX-C cell；Figure 19 C is each for describing The bar diagram of muscle cell blood capillary quantity.Notice the limb ischemia in the induction by the dyeing checking of special blood capillary After, rather than observe the capillary density of increase in PLX-C processes mice in control mice.

Figure 20 A-B describes the oxidative stress and endothelial inflammation reduced after PLX-C is administered.Figure 20 A is for describing oxidative stress The bar diagram of (nitrotyrosine dyeing)；And Figure 20 B is the bar diagram describing endothelial inflammation (VCAM evaluation).Notice with The mice that PLX-C processes observes oxidative stress and the endothelial inflammation of minimizing.

Invention embodiment description

The present invention is to use the adherent cell of Placenta Hominis or fatty tissue to increase blood vessels in tissue life in some embodiments Become and treat ischemia or need the method for medical condition of connective tissue regeneration and/or reparation.

By being better understood principle and the operation of the present invention with reference to accompanying drawing and supplemental instruction.

Before explaining at least one embodiment of the invention in detail, it should be understood that the application of the present invention be not only restricted to Listed by lower explanation or the detailed content of embodiment institute illustration.The present invention can have other embodiments or real in every way Execute or carry out.And, it is also contemplated that be the wording applied herein and term for descriptive purposes and is not construed as having limit Property processed.

When the present invention being put into practice, the inventor have discovered that the adherent cell being derived from Placenta Hominis or fatty tissue is increasing Blood vessels in tissue generates and treatment ischemia is effective with the medical condition camber needing connective tissue regeneration and/or reparation.

Embodiment 1-8 such as embodiment part hereafter and afterwards proves, the amplifiable substrate that comprises of the present inventor is done thin The adherent cell being derived from fat and Placenta Hominis of born of the same parents' character.Find that the cell expanded accordingly has vigor after freezing, as viscous Attached property and again proliferation assay are proved (see embodiment 1).The flow cytometric analysis of the adherent cell being derived from Placenta Hominis is taken off Show different marker expression types (see Fig. 3 A-B).Show further in the embodiment 6 of Examples below part, transplant source Significantly induce by the mice hip of artery ligation (ischemic hind limb model) and the blood flow (Figure 17) of foot from the adherent cell of Placenta Hominis, significantly Improve limb function (Figure 18), increase capillary density (Figure 19 A-C) and reduce oxidative stress and endothelial inflammation (Figure 20 A- B)。

Therefore, the method increasing blood vessels in tissue generation is provided according to an aspect of the present invention.The method is by making group Knit the adherent cell with the tissue selected from Placenta Hominis and fatty tissue and contact realization, thereby increase the angiogenesis in tissue.

Phrase used herein " increases the angiogenesis in tissue " and refers to generate new blood capillary in increase (induce, raise) tissue The process of pipe.

Phrase used herein " adherent cell " refer to anchor rely on i.e. need to adhere to from the teeth outwards with the homogenizing of growth in vitro or Foreign cell group.

Phrase used herein " fatty tissue " refers to comprise the connective tissue of adipose cell (lipocyte).

Terms used herein " placenta tissue " refer to for Uterus wall lining and period of gestation parcel fetus mammal female Any part of sexual organ, Placenta Hominis and fetus are connected by umbilical cord.Placental separation (referred to as postpartum placenta) after birth.Show at one In example embodiment, Placenta Hominis refers to complete Placenta Hominis.

The adherent cell being derived from Placenta Hominis or fatty tissue can use two dimension or three-dimensional cultivation condition propagation.

The condition breeding adherent cell in 2D cultivates is further described in embodiment part hereafter and afterwards.

Phrase used herein " dimensional culture " refers to be placed in cell and compatible with cell growth makes the cell can be many simultaneously Condition in one layer of upper growth.The most well recognize that cell in situ environment in live organism (or tissue) constructs in three-dimensional In.Cell by other cell around.It is fine that they are fixed on the nanoscale extracellular matrix so that setting up various local microenvironment In the composite network of dimension.The outer part of their born of the same parents not only mediates the attachment to basement membrane, but also can and multiple blood vessel and lymph Pipe.Oxygen, hormone and nutrient are transported to cell refuse and are then transported.Three-dimensional cultivation condition of the present invention is designed as simulating such as The environment of illustration further below.

It it should be understood that three-dimensional cultivation condition allows to expand adherent cell.

Terms used herein " amplification " and " amplification " refer to maintain cell and final cell growth the most undifferentiatedly, That is, make cell mass increase (such as at least 2 times) and there is not the differentiation with described increase.

Terms used herein " maintenance " and " maintenance " refer to the most undifferentiated cell turnover, the most substantially stablize cell Group and do not exist with described stable differentiation.

As discussed, this adherent cell on the one hand of the present invention is recovered from fat or placenta tissue.

Placenta cells can be obtained from mature or preterm labor placenta.Preferably upon external haemorrhage just collects Placenta Hominis.Preferably enough to remove Remove the time perfusion Placenta Hominis of residual cell.Terms used herein " irrigates " and refers to poured into by fluid or enable flow through organ or tissue Behavior.Placenta tissue may be from any mammal；The placenta tissue of such as people.Placenta tissue source is come self-produced easily Rear Placenta Hominis (such as 1-6 hour), but, the method for placenta tissue or the source of cell or separation placenta tissue is for the present invention Say unimportant.

The adherent cell being derived from Placenta Hominis may be from the fetus of Placenta Hominis (i.e. amniotic membrane or Placenta Hominis interior section, see embodiment 1) With parent (i.e. basal decidua and decidua parietalis) part.At physiological buffer, [such as phosphate buffered saline (PBS) (PBS) or Hank ' s buffer Liquid) middle washing tissue sample.By processing tissue (see below) with digestive enzyme or/and shred (mince) and use washing medium By flushed for tissue part NF or by aspirating (Falcon, Becton, Dickinson, San Jose, CA) gently, Prepare single-cell suspension liquid.

The adherent cell being derived from fatty tissue can be separated by multiple method well known by persons skilled in the art.Such as, Such method is set forth in U.S. Patent No. 6,153,432.Fatty tissue may originate from nethike embrane/internal organs, breast, gonad or other Adipose tissue site.A kind of source of fatty tissue is omental adipose.In the mankind, fat is generally separated by liposuction.

The separation adherent cell being derived from fatty tissue can obtain by processing tissue in following condition: with digestive enzyme (such as Collagenase, trypsin and/or Bacillus polymyxa Neutral proteinase；And/or the hyaluronidase of valid density or DNA enzymatic)；And ethylenediaminetetraacetic acid (EDTA)；At the temperature action 10 minutes to 3 hours of 25-50 DEG C.Then the nylon that cell passes through 20 microns-1 millimeter can be allowed Or garrha net filter.Then cell is allowed directly in the medium or to enter through Ficoll or Percoll or other particle gradient Row differential centrifugation.Cell to 1 hour (sees U.S. Patent No. in 1 minute the temperature of 4-50 DEG C with 100-3000xg centrifugation No. 7,078,230).

In addition to being derived from the adherent cell of Placenta Hominis or fatty tissue, it is also contemplated that be characterized as stromal stem cell phenotype (its To illustrate further below) the purposes of the adherent cell from other cell derived.Adherent cell can be extracted from which Tissue-derived include but not limited to: Cord blood, scalp, hair follicle [such as described in U.S. Patent Application No. 20060172304], testis Ball [such as Guan K. etc., Nature.2006 Apr 27；Described in 440 (7088): 1199-203], people's olfactory sensation mucosa [such as Marshall, CT. etc., Histol Histopathol.2006 Jun:21 (6): described in 633-43], embryonic yolk sac [such as Geijsen N, Nature.2004 Jan 8；Described in 427 (6970): 148-54] and amniotic fluid [Pieternella etc. (2004) Stem Cells.22:1338-1345], it is known that all of which all comprise mescenchymal stem cell.Tissue-derived from these Adherent cell can be by making these cells culture of isolated on the adhesive surface, and thereby the cell separation of other from primitive horde goes out Adherent cell.

No matter originate (such as Placenta Hominis or fatty tissue), the most aseptically extract cell.Once obtain separation Cell, just makes it adhere to, on adhesion material (such as constituting surface), thereby separate adherent cell.Cultivation can be such as embodiment portion Carry out under the conditions of the 2D that the embodiment 4 divided describes and cell can be further transferred to 3D condition.

" adhesion material " used herein refers to have chemical constitution (such as, the charged table that cell can be allowed to be maintained at surface Face exposed groups) synthetic, naturally occur or no cytotoxicity (i.e. bio-compatible) material of a combination thereof.

The example of the adhesion material that can be used for this respect of the present invention includes but not limited to: polyester, polypropylene, polyalkenes, poly- Fluorine vinyl chloride, polrvinyl chloride, polystyrene, polysulfones, cellulose acetate, glass fibre, ceramic particle, matrigel, the outer base of born of the same parents Matter component (such as fibronectin splicing variants, chondronectin, laminin，LN), collagen, poly-L-lactic acid and inert metal fiber.

Can realize being further purified or the step of enriched medium stem cell by the method for it is well known that

(such as by using the FACS of stroma stem cell marker expression, it illustrates further below).

Limiting examples for the basal medium of present invention cultivation includes that the limit must culture medium Eagle, ADC- 1, LPM (without bovine serum albumin), F10 (HAM), F12 (HAM), DCCM1, DCCM2, RPMI 1640, BGJ culture medium (are carried out Do not carry out Fitton-Jackson improvement), minimal medium Eagle (BME-adds Earle ' s base status), Dulbecco ' s Improvement Eagle culture medium (DMEM-serum-free), Yamane, IMEM-20, Glasgow improvement Eagle culture medium (GMEM), Leibovitz L-15 culture medium, McCoy ' s5A culture medium, culture medium M199 (M199E-s base status Han Earle '), culture medium M199 (M199H-s base status Han Hank '), the limit must culture medium Eagle (MEM-E-s base status Han Earle '), the limit must (MEM-NAA, containing nonessential amino for palpus culture medium Eagle (MEM-H-s base status Han Hank ') and necessary culture medium Eagle of the limit Acid), include in other culture medium numerous: culture medium 199, CMRL 1415, CMRL 1969, CMRL 1066, NCTC 135, MB 75261, MAB 8713, DM 145, Williams ' G, Ne μm an&Tytell, Higuchi, MCDB 301, MCDB 202, MCDB 501, MCDB401, MCDB 411, MDBC 153.Preferred culture medium for the present invention is DMEM.These and its Its useful culture medium is purchased from GIBCO, Grand Island, N.Y., USA and Biological Industries, Bet HaEmek, Israel etc..These culture medium a large amount of are summarized in Methods in Enzymology, the LVIII volume, " Cell Culture ", page 6272, William B.Jakoby and Ira H.Pastan edits, and Academic Press, Inc. publish.

Culture medium can add such as serum (fetal blood of such as cattle or other species is clear) and optional or alternative with pik/ml- The somatomedin of milligram/ml level concentration, vitamin (such as ascorbic acid), cytokine, salt (such as B-glycerophosphate), Steroid (such as dexamethasone) and hormone (such as growth hormone, erythropoietin, thrombopoietin, Bai Jie Element 3, interleukin 6, IL-7, M-CSF, c-kit part/stem cell factor, osteoprotegerin ligand, pancreas Island element, insulin like growth factor, epidermal growth factor, fibroblast growth factor, nerve growth factor, ciliary nerves are sought Support the factor, platelet derived growth factor and bone morphogenetic protein).

It should be further appreciated that other component can be added in culture medium.Such component can be antibiotic, antifongin, Albumin, aminoacid and other component cultivated for cell known in the art.Furthermore, it is necessary to time can add component with strengthen point Change process (sees set forth further below).

It it should be understood that, when adherent cell of the present invention gives people experimenter, cell and culture medium are (such as, containing above-mentioned training Support based additive) xenogeneic components should be substantially free of, i.e. there is no any asnimal pollution thing such as mycoplasma.Such as, culture medium can Add serum substitute, human serum and/or synthesis or the factor of restructuring generation.

As described herein, just can be passed on two dimension once acquirement adherent cell or three-dimensional environment (is seen following enforcement The embodiment 1 and 4 of example part).It would be appreciated that can the most described cell be transferred to 3D structure substrate in or Three-dimensional environment (as described above) is passed on after two-dimensional condition.

Therefore, the adhesion material configuring this respect of the present invention is cultivated for 3D, thereby provides and substantially increases for cell The growth substrate of the available attaching surface adhered to, in order to the base structure of simulated tissue (such as Placenta Hominis).

For large-scale production, can realize cultivating in 3D bioreactor.

Such thing reactor example includes but not limited to plug-flow bioreactor, continuous agitator tank biological respinse Device, fixed-bed bioreactor, CelliGenBioreactor system (New Brunswick Scientific ) or BIOFLO 310 bioreactor system (New Brunswick Scientific (NBS) (NBS).

As shown in the embodiment 4 of embodiment part, Celligen bioreactor can under controlled condition (such as, pH, temperature Degree and oxygen level) and utilize constant cell growth medium perfusion to carry out 3D amplification adherent cell.And, can directly monitor thin The concentration level of the glucose of born of the same parents' culture, lactate, glutamine, glutamic acid and ammonium.The glucose consumption speed of adherent cell Rate and lactate synthesis speed allow to measure cell growth rate and determine harvest time.

Other 3D bioreactor that can be used for the present invention includes but not limited to continuous agitator tank bioreactor, wherein makes Culture medium is continuously fed in bioreactor, is extracted out continuously by product, to maintain the time constant steady in reactor.With The stirred-tank bioreactor of fibre bed basket is purchased from such as New Brunswick Scientific Co.Edison, NJ, consolidates (wherein air is generally passed through the bottom of center conduit, flows up shape simultaneously for fixed bed bioreactor, airlift bioreactor Become bubble, at capital separation waste gas), cell containing Polyactive foam inoculation filling type bioreactor (such as Wendt, D. etc., Biotechnol Bioeng 84:205-214, (2003) are described), tubulose poly-L-lactic acid (PLLA) porous support radial flow Filling type bioreactor is [such as Kitagawa etc., Biotechnology and Bioengineering93 (5): 947-954 (2006) described].Other bioreactor that can be used according to the invention be set forth in U.S. Patent No. 6,277,151, No. 6197,575, No. 6139,578, No. 6132,463, No. 5902,741 and No. 5,629,186.

During inoculation, preferred cell inoculation reaches 100,000-1,500,000 cell/mm.An exemplary In, inoculation totally 150 ± 30x10⁶Individual cell, inoculates 3-5x10⁶Individual cell/gr carrier, or inoculation 0.015-0.1x10⁶Individual cell/ ml。

The harvesting and simultaneously avoid uncontrollable differentiation and aging when the cell proliferation of at least about 10%.

Cultivation carries out at least about 2 days, 3 days, 4 days, 5 days, 10 days, 20 days, 1 month or the most longer.It should be appreciated that giving birth to Cultivation in thing reactor may extend this time.Cultivating adherent cell in 3D cultivates can be real under continuously flowing into culture medium Existing.Also can carry out passing on to increase cell number.Will be appreciated that and can change culture medium to extend and to improve condition of culture.

The adherent cell of some embodiments of the invention comprises at least about 10%, 28%, 30%, 50%, 80% or more Proliferative cell (can by monitoring S and the G2/M phase FACS measure).

The adherent cell of some embodiments of the invention can comprise at least one " stromal stem cell phenotype ".

" stromal stem cell phenotype " used herein refer to be derived from the typical structure of substrate (i.e. mesenchyme) stem cell of bone marrow or Function phenotype.

Phrase used herein " stem cell " refers to the cell not having terminal differentiation.

It is therefoie, for example, cell is likely to be of spindle.Alternately or it addition, cell can express stroma stem cell typical case One or a group mark (such as surface markers).The example of stroma stem cell surface markers (positive and negative) includes but does not limits In CD105+, CD29+, CD44+, CD73+, CD90+, CD3-, CD4-, CD34-, CD45-, CD80-, CD19-, CD5-, CD20-, CD11B-, CD14-, CD19-, CD79-, HLA-DR-and FMC7-.Other stroma stem cell labelling includes but not limited to Tyrosine hydroxylase, nestin and H-NF.

Adherent cell according to the placenta tissue that present invention teach that generation has substantially such as the reality of Examples below part Execute the gene expression profile that example 4 describes.

Stroma stem cell typical function phenotype example includes but not limited to that T cell inhibitory activity (does not stimulate T cell and phase Instead it is suppressed), hematopoietic stem cell supports activity and becomes fat, becomes liver, skeletonization arbitrary with become Neural Differentiation.

Arbitrarily these structures or functional character can be used for proving the qualified (enforcement of the embodiment that sees below part of cell of the present invention Example 4).

The protein expression profile of uniqueness it is characterized as, such as the reality of embodiment part according to the cell mass that present invention teach that generation Execute shown in example 1.It is therefoie, for example, can express according to the adherent cell of the Placenta Hominis or fatty tissue that present invention teach that generation and/or The selected factor of secreting high levels.Such as, such cell is expressed or SCF, Flt-3, H2A histone family of secretion (H2AF) or aldehyde dehydrogenase X (ALDH X) be 2D cultivate in growth Placenta Hominis fatty tissue adherent cell express or secretion At least 2,3,4,5,6,7,8,9,10,11 or even 12 times.Additionally or as alternative, cell population secretes of the present invention or expression IL-6, eukaryotic translation elongation factor (EEEF2), net calbindin 3, EF-hands calcium binding structural domain (RCN2) or calcium conditioning egg The level of white 1 basic smooth muscle (CNN1) is the Placenta Hominis of growth in 2D cultivates or the adherent cell expression of fatty tissue or secretes At least 2,3 or 5 times.Additionally or as alternative, the feature of cell mass of the present invention be with 2D cultivate cell compared with various its Its protein expression level is relatively low.It is therefoie, for example, the life in 2D cultivates that secretion or expression are less than 0.6,0.5,0.25 or 0.125 Long Placenta Hominis or the adherent cell of fatty tissue are expressed or heterologous cells nuclear ribonucleoprotein H1 (Hnrph1) of secretion, CD44 resist Former isotype 2 precursor, 3 adenosine phosphates 5 phosphosulfate synthase 2 isotype a (Papss2) or ribosome protein L 7/L a's (rpL7a) Expression.

As shown in embodiment 3-4 of Examples below part, adherent cell especially 3D-adherent cell shows can suppress mixed The monocytic immunoreation of human cord blood in conjunction lymphocyte reaction (MLR) mensuration, therefore display is preferably for clinic Biological activity (such as, T cell inhibitory activity, hematopoietic stem cell support activity).

According to one embodiment of the invention, the adherent cell of the present invention can suppress the immunoreation in experimenter.

Phrase used herein " immunoreation in suppression experimenter " refers to reduce or suppress to occur response in experimenter anti- The immunoreation of former (such as, foreign cell or its part).The immunne response that can be attached Carbazole alkaloid includes that humoral immunization should Answering and cellullar immunologic response, it relates separately to be resisted by antibody and T-lymphocyte (propagation of T cell) special recognition pathogen body Former.

According to one embodiment of the invention, adherent cell of the present invention feature be grown in two dimension (2D) for ratio and cultivate In Placenta Hominis or the higher immunosuppressive activity of adherent cell of fatty tissue.

According to one embodiment of the invention, this immunosuppressive activity includes that T cell propagation reduces.

As described above with described in the embodiment 6 of Examples below part, the adherent cell of the present invention induces body vessel Generate (blood flow in such as hip and lower limb), significantly improve the limb function of artery ligation animal, increase capillary density and drop Suboxides stress and endothelial inflammation.And, the embodiment 7 such as Examples below part describes in detail, and adherent cell of the present invention shows Write and improve in rat model from the recovery of apoplexy.

Therefore, according to a further aspect of the invention, it is provided that treat the method for ischemia in the experimenter needing it.The method is passed through The adherent cell of the present invention giving described subject's effective dose realizes, and thereby treats the ischemia of this experimenter.

Terms used herein " ischemia " refer to be characterized as the not enough or relative any pathology of angiogenesis (disease, situation, Syndrome or disease).Example include but not limited to peripheral arterial disease (PAD) such as limb ischemia and critical limb ischemia (CLI), Ischemic heart desease, ischemic encephalopathy (such as apoplexy), wound healing delay, ulcer healing delay, reproduction associated conditions, tremulous pulse Hardening, ischemic angiopathy, ischemic heart desease, myocardial ischemia, coronary artery disease (CAD), Atherosclerotic cardiovascular Disease, left main coronary artery disease, arterial occlusive disease, periphery ischemia, peripheral vascular disease, renal vascular disease, peripheral arterial disease, Limb ischemia, lower limb ischemia, cerebral ischemia, cerebrovascular, retinopathy, retina reparation, reconstruction obstacle, vonHippel- Lindau syndrome, hereditary hemorrhagic telengiectasia ischemic vascular disease, Buerger are sick, ischemic nephropathy and Ischemia Placenta Hominis.

Terms used herein " is treated " and is referred to suppression or stop the development of pathology (such as ischemia) and/or cause subtracting of pathology Less, alleviate or regression.It will be appreciated by those skilled in the art that the development that various methodology and algoscopy can be used to evaluate pathology, and phase As, various methodologies and algoscopy can be used for evaluating the minimizing of pathology, alleviating or regression.Term " treat " also can refer to alleviate or Reduce the symptom relevant to this pathology.

Terms used herein " needs its experimenter " and refers to any experimenter (such as, mammal), such as, be diagnosed Or suffer from the human experimenter of this pathology.

As described above with described in the embodiment 8 of Examples below part, the inventor have discovered that the adhesion of the present invention is thin Born of the same parents can make connective tissue regeneration and/or reparation.

Therefore, according to a further aspect in the invention, it is provided that treat in the experimenter needing it and need connective tissue regeneration And/or the method for the medical condition repaired.The method realizes by giving the adherent cell of the present invention of subject's effective dose.

Term " connective tissue " refers to comprise collagen bundle, elastic fiber (such as, between muscle and blood vessel and around) and simply The support frame tissue of cell.The example of connective tissue includes but not limited to compact substance connective tissue, and (such as, ligament, tendon, periodontal are tough Band), areolar connective tissue's (such as, there is proteinaceous fiber such as collagen and elastin), reticular connective tissue, fat Tissue, blood, bone, cartilage, skin, intervertebral disc, dental pulp, dentin, gingiva, extracellular matrix (ECM)-formation cell, loose knot Form tissue and smooth muscle cell.

Phrase used herein " needs the medical condition of connective tissue regeneration and/or reparation " and refers to connective tissue impaired (i.e., Non-functional tissue, carcinous or precancerous tissue, rupture tissue, fracture tissue, fibrotic tissue or ischemic tissue) or lose The pathology that (such as wound, infectious disease, heredopathia etc. are rear) is characterized.The limiting examples of this pathology includes fracture, bone Cancer (such as osteosarcoma, osteocarcinoma transfer), burn wound, articular cartilage defect and deep wound.

Term " gives experimenter " and refers to cell of the present invention is introduced target tissue.These cells may originate from receiver or from same Plant allosome or xenogeneic.This phrase is also covered by " transplanting " cell of the present invention, in " cell replacement " or " immigration " experimenter.

This experimenter can be any mammal needing connective tissue regeneration and/or reparation, including such as people or domestic Animal, includes, but are not limited to horse (i.e. equine), cattle, goat, sheep, pig, Canis familiaris L., cat, camel, alpaca, yamma and yak.

According to one embodiment of the invention, the adherent cell of the present invention can be used for treating and includes following situation: soft Under bone, bone cyst, fracture, osteoporosis, osteoarthritis, degeneration bone, various connective tissue lose relevant cancer (such as, bone Cancer, osteosarcoma, Bone tumour), cartilage injury, articular cartilage defect, DDD, osteogenesis imperfecta (OI), burn, burn Wound surface, deep wound, wound healing delay, ligament are impaired and tendon is impaired, and such as horse needs its experimenter (as above with other Literary composition is described) in by the overworked tendon injury caused.

Include that the above-mentioned adhesion can cultivated in three-dimensional or two-dimensional environment is thin according to this cell on the one hand given of the present invention Born of the same parents and mesenchyme thereof and non-mesochymal part or the derivant of terminal differentiation.

From stroma stem cell of the present invention, the method for derivative pedigree specific cell is for it is well known that.See for example the U.S. special Profit No. 5,486,359, No. 5,942,225, No. 5,736,396, No. 5,908,784 and No. 5,902,741.

These cells can be (to see United States Patent (USP) Shen for experiment or genetically modified with derivative goal pedigree first Please No. 20030219423).

These cells can be the fresh or freezing (such as, freezing of autologous or non-autologous (i.e. allogeneic or xenogenesis) Preservation) prepared product.

According to medical condition, can give experimenter other chemicals (the most immunoregulatory, chemotherapy etc.) or Cell.

Because non-autogenous cell can induce immunoreation when giving health, so have developed several methods to reduce row Scold non-self somatic probability.These include suppress receptor immune system or before transplantation by encapsulated for non-autogenous cell In the semipermeable membrane of immunity isolation.

Encapsulated (encapsulation) technology is generally classified as micro encapsulation, and it relates to little spheroid carrier, and big glue Encapsulated (macroencapsulation), it relates to bigger flat board hollow-fibre membrane (Uludag, H. etc., Technology of mammalian cell encapsulation.AdvDrug Deliv Rev.2000；42:29-64).

The method preparing microcapsule is known in the art, the method including the most disclosed below: LuMZ etc., Cell encapsulation with alginate andalpha-phenoxycinnamylidene-acetylated poly (allylamine) .BiotechnolBioeng.2000,70:479-83, Chang TM and Prakash S.Procedures formicroencapsulation of enzymes, cells and genetically Engineeredmicroorganisms.Mol Biotechnol.2001,17:249-60 and Lu MZ etc., A novelcell encapsulation method using photosensitive poly(allylaminealpha- Cyanocinnamylideneacetate) .J Microencapsul.2000,17:245-51.

Such as, by by the collagen of improvement and HEMA (HEMA), methacrylic acid (MAA) and methyl Microcapsule is prepared in the ter-polymers shell complexation of acrylic acid methyl ester. (MMA), obtains the capsule thickness of 2-5 μm.Such micro-glue Capsule can be further with other 2-5 μm ter-polymers shell encapsulation, to provide electronegative smooth surface and to make plasma protein Absorption minimizes (Chia, S.M. etc., Multi-layered microcapsules for cellencapsulation Biomaterials.2002 23:849-56).

Other microcapsule is based on alginate, marine polysaccharide (Sambanis, A.Encapsulatedislets in Diabetes treatment.Diabetes Technol.Ther.2003,5:665-8) or derivatives thereof.Such as, can pass through In the presence of calcium chloride between polyanion sodium alginate and cellulose sodium sulfate and polycation poly-(methylene guanidine altogether) hydrochlorate Polyelectrolyte complex effect prepare microcapsule.

It will be appreciated that improve cell encapsulation when using less capsule.Therefore, it is reduced to from 1mm when capsule size During 400 μm, the quality control of encapsulated cell, mechanical stability, dispersing characteristic and external activity are all improved (Canaple L. etc., Improving cellencapsulation through size control.J Biomater Sci Polym compiles Collect .2002；13:783-96).It moreover has been found that aperture control well little to 7nm, surface chemistry treated (tailored) the nano-pore biological capsule with accurate micro-structure successfully isolates microenvironment (Williams for cellular immunization D.Small is beautiful:microparticle andnanoparticle technology in medical Devices.Med Device Technol.1999,10:6-9；Desai, T.A.Microfabrication technology For pancreatic cellencapsulation.Expert Opin Biol Ther.2002,2:633-46).

Immunosuppressant example includes but not limited to: methotrexate (methotrexate), cyclophosphamide (cyclophosphamide), ciclosporin (cyclosporine), Ciclosporin A (cyclosporin A), chloroquine (chloroquine), oxychloroquine, sulfasalazine (sulfasalazine, sulphasalazopyrine), gold salt, D-penicillium sp Amine (D-penicillamine), leflunomide (leflunomide), azathioprine (azathioprine), Antril (Synergen) (anakinra), infliximab (infliximab) (REMICADE), Embrel (etanercept), TNF α block Agent, the bio-pharmaceutical of targeting inflammatory cytokine, and nonsteroidal anti-inflammatory drugs (NSAID).NSAID example includes but does not limits In: aspirin, choline magnesium trisalicylate, diflunisal (diflunisal), magnesium salicylate, salsalate, sodium salicylate, Diclofenac (diclofenac), etodolac, fenoprofen (fenoprofen), flurbiprofen (flurbiprofen), Yin Diindyl U.S. pungent (indomethacin), ketoprofen (ketoprofen), ketorolac, meclofenamic acid ester, naproxen (naproxen), Nabumetone (nabumetone), Phenylbutazone (phenylbutazone), piroxicam (piroxicam), sulindac, tolmetin (tolmetin), acetaminophen (acetaminophen), ibuprofen (ibuprofen), Cox-2 inhibitor and tramadol (tramadol)。

In either method described herein, cell itself can be given, or pharmaceutically can connect preferably as comprising further Given by a part for the pharmaceutical composition of carrier.

" pharmaceutical composition " used herein refers to containing other chemical constituent (the most pharmaceutically acceptable carrier and excipient) The preparation of adherent cell of the present invention (i.e. selected from the adherent cell of tissue of Placenta Hominis and fatty tissue, it is available from dimensional culture). The purpose of pharmaceutical composition is conveniently to give experimenter by cell.

Term the most used " pharmaceutically acceptable carrier " refers to carrier or diluent, and it is bright that it will not cause experimenter Aobvious stimulation, does not eliminate biologic activity and the characteristic of given compound.The limiting examples of carrier be propylene glycol, saline, The mixture of emulsion and organic solvent and water.

Terms used herein " excipient " refers to be added in pharmaceutical composition further facilitate the inert material giving compound Matter.The limiting examples of excipient includes calcium carbonate, calcium phosphate, various sugar and all kinds starch, cellulose derivative, bright Glue, vegetable oil and Polyethylene Glycol.

According to the preferred embodiments of the invention, pharmaceutical carriers is aqueous saline solution.

Preparation and give the technology of medicine can be at " Remington ' s PharmaceuticalSciences ", Mack Publishing Co., Easton, PA, latest edition finds, it is by quoting and in herein.

Pharmaceutical composition (as described above) can be given with systemic fashion.Or, pharmaceutical composition can be administered locally to, such as Directly pharmaceutical composition is expelled to the tissue area of patient.

Can prepare pharmaceutical composition of the present invention by methods known in the art, such as by conventional mixing, dissolving, Pelletize, make dragee, levigation, emulsifying, encapsulated, bag load or the method for lyophilizing.

Therefore, available one or more of the physiologically acceptable carrier of excipient and auxiliary agent is comprised in a usual manner Being formulated for the pharmaceutical composition of the present invention, described carrier is convenient is processed into the system that can pharmaceutically use by active component Agent.Suitably preparation depends on selected route of administration.

For injection, can the active component of compounding pharmaceutical compositions in aqueous, preferably physical compatibility is slow Rush liquid such as Hank ' s solution, Ringer ' s solution, physiological saline buffer or the refrigerant comprising cryoprotective agent.For warp For mucosa delivery, preparation uses the penetrating agent being suitable to barrier to be infiltrated.Such penetrating agent is the most known in the art.

For any preparation in the inventive method, can cultivate from external and cell measuring and control according to a preliminary estimate Treat effective dose or dosage.Preferably in animal model allocating dosage to reach required concentration or titre.Such information can be used The dosage for people is determined in more accurately.

The poison of active component described herein can be measured in cell cultivation or laboratory animal by Standard in vitro method of pharmacy Property and curative effect.

And cell external from these is cultivated the data measured and obtain zooscopy and be can be used for allotment for the mankind's A series of dosage.Dosage visually dosage form used and route of administration used and change.Can consider that status of patient selects by individual physician Select definite preparation, route of administration and dosage and (see for example Fingl etc., 1975, The Pharmacological Basis of Therapeutics, the 1st chapter page 1).Such as, disturbances in patients with Parkinson disease can be shown the improvement of positive reaction to treatment The Symptom monitoring of motor function.

For injection, the slow of the active component of pharmaceutical composition, preferably physical compatibility can be allocated in aqueous Rush liquid such as Hank ' s solution, Ringer ' s solution or physiological saline buffer.

Dosage and dosing interval can be adjusted individually, so that active ingredient level be enough to effectively be regulated by the cell transplanted Neurotransmitter synthesizes.Reaching the dosage needed for ideal effect will be depending on personal feature and route of administration.Detection test can be used for Measure plasma concentration.

The order of severity according to situation to be treated and reactivity, can single or plural number time be administered, and the course for the treatment of continue some skies To some weeks, or reach the state of palliating a disease.

Certainly, the amount of compositions to be administrated by depending on just connect subject individuality, the ailing order of severity, administering mode, Depending on judgement of the doctor in charge etc..The individual state change of monitoring the most continuously should be made by dosage and arrangement of time Reaction.Such as, indication based on monitoring, the disturbances in patients with Parkinson disease being treated is enough to alleviate the cell concentration of disease symptoms.

Ligament injury model includes but not limited to the rabbit model [Jit-using mescenchymal stem cell to build anterior cruciate ligament again Kheng etc., Arthroscopy (2004) 20 (9): 899-910], the long-acting biological re-absorption for anterior cruciate ligament reparation is propped up The goat model [Altman etc., J AmAcad Orthop Surg. (2008) 16 (4): 177-187] of frame.The model that tendon is repaired Include but not limited to adult New Zealand white rabbit model [Awad etc., Tissue that the tendon that autologous mescenchymal stem cell mediates is repaired Eng. (1999) 5 (3): 267-77].Bone Defect Repari model is described in such as Stem Cells inEndocrinology, Humana Press (2005) 183-206, which depict operation room mesenchymal stem cells for Bone Defect Repari.

Cell of the present invention is preferably survived a period of time (e.g., from about 1 month) in affected areas after transplanting, in order to observes and controls Therapeutic effect.

Can be suitable through preparing, being placed in interior compositions including allotment invention formulation in pharmaceutical compatible carrier Container in；And labelling is for indicating the treatment of situation.

If necessary, the present composition can be stored in bag or distributor (dipenser device) such as FDA approval Test kit in, it can comprise one or more unit dosage forms containing active component.Described bag can such as comprise metal or Plastic tab, such as blister pack (blisterpack).Described bag or distributor can be with being administered explanation.Bag or distributor May also provide with by management drug manufacture, use or bulletin that the container of the form of governmental agency requirements sold is relevant, described Bulletin reflects that the composition forms of this mechanism approval or the mankind are administered or veterinary administration form.Such bulletin can be such as by The prescription drug label of United States food and drag administration's approval or the product description of approval.

Adherent cell of the present invention can suitably be configured to be packaged as suitably preparing the pharmaceutical composition of article.This is prepared Article include packaging material, it include for increase tissue blood vessel generate, treatment ischemia and/or treatment need connective tissue regeneration And/or the label of the pathology repaired；Adherent cell of the present invention packed by wherein said packaging material.

Will be appreciated that adherent cell of the present invention can induce the immunosuppressant in experimenter and/or tolerance.Therefore, this adhesion Cell can be used for treating needs any situation of immunosuppressant and/or tolerance, this situation to include but not limited to autoimmune disease With inflammatory diseases (including acute and chronic inflammatory diseases), include but not limited to cardiovascular disease, rheumatoid disease, adenopathy, gastrointestinal Tract disease, dermatosis, hepatopathy, neuropathy, myonosus, nephropathy, reproduction related disease, connective tissue disease and systemic disease.

The example of autoimmunity cardiovascular diseases include but not limited to atherosclerosis (Matsuura E. etc., Lupus.1998；7 Suppl 2:S135), myocardial infarction (Vaarala O.Lupus.1998；7 Suppl 2:S132), thrombosis Disease (Tincani A. etc., Lupus 1998；7 Suppl2:S107-9), Wegener ' s granulomatosis, Takayasu ' s tremulous pulse Scorching, Kawasaki syndrome (Praprotnik S. etc., Wien Klin Wochenschr 2000 Aug 25；112(15- 16): 660), anti-VIII factor autoimmune disease (Lacroix-Desmazes S. etc., SeminThromb Hemost.2000； 26 (2): 157), necrotizing small vessel vasculitis, microscopic polyangiitis, Churg-Strauss syndrome, few immunity (pauci- Immune) focal necrosis and crescentic glomerulonephritis (Noel LH.Ann Med Interne (Paris) .2000 May；151 (3): 178), antiphospholipid syndrome (Flamholz R. etc., J Clin Apheresis 1999；14 (4): 171), Antibody induction heart failure (Wallukat G. etc., Am J Cardiol.1999 Jun17；83 (12A): 75H), platelet subtracts Few property purpura (Moccia F.Ann Ital Med Int.1999Apr-Jun；14 (2): 114；Semple JW. etc., Blood 1996 May 15；87 (10): 4245), autoimmune hemolytic anemia (Efremov DG. etc., Leuk Lymphoma 1998 Jan；28 (3-4): 285；Sallah S. etc., Ann Hematol 1997 Mar；74 (3): 139), in Chagas disease Heart autoimmune (Cunha-Neto E. etc., J Clin Invest 1996 Oct 15；98 (8): 1709) and anti-auxiliary T Lymphocyte autoimmune (Caporossi AP. etc., ViralImmunol 1998；11 (1): 9).

The rheumatoid example of autoimmunity includes but not limited to rheumatoid arthritis (KrennV. etc., Histol Histopathol 2000 Jul；15 (3): 791；Tisch R, McDevitt HO.Proc Natl Acad Sci units S A 1994 Jan 18；91 (2): 437) and ankylosing spondylitis (Jan Voswinkel etc., Arthritis Res 2001；3 (3): 189).

The example of autoimmune gonadopathy includes but not limited to pancreopathy, type i diabetes, thyropathy, Graves disease, first Shape adenitis, SAT, Hashimoto ' s thyroiditis, idiopathic myxedema, ovary self Immunity, autoimmune anti-sperm is sterile, autoimmune prostatitis and I type autoimmune polyglandular syndrome.Disease Disease includes but not limited to the autoimmune disease of pancreas, type i diabetes (Castano L. and Eisenbarth GS.Ann.Rev.Immunol.8:647；Zimmet P.Diabetes Res ClinPract 1996Oct；34 Suppl: S125), autoimmune thyroid disease, Graves disease (Orgiazzi J.Endocrinol Metab Clin North Am 2000 Jun；29 (2): 339；Sakata S. etc., Mol Cell Endocrinol 1993 Mar；92 (1): 77), spontaneous Autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15；165 (12): 7262), Hashimoto ' s thyroiditis (Toyoda N. etc., Nippon Rinsho 1999Aug；57 (8): 1810), special Property myxedema (Mitsuma T.Nippon Rinsho.1999Aug；57 (8): 1759), ovarian autoimmunity (Garza KM. etc., J Reprod Immunol 1998Feb；37 (2): 87), autoimmune anti-sperm sterile (Diekman AB. etc., Am J ReprodImmunol.2000 Mar；43 (3): 134), autoimmune prostatitis (Alexander RB. etc., Urology 1997 Dec；50 (6): 893) and I type autoimmune polyglandular syndrome (Hara T. etc., Blood.1991 Mar1；77 (5): 1127).

The example of autoimmune gastrointestinal tract disease includes but not limited to chronic inflammatory bowel disease (Garcia Herola A. etc., Gastroenterol Hepatol.2000 Jan；23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y.Harefuah 2000 Jan 16；138 (2): 122), colitis, ileitis and Crohn ' s are sick.

The example of autoimmune skin disease includes but not limited to autoimmune bullous diseases, such as but not limited to Pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

The example of autoimmune liver disease includes but not limited to hepatitis, ACAH (Franco A. etc., Clin Immunol Immunopathol 1990 Mar；54 (3): 382), primary biliary cirrhosis (Jones DE.Clin Sci(Colch)1996 Nov；91 (5): 551；Strassburg CP. etc., Eur J Gastroenterol Hepatol.1999 Jun；11 (6): 595) and autoimmune hepatitis (Manns MP.J Hepatol 2000 Aug；33 (2): 326).

The example of Autoimmune neuropathies disease includes but not limited to multiple sclerosis (CrossAH. etc., J Neuroimmunol 2001 Jan 1；112 (1-2): 1), Alzheimer (OronL. etc., J Neural Transm Suppl.1997；49:77), myasthenia gravis (Infante AJ. and Kraig E, Int Rev Immunol 1999；18(1- 2): 83；Oshima M. etc., Eur JImmunol 1990 Dec；20 (12): 2563), neuropathy, motor neuron (Kornberg AJ.JClin Neurosci.2000 May；7 (3): 191)；Guillain-Barre syndrome and autoimmunity god Through sick (Kusunoki S.Am J Med Sci.2000 Apr；319 (4): 234), myasthenia, Lambert-Eaton myasthenia Syndrome (Takamori M.Am J Med Sci.2000 Apr；319 (4): 204)；Paraneoplastic sacred disease, cerebellum are withered Contracting, paraneoplastic cerebellar atrophy and stiff man syndrome (Hiemstra HS. etc., Proc Natl Acad Sci units S A 2001 Mar 27；98 (7): 3988)；Non-paraneoplastic stiff man syndrome, Progressive symmetric erythrokeratodermia cerebellar atrophy, encephalitis, Rasmussen ' s Encephalitis, amyotrophic lateral sclerosis, Sydenham chorea, coprolalia syndrome and autoimmune polyendocrinopathy (Antoine JC. and Honnorat J.Rev Neurol (Paris) 2000 Jan；156 (1): 23)；Dysimmune is neural Sick (Nobile-Orazio E. etc., Electroencephalogr Clin Neurophysiol supplementary issue 1999；50:419)；Obtain Obtain property neuromyotonia, congenital multiple arthrogryposis (Vincent A. etc., Ann N Y Acad Sci.1998 May13； 841:482), neuritis, optic neuritis (Soderstrom M. etc., J Neurol NeurosurgPsychiatry 1994 May；57 (5): 544) and neurodegenerative disease.

The example of autoimmunity muscle disease includes but not limited to myositis, autoimmune myositis and constitutional Sjogren ' s syndrome (Feist E. etc., Int Arch Allergy Immunol 2000Sep；123 (1): 92) and flat Sliding flesh autoimmune disease (Zauli D. etc., BiomedPharmacother 1999 Jun；53 (5-6): 234).

The example of autoimmune nephrosis includes but not limited to nephritis and autoimmune interstitial nephritis (Kelly CJ.J Am Soc Nephrol 1990 Aug；1 (2): 140).

The example of the autoimmune disease that reproduction is relevant includes but not limited to repeatability fetal loss (fetalloss) (Tincani A. etc., Lupus 1998；7 Suppl 2:S107-9).

The example of autoimmune connective tissue disease includes but not limited to otopathy, autoimmunity otopathy (Yoo TJ. Deng, Cell Immunol 1994 Aug；157 (1): 249) and autoimmune disease (Gloddek B. etc., the Ann N of internal ear Y Acad Sci 1997 Dec 29；830:266).

The example of autoimmunity systemic disease include but not limited to systemic lupus erythematosus (Erikson J. etc., Immunol Res 1998；17 (1-2): 49) and Systemic sclerosis (Renaudineau Y. etc., Clin Diagn Lab Immunol.1999 Mar；6 (2): 156)；ChanOT. etc., Immunol Rev 1999 Jun；169:107).

Additionally, adherent cell can be used for treating the disease that graft transplantation is relevant, include but not limited to transplant rejection, slow Property transplant rejection, subacute graft rejection, hyperacute graft rejection, acute transplant rejection and graft versus host disease.

Terms used herein " about " refers to ± 10%.

Investigating hereafter after non-limiting example, other objects of the present invention, advantage and new feature are to this area skill Will be apparent to for art personnel.Additionally, describe various with each present invention required by claims which follow part as above Embodiment and aspect can find experiment to support in examples below.

Embodiment

With reference now to following example, it illustrates the present invention together with described above in a non-limiting manner.

Experimental implementation used by nomenclature used herein and the present invention generally includes molecule, biochemistry, microbiology and restructuring DNA technique.These technology have detailed explanation in the literature.See for example: " Molecular Cloning:A laboratory Manual " Sambrook etc., (1989)；" Current Protocols in Molecular Biology " the I-III volume, Ausubel, R.M. edit (1994)；Ausubel etc., " Current Protocols in Molecular Biology ", John Wiley and Sons, Baltimore, Maryland (1989)；Perbal, " A Practical Guide ToMolecular Cloning ", John Wiley&Sons, New York (1988)；Watson etc., " Recombinant DNA ", Scientific American Books, New York；Birren etc. (editor) " Genome Analysis:A Laboratory Manual Series ", the 1-4 volume, Cold Spring Harbor Laboratory Press, New York(1998)；As U.S. Patent No. 4,666,828, No. 4,683,202, No. 4,801,531, No. 5,192,659 With No. 5,272,057 method proposed；" Cell Biology:A Laboratory Handbook ", the I-III volume Cellis, J.E. edit (1994)；" Current Protocols in Immunology " the I-III volume Coligan J.E. Editor (1994)；Stites etc. (edit), " Basic and ClinicalImmunology " (the 8th edition), Appleton& Lange, Norwalk, CT (1994)；Mishell and Shiigi (edits), " Selected Methods in Cellular Immunology ", W.H.Freeman and Co., New York (1980)；Useful immunoassay be extensively set forth in patent and In scientific literature, see for example U.S. Patent No. 3,791, No. 932, the 3rd, 839, No. 153, the 3rd, 850, No. 752, the 3rd, 850, No. 578, No. 3,853,987, No. 3,867,517, No. 3,879,262, No. 3,901,654, No. 3,935,074, No. 3,984,533, No. 3,996,345, No. 4,034,074, No. 4,098,876, No. 4,879,219, the 5th, No. 011,771 and No. 5,281,521；" Oligonucleotide Synthesis " Gait, M.J. edits (1984)；″ " Hames, B.D., and HigginsS.J. edit (1985) to Nucleic Acid Hybridization；″Transcription " Hames, B.D., and Higgins S.J. edits (1984) to and Translation；″Animal Cell Culture” Freshney, R.I. edit (1986)；" Immobilized Cells and Enzymes " IRL Press, (1986)；″ APractical Guide to Molecular Cloning " Perbal, B., (1984) and " Methods InEnzymology " the 1-317 volume, Academic Press；" PCR Protocols:A Guide ToMethods and Applications ", Academic Press, San Diego, CA (1990)；Marshak etc., " Strategies for Protein Purification and Characterization-ALaboratory Course Manual″CSHL Press(1996)；All above passing through as illustrated completely herein is quoted and in herein.Run through the literature and provide it The reference material that it is general.Think that method therein is for it is well known that, it is provided that they are readers for convenience.Included in it All information by quoting and in herein.

Embodiment 1

Produce from bone marrow, Placenta Hominis and fatty tissue and cultivate adherent cell

In the bioreactor system containing 3D carrier, cultivation adherent cell is to produce 3D-adherent cell, it is characterized by spy Different cell marking expresses overview.Growth efficiency is detected by cell counting.These are detected by cultivating in division culture medium The differentiation capability of cell.

Material and experimental implementation

Bone marrow adherent cell-from the breast of the healthy donor sucking-off of the hematology carrying out cardiac operation under direct vision or BM biopsy Bone bone marrow obtains bone marrow (BM) adherent cell.By Bone marrow aspirates with Hank ' s balanced salt solution (HBSS；GIBCO BRL/ Invitrogen, Gaithersburg MD) dilute three times and carry out Ficoll-Hypaque (Robbins Scientific Corp.Sunnyvale, CA) density gradient centrifugation.Afterwards, myelomonocyte (＜ 1.077gm/cm is collected³), in HBSS Wash three times and be resuspended in growth medium and [with the addition of 10%FCS (GIBCO BRL), 10-4M mercaptoethanol (Merck, White House Station, NJ), Pen .-Strep-nystatin mixture (100U/ml: 100 μ g/ml: 1.25un/ml； Beit Ha ' Emek), DMEM (Biological Industries, the Beit of 2mM L-glutaminate (Beit Ha ' Emek) Ha ' emek, Israel)].In tissue culture flasks, (Corning, Acton, MA) is in 37 DEG C of (5%CO₂) cultivate respectively from respectively The cell of individual donor, changes weekly culture medium.Every 3-4 days thin with 0.25% trypsin-EDTA (Beit Ha ' Emek) division Born of the same parents.After passing 2-40 generation, when reaching 60-80% and converging, collect cell for analyzing or for the cultivation in bioreactor.

(Bnei Zion cures to be derived from the inside part of the adherent cell of Placenta Hominis-aseptically cut term labor Placenta Hominis Center, Haifa, Israel), with Hank ' s buffer solution 3 times, in 37 DEG C, (1mg/ml organizes with 0.1% collagenase； Sigma-Aldrich, St.Lewis, MO) hatch 3 hours.Aspirate gently, then with the addition of 10%FCS, penicillin-strepto- The DMEM washing of element-nystatin mixture (100U/ml: 100 μ g/ml: 1.25un/ml) and 2mML-glutamine suspends Cell, is inoculated into 75em²At 5%CO in culture bottle and in 37 DEG C₂Hatch in tissue culture's incubator under humidification conditions.Hereafter Make cell adhere to 72 hours at frosting, then often within 3-4 days, change a subculture.(usual 10-is converged when reaching 60-80% 12 days) time, with 0.25% trypsin-EDTA from grown cultures bottle separation cell, and it is inoculated in new culture bottle.Hereafter collect The cell cultivated is for analyzing or for the cultivation in bioreactor.

It is derived from the adherent cell of fatty tissue-obtain from the human fat tissue (RambamHaifa, Israel) of liposuction operation Adherent cell.Thoroughly wash fatty tissue with isopyknic PBS, digest 30 minutes in 37 DEG C with collagenase (20mg/ml).Then With containing 10%FCS, Pen .-Strep-nystatin mixture (100U/ml: 100 μ g/ml: 1.25un/ml) and L-paddy The DMEM washed cell of glutamine, is centrifuged 10 minutes under room temperature (RT) with 1200rpm, with lysate resuspended (1: 10； Biological Industries, Beit Ha ' emek, Israel, to discard erythrocyte), centrifugal, with containing 10%FCS, green grass or young crops Mycin-streptomycin-nystatin mixture (100U/ml: 100 μ g/ml: 1.25un/ml) and the DMEM of L-glutaminate are resuspended. Then with 3-10x 10⁷Washed cell is inoculated into sterile tissue culture media culture bottle by individual cell/culture bottle.Second day use PBS washed cell is to remove RBC and the dead cell of residual.In 37 DEG C at 5%CO₂Under humidification conditions in tissue culture's incubator Keep cell.Within every 3-4 days, change culture medium.When 60-80% converges, with 0.25% trypsin-EDTA from grown cultures bottle Separate cell, and be inoculated in new culture bottle.Pass 2-40 generation after, when reaching 60-80% and converging, collect cell for analyze or For the cultivation in bioreactor.

PluriX^TMThe 3D of plug-flow bioreactor-fill with the 1-100ml that is made up of non-woven fiber polyester matrix Porrosive carrier (diameter 4mm) loads PluriX^TMPlug-flow bioreactor (Pluristem, Haifa, Israel；As Described in Fig. 1 G, referring also to U.S. Patent No. 6,911, No. 201).These carriers make to breed in relatively small volume in a large number Cell number.Glass drying oven is designed by Pluristem (Pluristem, Haifa, Israel) and is manufactured.Bioreactor is held in In 37 DEG C of incubators, and regulated by valve (6a in Fig. 1 G) and peristaltic pump (in Fig. 1 G 9) and monitored flow velocity.Bioreactor Comprise sampling and injection point (in Fig. 1 G 4) so that can vaccinization cell.Supply pH 6.7-from storehouse (Fig. 1 G 1) The culture medium of 7.4.To this storehouse supply air/CO containing different proportion₂/O₂(depending on the cell density in bioreactor) The admixture of gas of filtration (in Fig. 1 G 2,3).O₂Ratio be suitable for bioreactor outlet molten O₂Level, it is by monitoring Device (in Fig. 1 G 6) measures.Via silicone tubing or bubbler (Degania Bet, Emek Hayarden, Israel) by gas Mixture is supplied to described storehouse.Culture medium flows through the separation container (in Fig. 1 G 7) of the non-adherent cell can collected in circulation. By peristaltic pump (in Fig. 1 G 9), culture medium is circulated.The sample point that bioreactor is further configured with adding is (in Fig. 1 G 10) with for exchanging continuously the container of culture medium.

Produce 3D-adherent cell-non-converge primary people with what trypsin treatment was cultivated as described above and adhere to 2D cell Culture, washing, it is resuspended in and with the addition of 10%FBS, Pen .-Strep-nystatin mixture (100U/ml: 100ug/ml : 1.25un/ml) and the DMEM of 2mM L-glutaminate in, via injection point inoculate (10³-10⁵Individual cell/ml) to aseptic piston On 3D carrier in flowing bioreactor (seeing Fig. 1 G).Inoculation before by PBS-Ca-Mg (Biological Industries, Beit Ha ' emek, Israel) insert bioreactor, autoclaving (120 DEG C, 30 minutes), with heat-inactivated containing 10% Hyclone and the Dulbecco ' of Pen .-Strep-nystatin mixture (100U/ml: 100ug/ml: 1.25un/ml) S growth medium washs.Flow velocity is maintained at 0.1-5ml/ minute.Seeded process could involve ceasing to circulation 2-48 hour, thereby makes Cell deposition is on carrier.Bioreactor is maintained under controlled temperature (37 DEG C) and pH condition (pH=6.7-7.4)；According to Need to use supply filtrated air and CO₂Incubator.Displacement 2-3 secondary growth culture medium weekly.By fresh DMEM culture medium Within every 4 hours to 7 days, replace Cyclic culture base.It is 1x10 in density⁶-1x10⁷During individual cell/ml (after growth 12-40 days), from life Thing reactor removes total culture volume, bioreactor and carrier is washed 3-5 time with PBS.Then with trypsin-EDTA 3D-adherent cell is separated from carrier；(Biological Industries, Beit Ha ' emek, Israel；Vibrate 3-15 gently Minute, 1-5 time), hereafter it is resuspended in DMEM freezing.

3D-adherent cell quality biomass measures-thaws and count the 3D-adherent cell of freezing.For assessment cell Viability, by 2x10⁵Individual cell is inoculated into 150cm²Tissue culture's culture bottle in, assess its adhesive capacity in 7 days after inoculation Breed again.Hereafter 3D-is analyzed with fluorescent monoclonal antibody flow cytometer (Beckman Coulter, Fullerton, CA) Adherent cell membrane marker phenotype.

The cell membrane marker profile of the adherent cell that 3D and 2D cultivates is compared with flow cytometric assays.To train from 2D Support the 0.1ml cultivation that 100,000-200,000 adherent cell in thing and 3D running system culture is suspended in 5ml test tube In base, hatch with following each MAb of saturated concentration (4 DEG C, 30 minutes, dark situation): the anti-human CD90 that FITC-puts together Anti-human CD73 that (Chemicon International company, Temecula, CA), PE put together (Bactlab Diagnostic, Ceasarea, Israel), the PE anti-human CD105 (eBioscience, San Diego, CA), the FITC that put together put together anti-human It is anti-human that anti-human CD45 (eBiosience), the PE that CD29 (eBioscience, San Diego, CA), Cy7-PE put together puts together Anti-human CD14 Mab (IQProducts) that CD 19 (IQProducts, Groningen, The Netherlands), PE put together, It is anti-human that antihuman CD 34 (IQProducts) that anti-human CD11b (IQProducts) that FITC puts together and PE puts together or FITC put together HLA-DR Mab(IQProducts).After hatching, washed cell twice in the ice-cold PBS comprising 1% heat-inactivated FCS, resuspended In 500 μ l Q.5% formaldehyde, analyze with FC-500 flow cytometer (Beckman Coulter, Fullerton, CA).

The protein profiles of the adherent cell that 3D and 2D cultivates is compared with mass spectral analysis.Produce from Placenta Hominis as mentioned above and be derived from 2D With the adherent cell that 3D cultivates program.In short, by humidification 5%CO₂In 37 DEG C at 175cm under atmosphere²Culture bottle is cultivated 0.3-0.75x10⁶Individual cell 4 days is until reaching 60-80% and converging to produce 2D culture.By containing 2000 carriers Bioreactor is inoculated 2-10x10⁶Individual cell/gram and cultivate and over 18 days, produce 3D culture.Washed cell (X3) after results To remove all serum, precipitation is the most freezing.According to the scheme of manufacturer, [use Tri test kit from precipitate separation protein (Sigma, Saint Louis, USA), with trypsinization and with iTRAQ reagent labelling (Applied Biosciences, Foster City, CA)].In short, iTRAQ reagent is non-polymeric isobaric labelling reagent.By four kinds of commensurability different sequences Isotope-coded labelling in a kind of via the peptide in its N-terminal and/or each sample of lysine side-chain labelling.Mix four Labeled sample, uses analytical reagent composition peptide.After peptide fragmentation, the quality report ion that every kind of labelling release is unique, therefore, four Kind report ion ratio be given particular peptide in sample relative abundance (information is shown in: http: // docs.appliedbiosystems.com/pebiodocs/00113379.pdf)

Use at the Smoler Proteomics heart (department of Biology, Technion, Haifa, Israel) LC-MS/MS carries out being derived from the 2D training of the adherent cell of Placenta Hominis on QTOF-Premier (Waters, San Francisco, CA) Support the thing Proteomic analysis to 3D culture, and by Pep-Miner software [Beer, I. etc., Proteomics, 4,950- 60 (2004)] human segmental for nr data base identifies and analyzes.Analyzed protein has: foreign cell core ribose Nucleoprotein H1 (Hnrph1, GeneBank accession number NP_005511), H2A histone family (H2AF, GeneBank accession number NP_ 034566.1), eukaryotic cell translation elongation factor 2 (EEEF2, GeneBank accession number NP_031933.1), net calbindin 3, EF-hands calcium binding structural domain (RCN2, GeneBank accession number NP_065701), CD44 isotype 2 precursor (GeneBank accession number NP_001001389), calcium conditioning albumen 1 basic smooth muscle (CNN1, GeneBank accession number NP_ 001290), 3 adenosine phosphates 5 phosphosulfate synthase 2 isotype a (Papss2, GeneBank accession number NP_004661), ribosome Albumen L7a (rpL7a, GeneBank accession number NP_000963) and aldehyde dehydrogenase X (ALDHX, GeneBank accession number P47738).Each experiment carries out 2 times.Because the characteristic analyzed, each protein is according to occurring that peptide number in the sample divides Analysis (protein occurs 2-20 time in analyzing each time).

The protein-produced as above of secretion is compared in the adherent cell that 3D and 2D cultivates from Placenta Hominis with ELISA It is derived from 2D and 3D and cultivates the adherent cell of operation, and 3D cultivates and continues 24 days.Hereafter collection condition culture medium, independent at three In experiment, with ELISA (R&D system, Minneapolis, MN) to Flt-3 part, IL-6, thrombopoietin (TPO) and Stem cell factor (SCF) is analyzed.Result is standardized as 1x10⁶Individual cell/ml.

Osteoblast differentiation culture medium-by by with the addition of 10%FCS, 100nM dexamethasone, 0.05mM ascorbic acid The osteoblast differentiation culture medium that 2-phosphate, the DMEM of 10mM B-glycerophosphate form cultivates cell within 3 weeks, assess into Bone cell differentiation.By Alizzarin Red S dyeing display calcified matrix, by alkaline phosphatase assay test kit (all reagent From Sigma-Aldrich, St.Lewis, MO) detection of alkaline phosphatase.

Experimental result

PluriX^TMBioreactor system creates physiology sample microenvironment

In order to adherent cell provide effective condition of culture, with PluriX bioreactor (Pluristem, Haifa, Israel；Carrier is illustrated in Fig. 1 G, is shown in Figure 1B before inoculation) artificial creation's physiology sample environment (described in Figure 1A).As Shown in Fig. 1 C-F, bone marrow produce 3D-adherent cell in 3D substrate 20 days after inoculation (Figure 1B-C, respectively amplify x150 and 250) within and 40 days, (Fig. 1 C-D amplifies x350 and 500 respectively) is successfully cultivated and expands.

The cell being grown in PluriX bioreactor system is by the Placenta Hominis that is derived from of notable amplification-different production batch 3D-adherent cell grows in PluriX bioreactor system.Inoculum density is that 13,300 cell/carriers (are to total amount 2x10⁶Individual cell).After inoculating 14 days, cell density increases to 15 times, reaches about 200,000 cell/carrier (Fig. 2), or 30x10 in the bioreactor of 150 carriers⁶Individual.In different experiments, by cell with 1.5x10⁴The density of individual cell/ml Being inoculated in bioreactor, after inoculating 30 days, carrier contains the cell quantity having more than 50 times, the most about 0.5x10⁶Individual cell/load Body or 0.5x10⁷Individual cell/ml.Cell density on the carrier of various horizontal growth posts is consistent, illustrates that oxygen and nutrient are equal One is delivered to cell.Growth and long term maintenance offer that 3D culture systems is high density mesenchymal cell culture are thus provided Holding condition, described cell culture can effectively grow into be enough to be used in supporting to move into and the amount of successful implantation purpose.

The membrane marker feature of 3D-adherent cell display uniqueness-in order to determine secretion overview and the protein of shla molecule Produce the difference of (cultivating operation by the 3D of analog bone environment to implement), carry out FAC analysis.As shown in Figure 3A, cell mark The facs analysis of note describes, and 3D-adherent cell shows the labelling table different from the adherent cell of growth in 2D condition Reach pattern.With 3D cultivate cell compared with, 2D cultivate cell express the positive membrane marker CD90 of significantly higher level, CD105, CD73 and CD29 membrane marker.Such as, the cell that 3D cultivates shows that the CD105 of 56% expresses, and the cell that contrast 2D cultivates is 87%. The adherent cell of both 2D and 3D Placenta Hominis culture does not the most express any hemopoietic membrane marker (Fig. 3 B).

The unique soluble factor overview-hemopoietic tabernacle (niche) of 3D-adherent cell display include producing abundant cell because of Son, chemotactic factor and the support cell of somatomedin.In order to further determine that the difference between the adherent cell that 2D and 3D cultivates, The overview of four kinds of main hemopoietic secretory proteins in 2D and 3D adherent cell culture conditioned medium is obtained by ELISA.Figure 4A-C shows that the cell being grown in 3D condition produces containing higher levels of Flt-3 part (Fig. 4 A), IL-60 (Fig. 4 B) and SCF (figure Conditioned medium 4C), and in the conditioned medium of 2D culture, the IL-6 of reduced levels detected and close to zero level Flt-3 part and SCF.In two kinds of cultures, thrombopoietin (TPO) yield is the lowest and equal.

3D-adherent cell shows the protein profiles-in order to further determine that what 2D and 3D cultivated of uniqueness in mass spectral analysis Difference between adherent cell, with the protein profiles of these cells of mass spectral analysis.The adherent cell that Fig. 4 D display 2D and 3D cultivates Show visibly different protein expression profile.As shown in table 1 below, 3D cultivate cell demonstrate higher H2AF and ALDHX expression (respectively more than 9 and 12 times) and higher EEEF2, RCN2 and CNN1 protein level (be about 3 respectively, 2.5 and 2 times).It addition, the cell that 3D cultivates shows albumen Hnrphl and the CD44 isotype 2 precursor expression water of about half Papss2 and the rpL7a expression of gentle about 1/3rd.

Table 1

3D-adherent cell has the osteoblastic ability-in order to further characterize 3D-adherent cell, at skeletonization of being divided into Cell is cultivated 3 weeks by cell differential medium.Hereafter calcium deposit is implemented.The cell of differentiation shows and creates calcium (in Fig. 5 A-B Red described), and compared with control cells maintains fibroblast sample phenotype and shows do not have mineralising (Fig. 5 C-D).These result tables It is osteoblastic ability that the bright 3D-adherent cell being derived from Placenta Hominis has vitro differentiation.

Embodiment 2

The 3D-adherent cell being derived from Placenta Hominis improves the capability evaluation that HSC moves into

By detecting human hematopoietic cell in the immunodeficiency NOD-SCID mice through sub-Lethal irradiation or chemotherapy pretreatment (hCD45+) level, the support HSC assessing 3D-adherent cell moves into.

Material and experimental technique

Separation CD34+ cell-take cord blood sample (BneiZion Medical during giving a birth under aseptic condition Center, Haifa, Israel), by Lymphoprep (Axis-Shield PoCAs, Oslo, Norway) density gradient centrifugation Fractionated mononuclear cell freezen protective.Wash the mononuclear cell thawed and with AntiCD3 McAb 4 antibody incubation, use midi MACS (MiltenylBiotech, Bergish Gladbach, Germany) separates.Cell from more than one sample is merged and reaches To required amount (50,000-100,000 cell).

The cell of transplanting-raise 7 week old in aseptic open systems cage is detected male and female in mice via radiation NOD-SCID mice (NOD-CB17-Prkdcscid/J；Harlan/Weizmann Inst., Rehovot Israel), give Aseptic diet and autoclaved acid water.Sub-lethal (350cGy) radiation murine, hereafter (radiates latter 48 hours) and passes through vein It is injected to tail vein, transplants 50,000-100,000 hCD34+ cell, add or be not added with other being derived from Placenta Hominis or fat group Adherent cell (the 0.5x10 knitted⁶-1x10⁶) (often 3-7 mice of group).After transplanting 4-6 week, mice is put to death in dislocation, buffers with FACS Liquid (50ml PBS, 5ml FBS, 0.5ml 5% Hydrazoic acid,sodium salt) rinses at Thigh bone and tibia two and collects BM.Thin by streaming People's cell in born of the same parents' art detection mice BM, by by cell and anti-human CD45-FITC (IQ Products, Groningen, The Netherlands) hatch, obtain expressing people and the cell hundred of Mus CD45 hematopoietic cell markers in the NOD-SCID mice processed Proportion by subtraction.Clear and definite people moves into minimum threshold values and is appointed as 0.5%.

The detection of transplanted cells in the mice that chemotherapy processes-as male to 6.5 week old raised as described in radiation murine in above NOD-SCID mice (NOD.CB 17/JhkiHsd-scid；Harlan, Rehovot Israel) accept busulfan (25mg/kg- Continuous 2 days) peritoneal injection.Second time injection busulfan two days later, alone CD34+ cell or with produce from Placenta Hominis 0.5x10⁶Individual adherent cell injects mice together.After transplanting 3.5 weeks, put to death mice, above to measuring people as described in radiation murine The existence of hematopoietic cell.

Experimental result

3D-adherent cell improve in radiation murine HSC move into-in NOD-SCID mice via radiation co-transplantation people CD34 + hematopoietic cell and the 3D-adherent cell being derived from Placenta Hominis or fatty tissue.After co-transplantation 4 weeks assessment move into efficiency, and with list The mice transplanted with HSC is compared.As shown in table 2, with alone UCB CD34+ cell process mice compare, 3D-adherent cell and UCB CD34+ cell co-transplantation causes the people's cell in significantly higher immigration rate and higher levels of recipient mice BM.

Table 2

The cell transplanted	Average h-CD45	STDEV
			CD34	3.8	7.9
CD34+3D-adherent cell from Placenta Hominis	5.1	12.2
			CD34+3D-adherent cell from fat	8.7	9.6

3D-adherent cell improve chemotherapy mice HSC move into-by people's CD34+ hematopoietic cell be derived from the 500 of Placenta Hominis, 000-2D-adherent cell or 3D-adherent cell co-transplantation are in the NOD-SCID mice of chemotherapy pretreatment.In co-transplantation Assessment in 3.5 weeks afterwards moves into efficiency, and the mice transplanted with alone HSC is compared.As shown in table 3 and Fig. 6, with alone UCBCD34+ Cell is compared, and adherent cell and UCB CD34+ cell co-transplantation cause the immigration higher level in recipient mice BM.Additionally, As shown in table 3, the adherent cell (3D-adherent cell) being derived from Placenta Hominis being used in PluriX bioreactor system growth is common The mice transplanted, than the cell co-transplantation from same donor of growth in common static 2D condition of culture (culture bottle) Mice averagely move into higher level.

Table 3

The cell transplanted	Average h-CD45	STDEV
			CD34	0.9	1.1
CD34+ is from the conventional 2D culture of Placenta Hominis	3.5	0.2
			CD34+ is from the 3D-adherent cell of Placenta Hominis	6.0	7.9

Facs analysis result shown in Fig. 7 A-B proves the advantage of adherent cell and hHSC (Fig. 7 B) co-transplantation, and After HSC transplants, adherent cell improves the ability that hemopoietic system recovers.

These results integrate and show, after adherent cell can transplant (autologous or allogeneic) at HSC, conduct is supported Cell improves Radiation in jury.3D-adherent cell improves hematopoietic stem cell and/or the ability of CFU-GM immigration after HSC transplants May be from the cell that 3D-adherent cells secrete can improve the support HSC of the going back to the nest of transplanted cells, self renewal and multiplication capacity The ability of the factor, or go back to the nest from the transplantable HSC of those cellular reconstitutions and breed the ability of required impaired hematopoieticmicroenviron-ment.

Embodiment 3

The adherent cell suppression lymphocyte responses cultivated by 2D and 3D

The immunity of adherent cell especially 3D-adherent cell suppression Human Umbilical Cord's blood monocyte is found in MLR analyzes Reaction.

Material and experimental technique

Mixed lymphocyte reaction (MLR) algoscopy-by MLR algoscopy measure be derived from 2D and 3D cultural method from tire The immunosuppressant of the adherent cell that dish produces and immune privilege characteristic, MLR algoscopy measurement is positioned at the tissue compatible of HLA locus Property, come according to the rate of increase of incompatible lymphocyte in the mixed culture of responsive cell (propagation) and stimulation cell (non-proliferative) Realize.Use human cord blood (CB) mononuclear cell (2x10⁵) as responsive cell, its by with equivalent (10⁵) via radiation (3000Rad) be derived from human peripheral blood mononuclear cell (PBMC) or with 2D or 3D cultivate the adherent cell produced by Placenta Hominis Or the co-cultivation of adherent cell and PBMC combination stimulates.Each mensuration is in triplicate.Cell RPMI in 96-orifice plate 1640 culture medium are (containing 20%FBS at the 5%CO being humidified₂In 37 DEG C under atmosphere) in co-cultivation 4 days.Last 18 little cultivate Time with 1 μ C³H-this plate of thymidine pulse.Then collect cell through glass fibre filter, absorb with the quantitative thymidine of scintillation counter.

Experimental result

The immunne response of Fig. 8 A display CB cell, as when stimulating with PBMC, the propagation increase of these cells shows, Lower this is likely to relevant with the T cell propagation responding HLA incompatibility without being bound by any theory.But, when with the present invention When adherent cell is hatched, these cells demonstrate the lowest immunne response level.Additionally, when common with these adherent cell When hatching, the immunne response of PBMC is substantially reduced by CB.Therefore, in the way of similar to MSC, find that adherent cell has latent Reduce donor's cells T cell propagation (typically GvHD) ability.Although two kinds of cultures of 2D and 3D all reduce Lymphocyte immunity response, but consistent with the further advantage of 3D-adherent cell mentioned above, the immunosuppressant of 3D adherent cell Higher.

Embodiment 4

The adherent cell that the 3D adherent cell of relatively PLURIX production and CELLIGEN produce

In order to provide large-scale 3D adherent cell, apply the new production system of a kind of herein referred to as Celligen.

Material and experimental technique

PluriX^TMPlug-flow bioreactor-as described in Example 1.

3D-adherent cell (PLX cell)-as described in Example 1 is produced by Plurix.

Celligen^TMPlug-flow bioreactor-Celligen^TMProduce adherent cell (PLX-C cell) to be shown by Fig. 8 B The some key steps composition shown.The method originates in collects Placenta Hominis from planned mature cesarean.

Then from complete placenta separation adherent cell, make to be grown in tissue culture flasks (2D), gather in the crops and as 2D-cell Original seed (2DCS) is saved in liquid nitrogen, and thaw appropriate 2DCS, washs and is seeded on the carrier in bioreactor to enter one Step amplification is 3D-culture.Bioreactor grew after 1-3 week, harvesting and as PLX-C freezen protective at liquid nitrogen Gas phase in.

The reception of people's tissue

The Placenta Hominis of all acquisitions all under the allowance of Helsinki committee of these medical institutions available from obstetrical ward.Cause This, all of Placenta Hominis donor endorsed Informed Consent Form and carries out donor screening and donor detection (IPCI).Tire is obtained from donor It is placed on immediately in aseptic plastic bag after dish (in cesarean operates), is subsequently placed in the Styrofoam box of ice bag.Fortune Send Placenta Hominis and be immediately placed on isolation area until quality control (QC) and quality assurance (QA) let pass and use.Production step after all Rapid all carrying out in the cleanroom facilities of isolation arrives until the QC approval of detection of mycoplasma result, and then clearance cell is used for 2D Cell grows.

The recovery of adherent cell and processing

In order to start this process, by complete Placenta Hominis chopping under aseptic condition under Laminar Ventilation cupboard, delay with Hank ' s Rush liquid washing and hatch 3 hours in 37 DEG C with 0.1% collagenase (1mg collagenase/ml tissue).Add 2D cell culture medium (bag 2D-culture medium containing the DMEM that with the addition of 10%FBS, 0.25 μ g/ml amphotericin B and 50 μ g/ml gentamycins) and will be disappeared The tissue changed filters sterile metal filter screen roughly, be collected in sterile beaker and be centrifuged (10 minutes, 1200RPM, 4 DEG C).So After aspirate gently, with the addition of antibiotic 2D-culture medium washing suspend cell, be seeded in 80cm²In culture bottle and in group Knit in cultivation incubator in supplementing 5%CO₂Lower 37 DEG C of humidification conditions is hatched.After 2-3 days, cell adhesion to culture bottle surface, With PBS they washed and add 2D-culture medium.

Two dimension (2D) cell growth

Before passing on for the first time, the grown cultures based specimen of 10% total culture bottle number in isolation is merged and carries out propping up former (IPC2) is surveyed in health check-up.If it find that the mycoplasma of cell (EZ-PCR mycoplasma detection kits, Biological Industries, Israel) it is negative, cell is let pass from isolation area.After continuing to pass on 1-2 time, cell is transferred to 2D and produces toilet (2DP) in.Once entering 2DP room, cell continues to pass on 3-5 generation.Take IPC-3 sample after passing on for 4th time to examine for immunophenotype Survey.During whole, culture in tissue culture's incubator under 5%CO2 humidification conditions 37 DEG C be grown on without antibiosis In the 2D culture medium of element.After altogether passing on 6-8 generation (9-16 cell multiplication), collect cell and as 2D-cell stock (2DCS) freezen protective.

Generally carried out passing on for the first time after 10-15 days.Start to continue to 6-8 generation, generally after 3-5 days from 2nd generation (1.5-2 multiplication) reaches passage when 70-80% converges when culture.With 0.25% trypsin-EDTA (37 DEG C, 4 Minute) from culture bottle separation cell and with 3 ± 0.2x10³Individual cell/cm²Culture density inoculation.The size of tissue culture flasks Carry out along with passing on and increase.Incubation originates in 80cm²Tissue culture flasks, then at 175cm²Culture bottle in, then At 500cm²Culture bottle in (Triple bottle), finally cell is seeded to Cell Factory 10 dish (6320cm²In).

Before freezen protective, at the end of 2DCS trophophase, collect growth medium and prepare sample deliver to for Substance detection (IPC 4) certified GLP laboratory.

The freezen protective operation of 2D-cell-original seed product

For 2DCS freezen protective, aseptically collect, with 0.25% trypsin-EDTA, the cell that 2D-cultivates. By cell centrifugation (1200RPM, 10 ', 4 DEG C), count and be resuspended in 2D culture medium.

For freezing, with 2D-freezing (final concentration of 10%DMSO, 40%FBS and 50%2D-culture medium) 1: 1 Diluting cells suspension.About 1.5-2.5x10 is produced from a Placenta Hominis⁹Individual cell.By 4ml cell with final concentration 10x10⁶/ ml preserves In 5ml freezen protective polypropylene phial.By phial labelling and be transferred to have in the refrigerator of controlled rate and carry out gradually Temperature reduces process (1 DEG C/min), afterwards by they transfers to be saved in the gas phase of the liquid nitrogen freezer being positioned at freezen protective room. This material is referred to as 2D-cell stock (2DCS) batch.

Start three-dimensional (3D) and cultivate operation

Cultivate to start 3D, from the appropriate (150 ± 30x10 of 2DCS defrosting in 2DP room⁶) cell and use 3D culture medium (containing the DMEM of 10%FBS and 20Mm Hepes) washing is to remove before being inoculated into pre-prepd bioreactor system DMSO.The content of sucking-off each 2DCS phial also dilutes by (37 DEG C) 3D culture medium 1: 9 of pre-temperature.By cell centrifugation In (37 DEG C) 3D culture medium of (1200RPM, 10 ', 4 DEG C) the pre-temperature of 50-100ml that is again resuspended in 250ml aseptic bottle.Take Sample also counts cell to measure cell quantity and viability with Trypan Blue dye liquor.In Laminar Ventilation cupboard, just cell hangs Liquid is transferred in the inoculation bottle of 0.5L.Through sterile pipes, cell suspension is transferred to biological respinse from inoculation bottle by action of gravity In sundries.

The production of 3D adherent cell in Celligen bioreactor (PLX-C)

Bioreactor describes

Use the automatic CelliGen that Fig. 8 C describesOr BIOFLO 310 bioreactor system [(New Brunswick Scientific (NBS)] carry out 3D trophophase.This bioreactor system is used for cultivating cell culture, its In condition be suitable to high cell concentration.The bioreactor using fill-up mode implements incubation.Laboratory scale biology Reactor is made up of two main systems-control system and bioreactor self (tank and adnexa).The parameter of this process by Control station is monitored and controlled, and this control station includes the connector of probe, electromotor and pump, for dissolved oxygen (DO), pH, irrigates and shakes Swinging the control loop of (having electromotor), gas control system, water circulate and for temperature controlled heating system and operation circle Face.Controlled procedure parameter (such as temperature, pH, DO etc.) can be displayed on operation interface and by the monitoring control devices specified.

Growth of cell culture operation in bioreactor

As part is mentioned above, from the 2DCS defrosting 150 ± 30x10 of freezen protective⁶Individual cell, washs and is seeded to nothing In bacterium bioreactor.This bioreactor comprise carrier that 30-50gr is made up of polyester and polypropylene ( Disks, NBS) and 1.5 ± 0.1L 3D culture medium.Growth medium in bioreactor is maintained at following condition: 37 DEG C, 70% dissolved oxygen (DO) and pH 7.3.Gas (air, the CO being fed past filter is measured according to control system₂、N₂And O₂) to keep DO value is 70% and pH value is 7.3.For initial 24 hours, increase with 50 turns every point (RPM) shaken cultivation base and at the 2nd day To 200RPM.At 2-3 days started, cell is made to grow with batch mode.When the concentration of glucose of culture medium is reduced below 550mg/ starts perfusion when rising.Use aseptic silicone pipeline by culture medium from feeding container pump to bioreactor in.At laminar flow The lower aseptic joint of use carries out all of pipeline connection.Every day regulation perfusion with keep concentration of glucose constant be about 550 ± 50mg liter.Within every 1-2 days, sampling growth medium measures for glucose, lactate, glutamine, glutamic acid and ammonium concentration (BioProfile 400 analyser, Nova Biomedical).The glucose consumption rate of cell culture and lactate are formed Speed allows to measure cell growth rate.These parameters are for determining harvest time according to the experimental data of accumulation.

PLX-X cell from bioreactor results 3D growth

At the end of trophophase, (4-10 days) start cell harvesting process.Collect the sample of two growth mediums.Preparation One sample is also delivered to certified GLP laboratory and is carried out detection of mycoplasma according to USP and Eu standard, is shifted by another sample Carry out in the refrigerator of controlled speed gradually temperature reduce process (1 DEG C/min), afterwards by they transfer be saved in be positioned at cold In the gas phase of the liquid nitrogen freezer freezing preserving chamber, in case needing to repeat detection of mycoplasma.These media samples are considered as whole product A part for product detection of mycoplasma and result are considered as a part for product clearance standard.

The culture that 3D grows is gathered in the crops in Class-100 laminar region by 3DP room as follows:

Action of gravity is utilized to be emptied in waste canister by bioreactor tank through pipeline.Tank is opened by removing top board, Use aseptic nipper that from basket, carrier is transferred aseptically to top net (see Fig. 8 C).It is then shut off bioreactor tank and is further filled with The PBS (37 DEG C) of the pre-temperature of 1.5L.Hunting speed is increased to 150RPM persistently 2 minutes.Arranged through pipeline by pressure or gravity Go out PBS in refuse bottle.Washing operation is repeated twice.

In order to be discharged from carrier by cell, in bioreactor tank, add the trypsin EDTA of the pre-temperature of 1.5L to 37 DEG C (0.25% trypsin, EDTA 1mM) and by carrier in 37 DEG C, 150RPM vibrate 5 minutes.Cell suspension is collected to comprising In the 5L sterile chamber of 250ml FBS.Cell suspension divided to the sterile centrifugation tube of 4 500ml and take out detection of mycoplasma Sample.The centrifuge tube covered is transferred to 10 through 3DP active pass-through, 000 grade of bottling department (FR1), there It is PLX-C by cell sterile filling freezen protective.

Cell cycle analysis-by the PLX-C cell obtained by Celligen and pass through Plurix with 70%EtOH O.N The PLX cell obtained is fixed, centrifugal and be resuspended in comprise 2 μ g/ml PI (Sigma), 0.2mg/ml Rnase A (Sigma) and In propidium iodide (PI) solution of 0.1% (v/v) Triton (Sigma) and persistently 30 minutes.By facs analysis cell cycle.

Gene expression arrays (microarray)-adherent cell expands available from the mature Placenta Hominis of people and by Plurix or Celligen Increase.The cell of three different batches is obtained for inspection further from each amplification method.

From cell extraction RNA (Qiagen-Rneasy trace quantity reagent kit) and apply to Affymetrix complete genome group table Reach array.Chip usesHuman Exon1.0ST array (Affymetrix, Santa Clara, California, USA).

The facs analysis of membrane marker-with monoclonal anti somatochrome as previously described.In short, by 400,000- In dark place in 600,000 cell suspensions 0.1ml flow cytometry buffer in 5ml test tube and under room temperature (RT) The anti-human CD29MAb (eBioscience), the PE that put together with following monoclonal antibody (Mab) incubation 15 minutes: FITC-put together Anti-human CD73MAb (Becton Dickinson), the PE anti-human CD105MAb (eBioscience), the PE that put together put together anti- It is anti-human that anti-human CD45MAb (IQProducts), the PE-that people CD90MAb (Becton Dickinson), FITC-put together puts together The anti-human HLA-DR MAb that anti-human CD 14MAb (IQProducts) that CD19MAb (IQProducts), PE put together, FITC put together (IQProduct) the anti-human CD31MAb that antihuman CD 34 MAb (IQProducts), the FITC that, PE puts together puts together (eBioscience) anti-human KDR MAb (R&D systems) that, FITC puts together, anti-human fibroblast labelling (D7-FIB) Anti-human CD86MAb (BD), FITC-that anti-human CD80MAb (BD), the FITC-that MAb (ACRIS), FITC-put together puts together put together Isotype IgG1 (the IQ that anti-human HLA-ABC MAb (BD) that anti-human CD40MAb (BD), FITC-put together, FITC put together Products) the isotype IgG1 (IQ Products) that, PE puts together.

With flow cytometry buffer, cell is washed twice, be resuspended in 500 μ l flow cytometry buffer In and use FC-500 flow cytometry (Beckman Coulter) to analyze.Prepare with relevant isotype fluorescence molecule Negative control.

Mixed lymphocyte reaction (MLR)

2x10 is stimulated with the MNCs (from donor B) being derived from PB of (3000Rad) via radiation of equivalent⁵Individual it is derived from peripheral blood (PB) MNCs (from donor A).The PLX-C of the amount of being gradually increased is added in culture.By three repeated inoculations of each group in In 96 orifice plates.Cell is cultivated in RPMI 1640 culture medium comprising 20%FBS.That cultivated at 5 days uses 1 μ in last 18 hours C ³H-this plate of thymidine pulse.Then collect cell through glass fibre filter, absorb with the quantitative thymidine of scintillation counter.

CFSE is dyeed, PB-MNC cell dyeing is used for the CFSE (molecular probe) propagation before cultivating and measures.5 Cell the density by flow cytometry detection CFSE dyeing is collected after it.

ELISA

Carry out ELISA as described above.In short, with 5 μ g/ml ConA (Sigma), 0.5 μ g/ml LPS (SIGMA) or 10 μ g/ml PHA (SIGMA) stimulate MNC (to be isolatable from periphery in 37 DEG C in the presence of PLX-C under the 5%CO2 atmosphere of humidification Blood).Collect supernatant and carry out IFN γ (DIACLONE), TNF α (DIACLONE) and IL-10 by ELISA kit (DIACLONE) cytokine analysis.

Experimental result

Compared with Plurix, the change in producing with Celligen causes some main difference (being summarized in table 4 below).

Table 4:Plurix system and the comparison of Celligen system

Parameter	Cell according to embodiment 1 Growth	Present invention teach that	Improve
				Working volume (ml)	280	1500	By the popularization of the method.This Higher (the 2-8 of the level of production of teaching Secondary population doublings)

Parameter	Cell according to embodiment 1 Growth	Present invention teach that	Improve
				Vehicle weight (gr)	1.4	30	By the popularization of the method
Bed structure	Cone, 50ml post	Cylindrical shape Packed bed	This teaching-culture medium and nutrient substance Mobility more preferable. The teaching of embodiment 1-due to circular cone The outlet contraction of shape structure causes stream Dynamic property is not enough. Media flow uniformity is more preferable.Real Execute the connection in example 1 teaching.
				Cell concentration during inoculation (cell/gr carrier)	3x106Individual cell/gr carrier	5x106Individual cell/gr Carrier	In this teaching between cell and cell Interaction more preferable
Cell concentration during inoculation (cell/ml)	0.015x106Individual cell/ml	0.1x106Individual cell/ ml	In this teaching between cell and cell Interaction more preferable
				Inoculation operation	Low culture volume is inoculated, Culture medium is added extremely after 24 hours Final working volume	At final working volume Vibration limit, middle limit is inoculated	The teaching of embodiment 1-cell is cultivated Thing divides at the internal heterogeneous body of carrier bed Cloth.24 little first of operating Time interior culture volume not enough.Cause Inappropriate (the acyclic acidic of working condition Border)
The production period persistent period	14-21 days	4-10 days	In this teaching product quality more preferably, receive The process that obtains is effective, productivity is higher, one-tenth This is lower

Parameter	Cell according to embodiment 1 is raw Long	Present invention teach that	Improve
				Operator scheme	Repeated batch-change weekly two Subculture	Fill-up mode-basis Concentration of glucose regulates Flow velocity (works as glucose Concentration is 550 ± Training is changed during 50mg/L Support base)	This teaching-in whole operating is just Moderate is there is for nutrient media components Condition change.Persistently remove by The toxicant that cell produces. In batch mode-Major Nutrient Constituent concentration relatively low (limit because of Element).Cell debris is less
Results operation	Results Trypsin in 50ml pipe Enzymic digestion 3 circulation	In bioreactor Results trypsin disappears Change 1 circulation	This teaching-more effective way.? Closed system is gathered in the crops.1 time Trypsinization circulation-cell Better quality
				Vibration	With peristaltic pump carry out storage capacity device and Culture medium circulation between pillar	Cell hoisting type impeller	This teaching-culture medium flows through filling Bed-preferably supply to culture Nutrient substance and oxygen.Culture medium Uniformity.Improve other controls Loop processed (temperature, DO, pH)
Temperature controls	Produce and carry out in incubator. Indirect temperature controls (incubator Room).Through Air Interface transmission Heat	Online directly control. Heat is transmitted through water leg Amount.	This teaching-measure training more accurately Support the temperature of thing.Reaction is fast.Reach The time of set point is short.
				Temperature monitoring	Manual monitoring.Indirectly water temperature Monitoring.	Online directly monitoring.	This teaching-preferably monitor and control Make this process.Fast to fault reaction Speed.

Parameter	Cell according to embodiment 1 is raw Long	Present invention teach that	Improve
				DO monitors	Nothing	On-line monitoring	This teaching-preferably monitor and control Make this process.Fast to fault reaction Speed.
DO controls	Nothing.Only introduce air	With air, O2And N2。 Directly control spy online Fixed set point	This teaching-preferably control DO water Flat.Preferably maintain specific work Make condition.
				PH is monitored and controlled	Only visual monitoring (phenol red conduct A part for culture medium)	On-line Control and monitoring	This instructs-better controls over pH water Flat. Preferably maintain the bar that specifically works Part.
Ventilation	Only bubbling	Cover (Overlay) (bubbling is as one Select)	The teaching of embodiment 1-pass through bubbling Ventilation produces the bubble that can damage cell Foam.

The change of production method causes the characteristic changing of gained 3D adherent cell.These differences are summarized in hereafter.

The cell cycle-compare and pass through of the PLX that Plurix produces is analyzed compared with the PLX-C that Celligen produces PLX-C cell and the PLX cell obtained by Plurix that Celligen obtains with detect cell cell cycle not same period it Between distribution.As Fig. 9 A-B is explicitly shown, Celligen the PLX-C cell expanded demonstrates typically breeds overview (cell Cycle asynchronous cell distribution).Specifically, the cell of 28% is at S and G2/M phase (Fig. 9 A).These results show in propagation During cell harvesting and Celligen bioreactor condition supported cell growth.

Microarray between the obtained cell of Plurix and Celligen compares-and gene expression arrays allows to monitor simultaneously By Plurix (PLX) or the full-length genome table of the adherent cell of the mature Placenta Hominis of derived from human that expanded by Celligen (PLX-C) Reach overview.These results make to assess these different growing methods obtain phenotypic difference between cells molecular mechanism (see Table 5 below).

Table 5: compare the gene expression in Plurix and Celligen cell

Gene C elligen p-value (process)

To Plurix

(multiple change)

The interferon inducible protein 17.52 0.0401812 repeated containing three tetradecapeptides

Aldehyde dehydrogenase 1 family, member A1 16.76 0.00145807

Leukocyte derives arginine aminopeptidase 13.99 3.88E-06

Keratin 27 pseudogene 27 12.25 0.000224998

Similar keratin, I type cytoskeleton 18 (cytokeratin 11.83 0.000304949

G protein coupled receptor, family C, organizes 5, member A 10.35 3.39E-05

Integrin, α 6 9.84 0.0411667

G protein coupled receptor 126 8.73 0.00197635

Thromboplastin (Thromboplastin, tissue factor) 7.36 0.012192

Rho GDP dissociates inhibitive factor (GDI) β 7.36 0.00200066

Signal peptide, CUB territory, EGF-sample 3 7.20 0.0255115

The interferon inducible protein 7.09 0.0139777 repeated containing three tetradecapeptides

Dickkopf homologue 1 (Africa xenopus) 7.06 3.06E-07

NAD (P) H dehydrogenase, quinone 1 6.63 0.000282423

Keratin 18 6.46 0.000514523

Opioid growth factor receptors-sample 1 5.96 0.00114551

Mal, T-cell differential protein-sample 5.95 0.00664216

Neurofilament, medium polypeptide 150kDa 5.86 0.0190611

Containing DEP territory 1 5.82 0.000370513

Cathepsin C 5.72 0.00532262

WAS 5.47 0.00178153

Serpin peptidase inhibitors, clade B (ovalbumin), 5.44 0.0190218

Member

Solute Carrier family 7, (cationic amino acid transporter albumen 5.33 0.00688017

The interferon inducible protein 5.18 0.00357376 repeated containing three tetradecapeptides

NUF2, NDC80 kinetochore complex component, homologue (S.cere 5.05 0.00276524

SHC SH2-territory Binding Protein 1 4.95 0.00430878

Thioredoxin reductase 1 4.86 0.000197486

Lung cancer metastasis related protein 4.85 0.00148024

Rho gtpase activating protein 29 4.85 0.0466211

Cell division cycle 20 homologue (saccharomyces cerevisiae) 4.80 0.00514206

Sequence similarity family 11 1, member B 4.63 0.000125819

PDZ combines kinases 4.54 0.00784983

Determine and cohere 1 homologue 2 (saccharomyces cerevisiae) 4.53 0.000773033

Guanine nucleotide binding protein 4 4.47 0.000215944

Lipase A, lysosomal acid, cholesteryl esterase (Wolman sick 4.42 0.0167385

Kinesin family member 20A 4.39 0.00582352

KIAA0101 4.28 0.0105909

(the CDK2-association double 4.25 0.000732492 of cyclin-dependent kinase initiator 3

Thymidylate synthetase 4.23 0.00685584

Chromosome 13 open reading frame 3 4.18 0.000548296

BTAK 4.16 0.00632571

Nei endonuclease V III-sample 3 (escherichia coli) 4.14 0.00115606

Centrosome protein 55kDa 4.13 0.0021952

OxLDL ELISA (agglutinin-sample) receptor 1 4.11 0.0205198

Without little tooth homologue (fruit bat) 4.05 0.00141153

Born of the same parents' cyclase protein, actin binding protein 4.01 0.010923

Ribonucleotide reductase M2 polypeptide 3.98 0.00834059

Ankyrin duplicate domain 1 (myocardium) 3.93 0.00911953

Transcription factor 19 (SC1) 3.89 0.00109627

Keratin 18 3.89 0.000112551

Non-SMC cohesion protein I complex, subunit G 3.88 0.00537097

Cyclin E2 3.87 0.000203389

Trypsinogen C 3.86 0.00416276

Little nucleolar RNA, C 3.81 0.0334484

Tight junction protein 2 (zonula occludens 2) 3.81 0.00012562

Kinesin family member 18A 3.78 0.00134108

Kinesin family member 2C 3.77 0.0059888

Shugoshin-sample 1 (fission yeast) 3.76 0.00101318

Polo-sample kinases 1 (fruit bat) 3.75 0.0140309

Thymidine kinase 1, solvable 3.73 0.00124134

Transcription factor 19 (SC1) 3.73 0.00124327

Transcription factor 19 (SC1) 3.73 0.00124327

Claspin homologue (Africa xenopus) 3.71 0.00683624

GINS complex subunit 1 (Psf1 homologue) 3.69 0.00104515

Microsome glutathione S-transferase 1 3.67 0.041701

Virtue acetamide deacetylase-sample 1 3.67 0.000902645

SPC25, NDC80 kinetochore complex component, homologue (S.ce 3.65 0.00568662

Integrin, α 4 (antigen CD4 9D, VLA-4 receptor alpha 4 subunit 3.62 0.0158411

Catenin (cadherin related protein), α-sample 1 3.57 7.46E-05

Discs, big homologue 7 (fruit bat) 3.56 0.0317074

V-myb Myeloblastosis Virus oncogene homologue (birds)-sample 3.55 0.0043878

Serglycan 3.54 0.0443487

Centromere protein N 3.53 0.000540143

Cyclin A 2 3.53 0.00965934

Heat shock 22kDa albumen 8 3.52 0.0219583

Sema territory, immunoglobulin domain (Ig), short alkalescence territory 3.49 0.008548

Rho gtpase activating protein 11A 3.49 0.00834174

Fanconi anemia, complementary group I 3.43 0.00464532

Benzimidazole 1 homologue (yeast) releases BUB1 and goes out bud inhibition 3.42 0.0108258

The special acidic protein of ovary 3.42 0.00334641

Cholinoceptor, muscarine 2 3.41 0.0320078

Cell division cycle 2, G1 to S and G2 to M 3.41 0.0017111

The cytokinesis protein regulation factor 1 3.39 0.0325664

Minichromosomes maintains complex component 5 3.38 0.00475504

Sperm closes associated antigen 5 3.37 0.00906321

Maternal embryo leucine zipper kinases 3.34 0.00908391

Little nucleolar RNA, C 3.33 0.0298703

Carnitine palmityl transferase 1A (liver) 3.33 0.00170894

Similar Ubiquitin-conjugation enzyme E2S (ubiquitin 3.33 0.000415822

Kinesin family member 11 3.33 0.00915145

NIMA is (forever from mitotic gene a) associated kinase 7 3.33 0.00159114

Containing the ADAM metallopeptidase of platelet factor4 type motif, 3.32 0.0102751

Convert, acid containing coiled-coil protein 3 3.31 0.0014577

Cyclin B1 3.29 0.0103092

MAD2 mitotic arrest defect sample 1 (yeast) 3.28 0.00488102

Dihydrofolate reductase 3.28 0.00178879

Containing NIPA-sample territory 3 3.27 0.00164708

Cell division cycle association 2 3.26 0.0122226

Apolipoprotein B mRNA editing enzymes, catalytic polypeptide 3.26 0.00308692

Cyclin B2 3.25 0.016544

Containing Cobra venom endonuclease territory 1 3.24 0.000429245

Dihydrofolate reductase pseudogene 3.23 0.00141306

ATPase, Na+ 3.23 0.000381464

Replication factor C (activity factor 1) 3,38kDa 3.23 0.00109668

WD duplicate domain 76 3.22 0.0023531

Platelet leukocyte C kinase substrate 2 3.17 0.0304429

Rac gtpase activating protein 1 3.17 0.00381613

PHD finger protein 19 3.17 0.000177604

Lymphocytic leukemia lacks, and 2 3.15 0.0109528

Centromere protein I 3.15 0.0106816

BRCA1 associates annulus 1 3.14 0.000540414

G-protein signaling regulatory factor 4 3.13 0.00781061

STAM associated proteins-sample 1 3.11 0.0181743

Thioredoxin 1 homologue (saccharomyces cerevisiae) 3.10 5.14E-05

Chromosome 15 open reading frame 23 3.08 0.000147331

TTK protein kinase 3.08 0.0112171

Non-SMC cohesion protein I I complex, subunit G2 3.08 0.0130322

Villin 2 (ezrin (ezrin)) 3.07 0.0131934

stomatin 3.06 0.00387095

Containing Protein-tyrosine-phosphatase-sample A territory 3.06 0.0419644

Serpin peptidase inhibitors, clade B (ovalbumin), 3.05 0.0030439

Member

Kinesin family member 4A 3.05 0.0114203

Predicted protein DKFZp762E1312 3.05 0.00726778

Ubiquitin-conjugation enzyme E2S 3.04 0.00118205

Hydroxysteroid dehydrogenase sample 2 3.03 3.71E-05

ATP enzyme family, containing AAA territory 2 3.01 0.00415258

TPX2, micro-pipe associates, homologue (Africa xenopus) 3.00 0.0253137

Histone group 1, H4d 3.00 0.030183

Kinesin family member 23 2.99 0.00790585

Heat shock 70kDa albumen 2 2.99 0.0215102

Origin recognition complex, subunit 1 sample (yeast) 2.99 0.00207753

Dihydrofolate reductase 2.98 0.00307793

Cell migration receptor (RHAMM) 2.97 0.00467816 of hyaluronic acid mediated

3 '-adenosine phosphate, 5 '-phosphosulfate synthase 2 2.97 1.43E-05

Glycerol-3-phosphate dehydrogenase 2 (mitochondrion) 2.95 0.00211969

Kernel and spindle related protein 1 2.95 0.00520875

Transparent homologue 3 (fruit bat) 2.95 0.00107709

Kinesin family member 14 2.94 0.00947901

Histone group 1, H1b 2.93 0.0470898

Guanine-nucleotide-binding protein (G-protein), α inhi 2.92 0.00184597

Minichromosomes maintains complex component 8 2.92 0.000841489

The susceptible candidate gene of cancer 5 2.92 0.0330594

Leukotriene B4 12-hydroxyl dehydrogenase 2 .92 0.000685452

Glutamate-cysteine ligase, modifies subunit 2.91 0.00378868

Jaw frame M1 2.91 0.0203154

Adipose Differentiation related protein 2.90 0.000331751

O-acyltransferase territory 1 2.90 0.01185 is combined containing film

Ubiquitin-conjugation enzyme E2T (presumption) 2.90 0.00741886

Cell division cycle association 3 2.89 0.006289

Integrin, α 3 (antigen CD4 9C, VLA-3 receptor alpha 3 subunit 2.88 0.00574148

Factor XIII, B polypeptide 2.88 0.0294465

RAD51 homologue (RecA homologue, escherichia coli) (saccharomyces cerevisiae) 2.87 0.000854739

ATP-binding cassette, subfamily C (CFTR 2.87 0.00382491

Sequence similarity family 29, member A 2.85 0.00111165

Containing SH2 territory 4A 2.84 0.0323646

Memebrane protein, palmitoylation 1,55kDa 2.84 0.000396285

CDC28 pka regulatory subunit 1B 2.84 0.0107391

PSMC3 interaction protein 2.84 0.00766442

The elastin laminin microfibril interface factor 2 2.84 0.0192072

Topoisomerase (DNA) II α 170kDa 2.83 0.0321109

Transmembrane protein 106C 2.82 0.000214223

Histone group 1, H3b 2.80 0.0304598

Chromosome 18 open reading frame 24 2.80 0.00347442

EGF-R ELISA approach substrate 8 2.79 0.0194949

High mobile group nucleosome binding domain 2 2.78 0.0030536

SCL 2.78 0.00390288

Hundred territories (hecto domain) and RLD 4 2.78 0.00679184

The anti-reticent function 1 homologue B (saccharomyces cerevisiae) 2.77 0.00543408 of ASF1

Pth receptor interaction factor 13 2.76 0.0118319

Cell division cycle association 8 2.75 0.00619878

Kinesin family member C1 2.74 0.00821937

High mobile group nucleosome binding domain 2 2.73 0.00384071

Ornithine decarboxylase 1 ODC1 2.73 0.00144868

V-myb Myeloblastosis Virus oncogene homologue (birds)-sample 2 2.71 0.00989416

KIT part 2.70 0.00641955

Dual tyrosine (Y) phosphorylation ki 2.70 0.0234606

Interior flagellum transports 80 homologues (clothing infusorian) 2.70 0.0247286

Transmembrane protein 48 2.69 0.00458248

EBNA1 associated proteins 2 2.69 0.00296292

ZW10 interactant 2.69 1.88E-05

Exonuclease 1 2.68 0.00739393

Transketolase (Wernicke-Korsakoff syndrome) 2.68 1.92E-05

Growth hormone suppression protein receptor 1 2.68 0.0144901

Isocitrate dehydrogenase 3 (NAD+) α 2.67 0.00297129

Cytoskeleton related protein 2 2.67 0.0030499

Minichromosomes maintains complex component 4 2.67 0.00342054

DNA binding inhibitor 1, dominant negative helical ring hel 2.66 0.036485

CDC28 regulated protein kinases subunit 1B 2.66 0.0145263

Keratin 18 2.66 8.40E-05

CD97 molecule 2.66 0.00994045

Chromosome 6 open reading frame 173 2.64 0.00222408

Containing BTB (POZ) territory 3 2.62 0.0166824

Deafness, autosomal dominant 5 2.62 0.00235481

KIAA0286 albumen 2.62 0.00130563

Fanconi anemia, complementary group D2 2.61 0.0281405

Polo-sample kinases 4 (fruit bat) 2.60 0.00209633

Ribonucleotide reductase M1 polypeptide 2.60 0.000170076

Malate dehydrogenase 1, NADP (+)-rely on, cytosol 2.59 0.0435444

Non-SMC cohesion protein I complex, subunit H 2.59 0.0216752

S100 calbindin A3 2.58 0.0324073

Ubiquitin-conjugation enzyme E2L 3 2.57 0.00343347

Benzimidazole 1 homologue β releases BUB1 and goes out bud inhibition 2.56 0.0166047

Glycerol kinase 2.55 2.66E-05

TAF9B rna plymerase ii, TATA frame associated proteins (TBP)-as 2.54 0.0170365

TAF9B rna plymerase ii, TATA frame associated proteins (TBP)-as 2.54 0.0170365

Histone group 1, H2bg 2.52 0.000180822

High mobile group frame 2 2.52 0.0196872

NIMA is (forever from mitotic gene a) associated kinase 2 2.50 0.00289469

Proline rich 11 2.50 0.0357125

myopalladin 2.49 0.0255088

Containing brix territory 1 2.49 0.00471977

Cell division cycle association 5 2.49 0.01021

Fucosidase, α-L-2, blood plasma 2.49 0.00540929

Cyclin-dependent kinase 2 2.49 0.00250724

Lamin B receptor 2.49 0.000151784

Hypoxanthine phosphoribosyltransferase 1 (Lesch-Nyhan syndrome 2.49 0.000634057

Containing three symbasis units 25 2.47 0.0456344

Proteasome (proteasome, macropain) subunit, β type, 9 (lar 2.46 0.0202595

Proteasome (proteasome, macropain) subunit, β type, 9 (lar 2.46 0.0202595

Proteasome (proteasome, macropain) subunit, β type, 9 (lar 2.46 0.0202595

Sphingomyelins synthase 2 2.46 0.0020701

Transmembrane protein 62 2.45 0.00761064

Glucose-6-phosphate dehydrogenase (G6PD) 2.44 0.00278311

PHD finger protein 1 2.44 0.010191

Retinoblastoma-sample 1 (p107) 2.44 0.00319946

KIAA1524 2.43 0.0380688

ST6 (α-N-acetyl group-nerve amido-2,3-beta galactose base-1,2.43 0.00830766

Cofilin 2 (muscle) 2.43 0.0459235

Predicted protein LOC201725 2.42 0.000313319

Cell division cycle 25 homologue A (fission yeast) 2.42 0.000341692

Mammary cancer 1, early sends out 2.41 0.0180553

Transaldolase 1 2.41 0.00199537

MRNA has enough to meet the need 4 homologues (saccharomyces cerevisiae) 2.41 0.00373104

Glycosamine (N-acetyl) transferring enzyme 1, core 2 (β-1,6-N-2.41 0.0197148

Rich in half Guang acid cross-film BMP modulator 1 (desmin sample) 2.41 0.0267286

Tissue factor approach inhibition factor (lipoprotein association 2.40 0.0356227

Chromosome 16 open reading frame 59 2.40 0.00185191

Glycogen protein 1 2.39 0.0224317

Transmembrane protein 154 2.39 0.0045589

Tubulointerstitial nephritis antigen sample 1 2.39 0.00510812

CTP synthase 2.38 8.80E-05

Phenylalanyl-tRNA synthetase, β-subunit 2.38 0.000245973

Geminin, DNA replication dna inhibitive factor 2.38 0.00167629

Lamin B 1 2.37 0.0477748

SPC24, NDC80 kinetochore complex component, homologue (S.ce 2.36 0.00287227

Glutathion reductase 2.36 0.00353875

Ribosomal protein L 2 2-sample 1 2.36 0.00335381

Fumaroyl acetoacetate hydrolytic enzyme fumaroyl acetoacetate enzyme) 2.36 3.88E-05

Little nucleolar RNA, C 2.35 0.0188991

Sequence similarity family 64, member A 2.35 0.0019785

Epithelial cell transforming sequence 2 oncogene 2.35 0.000571152

Polymerase (DNA guiding), ε 2 (p59 subunit) 2.34 0.00479612

Glycerol kinase 2.34 3.37E-06

Glutathione S-transferase M2 (muscle) 2.33 0.0402076

Elongation factor, rna plymerase ii, 2 2.33 0.0130017

Thioredoxin 2.33 0.009636

Polymerase (DNA guiding), α 2 (70kD subunit) 2.32 0.0033903

Breast carcinoma 2, early sends out 2.32 0.00586847

CDC45 cell division cycle 45-sample (saccharomyces cerevisiae) 2.32 0.00735977

H2A histone family, member Z 2.32 0.0129697

Transport protein 1, ATP-binding cassette, subfamily B (MDR 2.31 0.0164234

Transport protein 1, ATP-binding cassette, subfamily B (MDR 2.31 0.0164234

Transport protein 1, ATP-binding cassette, subfamily B (MDR 2.31 0.0164234

Kernel complex associates 3 homologues (saccharomyces cerevisiae) 2.30 0.000373346

ATPase, Ca++ transport, plasma membrane 4 2.30 0.023011

Minichromosomes maintains complex component 7 2.30 0.0457691

TIMELESS interaction protein 2.29 0.00771062

Von Hippel-Lindau interaction protein 1 2.28 0.00329061

Ras is correlated with C3 Botulinum toxin substrate 2 (rho family, sma 2.28 0.0292466

Thymopoietin 2.28 0.0223176

Peptide acyl prolyl isomerase F (cyclophilin F) 2.28 0.00093846

Activated leukocyte cell adhesion molecule 2.27 0.00242163

Many comb basic rings refer to 5 2.27 0.000294142

Ran gtpase activating protein 1 2.27 9.68E-05

Replication factor C (activation factor 1) 4,37kDa 2.26 0.00164152

Tubulin, β 2C 2.26 0.000346744

Minichromosomes maintains complex component 10 2.26 0.0037925

H2B histone family, member S 2.25 0.000885505

Gamma-Glutamyl hydrolase (conjugated enzyme, folic acid poly γ glutamy 2.25 0.0195219

Transcription termination factor, rna plymerase ii 2.25 0.000393489

Polymerase (DNA guiding), δ 2, regulate and control subunit 50k 2.25 0.0123823

Transport protein 1, ATP-binding cassette, subfamily B (MDR 2.25 0.00859077

Transport protein 1, ATP-binding cassette, subfamily B (MDR 2.25 0.00859077

Transport protein 1, ATP-binding cassette, subfamily B (MDR 2.25 0.00859077

Histone group 1, H2bf 2.25 0.0124279

Eukaryotic translation initiation factor 1A, X-chain 2.24 0.00330183

Glucophosphomutase 2 2.24 0.00818204

Peroxisome D3, D2-enoyl--CoA isomerase 2.24 0.00148722

The interferon inducible protein 2.24 0.0177928 repeated containing three tetradecapeptides

G-2 and the S-phase expresses 1 2.23 0.0241887

Minichromosomes maintains complex component 2 2.23 0.0021347

Sequence similarity family 72, member A 2.23 0.00143248

The genomic instability 1 of RMI1, RecQ mediation, homologue (saccharomyces cerevisiae 2.23 0.00294705

FLJ20105 albumen 2.23 0.0127979

Multiple clotting factor deficiencies 2 2.22 0.0116892

Dihydrophytoceramide enzyme, alkalescence 2.22 0.0157729

Containing coiled coil domain 68 2.22 0.00227586

Cytokinesis acting factor (dedicator) 11 2.21 0.00697577

Platelet derived growth factor α polypeptide 2.21 0.00176418

ASAH1 (non-lysosomal ceramidase 2.20 0.00728536

S-phase kinases related protein 2 (p45) 2.20 0.00230153

Polymerase (RNA) III (DNA guiding) polypeptide G (32kD) 2.20 0.0298794

ADP-ribosylation factor-sample 6 interaction protein 1 2.20 0.00139745

Histone group 1, H2bh 2.19 0.0377748

Origin recognition complex, subunit 5-sample (yeast) 2.19 0.049697

CDC28 regulated protein kinases subunit 2 2.19 0.0128024

Histone group 1, H4c 2.19 0.0112695

Predicted protein LOC729012 2.19 0.000446087

DEAD (Asp-Glu-Ala-Asp) frame polypeptide 39 2.19 0.000340561

The Chromatin assembly factor 1, subunit B (p60) 2.18 0.0119687

MLF1 interaction protein 2.18 0.0177203

Micro-pipe association serine 2.18 0.00536974

MHC class I polypeptide relating sequence B 2.18 0.0165406

Shugoshin-sample 2 (fission yeast) 2.18 0.000852557

COP9 composing type photic form generation homologue subunit 6 (Arab 2.18 0.000793512

Methylenetetrahydrofolate dehydrogenase (NADP+ dependence) 2.18 0.00119726

Chromosome 6 open reading frame 167 2.18 0.0011095

Pituitary tumor converts 1 2.17 0.0485166

Ribonuclease H 2, subunit A 2.17 0.00669936

China's ham X-ray reparation supplements defect repair 2.16 0.0369865

Memebrane protein, palmitoylation 5 (MAGUK p55 subfamily member 2.16 0.00211873

Core peripheral proteins α 2 (RAG group 1, inputs protein alpha 1) 2.16 0.000650645

Containing platelet leukocyte C kinase substrate homologue territory, family A (phosphoric acid 2.15 0.0256434

Ribosomal protein L 39-sample 2.15 0.00429384

Core peripheral proteins α 2 (RAG group 1, inputs protein alpha 1) 2.15 0.000700649

Amyloid-beta (A4) precursor protein combines, family B, m 2.15 0.00201004

Minichromosomes maintains complex component 3 2.14 0.0018389

Histone group 1, H2ai 2.14 0.0129155

Chromosome 13 open reading frame 34 2.14 0.000702936

RAD18 homologue (saccharomyces cerevisiae) 2.14 0.0016685

WD repeats and HMG frame DBP 1 2.13 0.0034833

Sulfide quinone reductase-sample (yeast) 2.13 0.0473641

Chromosome 16 open reading frame 63 2.12 0.000804179

M-phase phosphoprotein 1 2.12 0.0271814

Minichromosomes maintains complex component 6 2.12 0.0161279

Homology frame A9 2.11 0.00520942

FGF9 (the glial activation factor) 2.10 0.0475844

Cell division cycle 25 homologue C (fission yeast) 2.10 0.0169914

Chromosome 9 open reading frame 64 2.10 0.0265979

U2AF homologue motif (UHM) kinases 1 2.09 0.0255167

Replication factor C (activity factor 1) 2,40kDa 2.09 0.00768959

Predicted protein LOC440894 2.09 0.0103358

Minicell nuclear ribonucleoprotein D1 polypeptide 16kDa 2.09 0.0334665

CSE1 chromosome separation 1-sample (yeast) 2.09 0.0013662

Glypican anchor biosynthesis, class W 2.09 0.0151967

Centromere protein O 2.09 0.00397056

Sequence similarity family 20, member B 2.09 0.00460031

Predicted protein FLJ40869 2.09 0.00444509

Guanine-nucleotide-binding protein (G-protein), γ 11 2.08 0.00140559

Calcyclin associated proteins 2.08 0.00524566

ATP-binding cassette, subfamily E (OABP), member 1 2.08 0.00454751

CD44 molecule (India's blood group) 2.08 0.000651436

Exosome composition 8 2.08 0.00132017

Sequence similarity family 102, member B 2.08 0.025743

Histone group 2, H3d 2.07 0.0102932

Sequence similarity family 33, member A 2.07 0.000318673

Fanconi anemia, complementary group B 2.07 0.000255109

Kinesin family member 22 2.07 0.0192406

Histone group 1, H2ai 2.07 0.0161621

Cowpox associated kinase 1 2.06 0.0233182

Integration factor complex subunit 7 2.06 0.000841371

The special endonuclease of laminated structure 1 2.06 0.006882

Predicted protein FLJ25416 2.06 0.000177531

Ecotropic virus integration site 2B 2.06 0.0171408

Retinitis pigmentosa 2 (X-linked recessive) 2.05 0.0264185

Centromere protein L 2.05 0.000880856

The cofactor that Spl transcriptional activation is required, subu 2.04 0.00141809

Chromosome 20 open reading frame 121 2.04 0.0146323

Sequence similarity family 72, member A 2.04 0.00162905

Sequence similarity family 72, member A 2.04 0.00165234

Eukaryotic translation initiation factor 1A, X-chain 2.04 0.00520549

Elongation factor, rna plymerase ii, 2 2.03 0.0458007

ATP enzyme, Na+ 2.03 0.0189108

Histone group 1, H3a 2.03 0.0244273

Containing brix domain 1 2.03 0.00981178

Containing sushi domain 1 2.03 0.0258164

Outer ribonucleoside triphosphote diphosphonic acid hydrolytic enzyme 6 (putativ 2.03 0.00423628

Fructosamine 3 kinases 2.03 0.00470972

Bloom syndrome 2.02 0.0209259

Tubulin, α 1c 2.01 0.00862586

E2F transcription factor 2 2.01 0.0496479

Exosome composition 2 2.01 0.00649147

Kinesin family member 22 2.01 0.0242075

LTV1 homologue (saccharomyces cerevisiae) 2.01 0.00812652

Dihydrolipoamide S-Acetylase (the E2 composition 2.01 0.00179011 of pyruv

(ras is correlated with 2.01 0.012225 to v-ral ape leukemia viral oncogene homologue B

Fourth finger and WD duplicate domain 3 2.01 0.0013797

Annexin A1 2.01 0.0173578

ElaC homologue 2 (escherichia coli) 2.00 0.00266504

Aldehyde dehydrogenase 9 family, member A1 2.00 0.00911609

Tubulin, α 4a 2.00 0.0435427

Nuclear pore complex interaction protein-2.00 0.00111223

oculomedin -2.01 0.00778869

Similar PI-3 kinases associated kinase SMG-1-2.01 0.0356628

Gorky's autoantigen, Golgi apparatus protein subfamily a sample pseudogene-2.01 0.00770626

Repeat containing spectrin, nuclear envelope 1-2.01 0.00438469

Nuclear pore complex interaction protein-2.01 0.00117582

Sushi, nestin and EGF-sample territory 1-2.01 0.00161129

Integrin, alpha V (Vitronectic receptor, α polypeptide-2.02 0.00252702

Cyclin-dependent kinase inhibitive factor 2B (p15 suppresses CDK4)-2.04 0.0150268

Lysyloxidase sample 4-2.04 0.0120148

Nuclear pore complex interaction protein-2.04 0.000213956

Calcium-2.04 0.00657494

calsyntenin3 -2.04 0.00300887

Cell adhesion molecule 1-2.05 0.0261129

Solute Carrier family 22 (organic cation transporter) ,-2.05 0.0137275

Containing RUN and FYVE territory 3-2.05 0.00387265

Glucosidase, α；It is acid that (Pompe is sick, glycogen storage disease-2.05 0.000418401

Nuclear pore complex interaction protein-2.05 0.00988632

Proline rich nuclear receptor coactivator albumen 1-2.06 0.0039587

Film Zinc metalloproteinase-2.06 0.0152684

PHD finger protein 21A-2.06 0.00980401

Rho GTP enzyme-active protein2 .06 0.00705186

Homology frame B6-2.06 0.00301714

Nuclear pore complex interaction protein-2.07 0.00032839

Phospholipase A2 receptor 1,180kDa-2.07 0.00069343

Nuclear pore complex interaction protein-2.08 0.000352007

Slit homologue 3 (fruit bat)-2.08 0.02844

Nuclear pore complex interaction protein-2.09 0.000414309

Cyclin-dependent kinase 6-2.09 0.0456892

Dynamin 1-2.09 0.00139674

Jumonji, rich in AT binding domain 1B-2.09 0.00861002

Calcium combines and coiled coil domain 1-2.09 0.00370041

Type-1 insulin like growth factor receptor-2.09 0.00114467

Nuclear pore complex interaction protein-2.10 0.000377834

CD82 molecule-2.10 0.0175517

Adjacent zinc refers to the Bu Luomo domain in territory, 2B-2.10 9.88E-05

--- -2.10 0.00666187

Synaptotagmin XI-2.11 0.0129428

KIAA1546 -2.11 0.000255634

Jun B proto-oncogene-2.12 0.0120169

CXXC refers to 6-2.12 0.0277527

Nuclear pore complex interaction protein-2.14 0.00282604

Cdon homologue (mice)-2.15 0.0350357

B-cell CLL-2.15 0.00343507

Nuclear pore complex interaction protein-2.15 0.00263888

V-abl Abelson Muridae leukemia virus oncogene homologue 1-2.16 0.0136688

Nuclear pore complex interaction protein-2.16 0.00583397

FAT tumor-inhibiting factor homologue 1 (fruit bat)-2.18 0.0158766

Transforming factor 2 α-2.18 0.012256

Chimeric protein 1-2.18 0.0287031

Butter oil ball-EGF the factor 8 albumen-2.18 0.000987073

Vitamin D (1,25-dihydroxyvitamin D3) receptor-2.19 0.000192208

Neuroblastoma, suppression tumor occurs 1-2.20 0.00090639

Containing jumonji territory 1A-2.20 0.0188513

WNK lysine defect protein kinase 1-2.21 1.57E-05

Protocadherin β 14-2.21 0.0103892

Cortactin associated proteins 2-2.21 2.28E-05

Containing WW territory transcription regulatory factor 1-2.22 0.0379899

Cyclin L1-2.22 0.00831474

Nuclear factor of activated T cells, Cytoplasm, calcine-2.22 0.00786451

Pellino homologue 1 (fruit bat)-2.23 0.00939357

Gorky's autoantigen, Golgi apparatus protein subfamily a sample pseudogene-2.24 0.00603583

Chromosome 7 open reading frame 10-2.26 0.00738442

Gorky's autoantigen, Golgi apparatus protein subfamily a sample pseudogene-2.27 0.00320764

Little Cajal body-special RNA 17-2.27 0.0301336

Latent transforming growth factor-beta associated proteins 2-2.29 4.08E-05

Gorky's autoantigen, Golgi apparatus protein subfamily a, 8A-2.29 0.0111179

Suppression albumen, β A (activator protein A, activator protein AB α polypeptide)-2.29 0.00877271

Solute Carrier family 41, member 2-2.30 0.00453672

Jaw frame P1-2.30 0.0463138

Matrix Metallopeptidase 14 (film insertion)-2.31 1.93E-05

Transcription factor 4-2.31 0.0367869

Jun oncogene-2.32 7.21E-05

Neuroepithelial cell transformed gene 1-2.33 0.0109689

asporin -2.33 0.000659873

V-fos FBJ Muridae osteosarcoma virus oncogene homologue-2.35 0.0138624

Ephrins-B2-2.36 0.00611474

Repeat and SOCS frame 1-2.36 0.0387851 containing WD

Similar dJ402H5.2 (the new albumen-2.36 0.00621503 of similar wo

PX territory-2.38 0.000927628 containing serine

Collagen, type VII, α 1 (kabner's disease, dystr-2.38 0.00109233

AE Binding Protein 1-2.39 0.000105628

Peroxidasin homologue (fruit bat)-2.40 0.00219049

Calcium channel, voltage relies on, L-type, α 1C subunit-2.41 0.0189661

Prader-Willi syndrome chromosomal region 1-2.45 0.0415526

Center line 1 (Opitz-2.45 0.00130803

Nuclear pore complex interaction protein-2.45 0.00354416

Chromosome 1 open reading frame 54-2.47 0.0186089

Transmembrane protein 16A-2.48 0.0481085

Containing basic helix-loop-helix territory, class B, 2-2.49 0.00270257

Nuclear pore complex interaction protein-2.50 0.00316496

Short and small associated transcription factor 1 (acute myeloid leukaemia-2.50 0.000607387

Zinc finger protein 29 2-2.50 0.029832

Fibronectin is rich in leucine transmembrane protein 2-2.51 0.0135122

Nuclear pore complex interaction protein-2.51 0.00283418

Potassium voltage-gated channel, subfamily G, member 1-2.54 0.0244306

Interleukin-11 9-2.54 0.0310328

Transforming growth factor, β 3-2.54 0.0287865

Dihydropyrimidinase-sample 3-2.55 0.0165203

Gorky's autoantigen, Golgi apparatus protein subfamily a, 8B-2.56 0.0121417

Predicted protein PRO2012-2.57 0.00756704

SATB homology frame 2-2.57 0.039781

T-complex 11 (mice)-sample 2-2.57 0.0324227

Ring finger protein 122-2.57 0.0236621

Chromosome 8 open reading frame 57-2.59 0.00261522

ADAM metallopeptidase-2.60 0.0113968 containing platelet factor4 type motif

Sushi, von Willebrand factors A type, EGF and PTX-3 territory-2.63 2.23E-05

ST6 beta galactose amide α-2,6-sialyltransferase 2-2.64 0.0216987

Sortilin is relevant containing VPS10 domain receptor 2-2.65 0.00936311

Protocadherin β 9-2.66 0.0285124

Chromosome 5 open reading frame 13-2.67 0.00410172

Enah -2.68 0.0077547

Decarboxylase territory 2-2.69 0.00683647 is relied on containing 2-methyl-3-hydroxy-4-formyl-5-hydroxymethylpyridine.

Similar nuclear pore complex interaction protein-2.70 0.0187322

Nuclear pore complex interaction protein-2.70 0.00368967

Transmembrane protein 119-2.70 0.00801387

Chromosome 14 open reading frame 37-2.70 0.0182453

Containing sushi-repetitive proteins, X-chain 2-2.71 0.0253856

Containing pdz domain fourth finger 3-2.71 0.00931014

Collagen, type XII, α 1-2.72 0.000204664

Matrix remodeling association 5-2.72 0.000317637

Collagen, type V, α 1-2.72 0.0166427

Dystrophin related protein 2-2.72 0.0137557

ATP-binding cassette, subfamily A (ABC1), member 1-2.73 0.00131361

Nourish albumen-2.77 0.00298044

Cornichon homologue 3 (fruit bat)-2.78 0.0261738

Formin Binding Protein 1-sample-2.78 0.00290401

Brain and acute leukemia, Cytoplasm-2.78 0.0476919

Protein-tyrosine-phosphatase, receptor type, U-2.80 0.0270428

Predicted protein MGC24103-2.82 0.0346673

Containing unwindase C territory 1 interferon-induced-2.83 0.0024839

Phospholipid transfer protein-2.84 0.00999206

The most early response 3-2.87 0.0152127

The most early response 3-2.87 0.0152127

ADAM metallopeptidase territory 12 (meltrin alpha)-2.87 0.000870288

Synaptic vesicle glycoprotein 2A-2.88 0.00704212

Chromosome 9 open reading frame 3-2.88 0.00410177

Thioredoxin interaction protein-2.90 0.0135494

Early growth response 1-2.93 0.000425035

Little nucleolar RNA, C-2.94 0.00666866

Little nucleolar RNA, C-2.95 0.00765575

The most early response 3-2.99 0.0167309

Low density lipoprotein, LDL associated protein 1 (α 2macroglo-2.99 4.26E-05

Two tail C homologues 1 (fruit bat)-2.99 0.0347162

Homology frame B2-3.03 0.00665994

Little nucleolar RNA, C-3.10 0.0274043

Little nucleolar RNA, C-3.10 0.0274043

Matrix Metallopeptidase 2 (gelatin enzyme A, Gelatinase A ,-3.13 5.59E-05

KIAA1641 -3.14 0.00659194

Collagen, type VI, α 3-3.14 2.09E-06

Homology frame A2-3.15 0.0435423

SH3 and PX territory 2B-3.15 0.0244357

Collagen, type VI, α 2-3.16 0.0149554

Chromosome 9 open reading frame 3-3.21 0.0233723

Little nucleolar RNA, C-3.24 0.0104491

Little nucleolar RNA, C-3.24 0.0104491

---- -3.27 0.00488845

UDP-N-acetyl-α-D galactosamine: polypeptide N-acetyl group ga-3.35 0.00964109

Cholesterol 25-hydroxylase-3.38 0.0445558

KIAA1641 -3.40 0.013175

Ring finger protein 144-3.40 0.0135334

Versican-3.41 0.023885

Angiopoietin-like 2-3.42 0.0245161

KIAA1641 -3.44 0.0170531

FBJ Muridae osteosarcoma virus oncogene homologue B-3.54 0.00025573

Similar RIKEN cDNA 1110018M03-3.59 0.00516476

Early growth response 2 (Krox-20 homologue, fruit bat)-3.62 0.00821813

Dachsous 1 (fruit bat)-3.63 0.00697244

Kinesin family member 26B-3.64 0.00363199

Frame without terminal homologous 5-3.66 0.000640157

Analogous protein KIAA0220-3.69 0.0302619

Type-1 insulin like growth factor receptor-3.71 3.42E-05

Protein Tyrosine Phosphatases, receptor type, N-3.77 0.0294569

KIAA1641 -3.85 0.0191782

Containing sushi repetitive proteins, X chain-3.85 0.00370941

Microfibre related protein 2-3.91 0.0152901

Complement component 1, s subconstiuent-3.97 0.0395863

CD24 molecule-3.99 0.0340122

Homology frame B3-4.02 0.0354368

Hair-nose-refer to syndrome i-4.02 0.00557712

Kallmann syndrome 1 sequence-4.04 0.000548703

17-4.09 0.0263961 are repeated containing rich in leucine

Containing clump albumen territory 2-4.32 0.031799

PTK7 protein tyrosine kinase 7-4.42 0.000116114

supervillin -4.43 0.0412717

Zinc finger protein 521-4.58 0.00668815

Calbindin 2,29kDa (calretinin)-4.77 0.0290743

Ras homologue gene family, member J-4.79 0.00197982

Integrin, α 11-4.80 0.000390317

Odz, strange Oz-5.05 0.00172671

F-frame protein 32-5.52 0.0212957

Raftlin family member 2-5.72 0.0260454

Clusterin-5.74 0.0303973

neurotrimin -5.79 3.78E-06

WNT1 can inducement signal pathway albumen 1-5.86 0.000672342

IGFBP (insulin-like growth factor binding protein) 5-6.34 0.011614

Sulfatase 2-6.34 5.88E-05

Microfibre related protein 4-6.93 0.00155578

Connect adhesion molecule 2-7.07 0.0306758

Containing fi-bronectin type III territory 1-7.29 0.0334696

Myogen polysaccharide, δ (35kDa dystrophin-association glycoprotein-7.37 0.000881984

hephaestin -7.53 0.0123141

Serine protease inhibitor peptide enzyme inhibitor, clade F (α 2 antiplasmin-7.66 0.00362941

CST1 Cystatin 1-7.96 0.0496433

hemicentin1 -8.18 0.0461603

Tenascin C (six arms)-8.32 8.26E-05

Biglycan-8.62 0.00161284

Cross-film, prostatic androgen induction RNA-11.20 0.000100935

CPE-11.22 0.00738131

The expression of cell marking on PLX-C cell-detect the surface antigen that PLX-C expresses by monoclonal antibody.Result table Bright PLX-C is with positive mark: CD73, CD29 and CD105 and negative marker: CD34, CD45, CD19, CD14 and HLA-DR (number According to not showing) it is characterized.Values of immunophenotyping specification is set as all positive mark >=90% and all negative marker≤3%.

And, as shown in Figure 10 A-B, endothelial marker do not expressed by PLX-C culture, such as two endothelial marker CD31 and KDR Negative staining shown in.But, it is significantly (expression of D7-fib, Figure 10 C) that the PLXC of fibroblast typical marks expresses.

The immunogenicity of PLX-C cell and immuno-modulating properties-be made up of the adherent cell being derived from Placenta Hominis due to PLX-C, HLA I quasi-molecule is expressed in its expection, and these developed by molecule are in all of soma and known induction alloreactivity Immunne response.HLA II quasi-molecule and other costimulatory moleculeses are the most only expressed in antigen presenting cell (APCs) surface.

In order to detect the immunogenicity of gained PLX-C cell, carry out the table of costimulating factor on the surface of these cell membrane Reach.Facs analysis proves to there is not CD80, CD86 and CD40 (Figure 11 A-C) on PLX-C cell membrane.Additionally, as by HLA A/ Shown in B/C dyeing (Figure 11 D), PLX-C expresses low-level HLA I quasi-molecule.The expression class of stimulation molecule and costimulatory molecules It is similar to be derived from the MSC (as shown in Figure 11 A-D) of bone marrow.

In order to study immunogenicity and the immuno-modulating properties of PLX-C cell further, carry out mixed lymphocyte reaction (MLR) detection.As shown in Figure 12 A-B, measuring as mixed by thymus pyrimidine, PLX-C cells escape allogeneic identification is also dropped Low t cell response.And, along with increase (in a dose-dependent manner) the lymphopoietic minimizing of PLX-C cell quantity is (logical Cross CPM and measure assessment) higher.PLX-C is at mitogenstimulated such as Concavalin A (ConA, Figure 12 B) and phytohemagglutinin (PHA) and lymphopoiesis is also reduced after the non-specific stimulation of anti-CD3 and anti-CD28 (data do not show).

In order to study PLX-C immunoregulatory lymphocytes propagation mechanism of action and judge this effect whether by cell with Intercellular interaction or cytokine secretion mediation, (it prevents contacting still of cell and cell to use transwell method Allow the diffusion of cytokine between two compartments) mononuclear cell being derived from PB is stimulated with PHA.Result shows even when inhibiting Proliferation Ability (data do not show) is still maintained when cell is with intercellular contact.

Cytokine secretion-as described above, PLX-C reduces the propagation speed of lymphocyte likely via soluble factor Rate.The cytokine of research lymphocyte response PLX-C secretion is to illustrate the mechanism of action of PLX-C further.Such as Figure 13 A-B institute State, cultivate mononuclear cell together with PLX-C and reduce slightly the secretion of proinflammatory cytokine INF γ and substantially reduce TNF α Secretion (even in the presence of a small amount of PLX-C).Additionally, after lipopolysaccharide (LPS) stimulates, the IL-10 of the MNC being derived from PB divides Secrete and increase in the presence of PLX-C, and the secretion level of TNF α reduces (Figure 13 C) in a dose-dependent manner.

Embodiment 5

The bio distribution of PLX-C

Material and experimental technique

PLX-C cell is transfected with luciferase expression vector

Lentivirus construct (Figure 14) stable transfection PLX-by the luciferase genes expressed under the control of a cmv promoter C cell.

Infect the generation of virus

293TN is made to produce cell growth 2-3 in the DMEM culture medium (Gibco) that with the addition of serum and antibiotic before transfection My god (50-70% converges).By 10 μ g packaging plasmids and 2 μ g expression construct andμl Plus^TMReagent (Invitrogen) Mixture addμ l is without in the DMEM of additive.By mixture and Lipofectamine^TM(addIn μ lDMEM 30 μ l dilutions) under room temperature (RT), hatch 15 minutes.Mixture is hatched 15 minutes at RT.Washing 293TN cell also turns Move to 2% blood serum medium and add transfection mixture.By cell at CO₂37 DEG C of overnight incubation after infection in incubator Within 24-60 hour, collect culture medium.Reach peak virus after 48 hours to produce.Collect culture medium, be centrifuged 5 minutes in room temperature 3000rpm Precipitate cell debris.After Li Xin, supernatant is filtered Millex-HV 0.45 μm PVDF filter (Millipore, Cat.# SLHVR25LS)。

The infection of PLX-C

Virus infects first 24 hours with 0.6-1x10⁵PLX-C cell is inoculated in 24 orifice plates by the density of individual cell per well Complete medium in.After 24 hours, add 0.5ml viral suspension and (be diluted in containing Polybrene's with final concentration 5-8 μ g/ml In complete medium).By cell incubation 24 hours, then with complete DEME culture medium replacement medium and by cell in 37 DEG C, 5%CO₂Overnight incubation.4th day, culture reached to converge and 1: 3 to 1: 5 divisions, makes cell grow 48 in complete DEME little Time, then analyze the luciferase expression of cell.

The effective percentage infected is close to 100%.Sending out in living cells and intravital mouse is carried out by IVIS Lumina imaging system Light is assessed, and this system includes the highly sensitive CCD camera capturing luciferase luminous signal.

Infect two weeks after, by 2x10⁶Individual cell IM or IV is expelled to SCID/Beige, NOD/SCID, SCID and Balb/C Mice.Cell with the injection of described IVIS system monitoring.

Result of the test

As from result finding, during after infection, CXL cell continues differentiation and growth cell, the expression of luciferase keeps Strong and stable (Figure 15).

Once PLX-C cell is injected Balb/C mice, detect bio distribution pattern.As after result finding, IM injection 72 hour cells disappear (data do not show).But, PLX-C cells in vitro keeps the constant high level luciferase more than three weeks Express (data do not show).

As depicted in figures 16 a-d, IM is expelled to the cell of SCID/Beige immunodeficient mouse and remains in injection site many Reach 5 days and do not observe afterwards.IV is expelled to the CXL cell of SCID/Beige mice and migrates to lung after 24 hours, then to note Penetrate site (supposition is gone back to the nest to damage location).Cell fades away and 3-4 Zhou Houwei observes afterwards.

Embodiment 6

Adherent cell can interior therapeutic limb ischemia

Whether the adherent cell being derived from Placenta Hominis in order to measure transplanting can reduce ischemic injuries and improve clinical and motion merit Can, use posterior-limb ischemia model as follows.

Material and experimental technique

Posterior-limb ischemia model-little at 20 male Balb/c of 8-10 week old body weight about 25g ± 20% of nonimmune defect Mus is induced posterior-limb ischemia.Assess and IPCA (AAALAC) according to NIH (NIH) and management of laboratory animal Carry out animal process.Letting animals feed under standard laboratory conditions.By animal feeding in the environment that weather is controlled.Temperature range Between 20-24 DEG C and relative humidity (RH) is between 30-70%, illumination in 12 hours and 12 hours dark cycle.

The randomization program " Research Randomizer " generated with computer by animal randomization and is divided into 2 groups, often Organize 10 animals.One group accepts intramuscular (IM) injection 1x10⁶The individual adherent cell (PLX-C) being derived from Placenta Hominis, another organizes conduct Compare and inject PBS.

Operation technique-at the skin up 1-1.5cm otch of inguinal region.With 6-0 silk thread by femoral artery ligation twice And cut off ligature far-end.With 3-0 silk suture wound and make mice recover.Excision one end femoral artery after 5 hours two Individual site of administration is to the 1x10 that mice IM injection cumulative volume is 50 μ 1⁶The individual adherent cell (PLX-C) being derived from Placenta Hominis.Matched group moves Thing is injected with PBS (Gibco) equally, see table 6.

Table 6: the preliminary study of PLX-C in mouse hind leg ischemia model

Tracing study-Post operation immediately with Post operation the 6th, 9,14 and 21 days with non-contact laser doppler instrument continuous 3 times Measure the blood flow on lower limb both sides, and be expressed as the ratio [Tokai-J. etc.] of ischemic limb and normal limb blood flow.

The classification morphology scale of the visual assessment of ischemia seriousness-use necrotic zone was at the 1st, 6,9,14,21 days naked eyes Assessment ischemic limb is until research terminates；0 grade: without downright bad, I level: be only limitted to the necrosis (toe forfeiture) of toe, II level: extend to The necrosis (foot is lost) on instep；III level: extend to the necrosis (knee joint forfeiture) of foot, IV level: extend to the necrosis of thigh (after Wan Quan Limb is lost) [Tokai.J. etc.].

Internal assessment limb function and ischemic injuries-following series carry out the semi-quantitative assessment of the impaired purposes of ischemic limb: 3= Dragging foot, 2=is without dragging foot but without plantar flexion, and 1=plantar flexion is, and 0=bending toe is with the opposing mild traction to tail (Rutherford etc., 1997).

Molecule and biochemical analysis-and except clinical assessment, obtained molecule and biochemical samples at the 21st day and carry out Analyze to be best understood from causing injection to be derived from the molecular mechanism that adherent cell (PLX-C) the group healing of Placenta Hominis improves.

Experimental result

Transplant hip and the blood flow-in order to detect adhesion of foot of the adherent cell notable inducing ischemia hind limb model being derived from Placenta Hominis The in vivo efficacy of cell, after being ligatured by rat aorta, intramuscular injection is derived from the adherent cell of Placenta Hominis and after the treatment in the scheduled time Section uses non-contact laser doppler instrument to measure hip and the blood flow of foot (health both sides).As shown in figure 17, injection PLX-C shows Write improve to impaired limb blood flow (BF) (as measured by blood flow assessment), strengthen limb function, increase capillary density, Reduce oxidative stress and endothelial injury.For blood flow, inject latter 9 days and demonstrate this effect and can observe in whole experiment Arrive.In PLX-C process group, BF increases to 80 ± 4.7% from 24 ± 2.3, and in comparison, the BF of vehicle treated group 35 ± In the range of 2 to 54 ± 4.5%-hip/graft area (within respectively the 0th day, contrasting the 21st day).Similar to hip region, but degree Less, the pawl region of PLX-C process mice also show the increase of BF.Therefore, in vehicle treated group, BF increases from 12 ± 0.6 To 46 ± 4.9%, and BF increases to 52 ± 5.5% (contrasting the 21st day for respectively the 0th day) from 10 ± 0.7 in PLX-C group, as Shown in Figure 17.

Adherent cell internal can improve limb function-in order to evaluate the internal work of the adherent cell being derived from Placenta Hominis further With, assess the limb function in processed mice with the marking system described in material above and experimental technique.As shown in figure 18, Mice display the significantly improving of limb function (respectively 2.5 ± 0.2 contrast 2.1 ± 0.2 comparison contrasts processed with adherent cell PLX-C group, it is noted that the remarkable effect of the 21st day after process).But, the improvement degree of the limb function in observing at 21 days is Comparable, show that PLX-C does not shows the Main change of functional rehabilitation under this experiment condition.

The visual assessment of ischemia seriousness discloses in comparison i.e. vehicle treated group, sees in two animals at the 6th day Observe the necrosis being limited to toe.In PLX-C process group, the most only demonstrate in an animal and be limited to the bad of toe Extremely.Show to supply the number of the new blood capillary (blood vessel) of limbs with the after death immunohistochemical analysis of the limbs of PLX-C process Amount dramatically increases, and shows that PLX-C has the ability (Figure 19) promoting angiogenesis.

Finally, process in mice at PLX-C and observe in process animal that oxidative stress reduces and endothelial inflammation reduces that (it is The alternate parameter that endothelial function improves) (Figure 20 A-B).This be likely due to PLX-C cell process mice in rather than Increase with oxygen supply in the control mice of PBS process.

In a word, compared with comparison i.e. PBS-injection mice, there is no a PLX-C injection mice display response intramuscular (i.m.) cell is used any bad clinical sign or symptom.Therefore, PLX-C induced blood flow increases, it is likely that by the most impaired The angiogenesis that the Histological assessment of limbs supports causes.Additionally, the difference of the delay of downright bad development and size of animal of getting involved is equal Show clinical response.

It is derived from the transplanting of the adherent cell of Placenta Hominis

Implementing the experiment of another effectiveness in Balb/C mice, it includes the peace as described in material above and method part Full terminal (the overall downright bad and histopathological analysis of i.e. selected organ).

In this experiment, use the seven groups of mices being respectively made up of 10 male Balb/c mices (ischemic hindlimb), as follows Literary composition table 7 describes in detail.One Dan Zuwei inducing ischemia of 10 mices is (to detect overall in normal, healthy animal of PLX-C cell Safety and toleration).After inducing ischemia, limb of comparison buffer or PLX-C cell i.m. being got involved is little to observation post administration Mus was to 1 month.The mice of one single group is got involved individually the injecting for twice of limb, between be separated by 1 week (the 1st and the 8th day).Pass through Laser doppler seanning research and application blood flow, by naked eyes and behavior evaluation ischemia seriousness to being administered latter 30 days, now by mice Put to death and retain tissue for histologic analysis.

Table 7: the Efficacy experiments of PLX-C in mouse hind leg ischemia model

In this experiment, use the different PLX-C batches of three concentration.Result display 0.1x10⁶And 0.5x10⁶PLX-C There is the least treatment benefit.With 1x10⁶The animal processed observes the notable of blood flow the 29th day (at the end of experiment) Improvement.Compared with comparison i.e. vector injection mice, in 2M group (batch G.C25), the improvement of this blood flow is significant (p ＜ 0.05).Additionally, compared with single injection, the cell of second time injection same batch significantly improved BF (respectively 55 at the 15th day ± 24 contrasts 31 ± 12.9 and 27 ± 12.5%).The visual assessment of ischemia seriousness shows and control vector process group (6M) phase Ratio is accepting 1x10⁶(1M&2M) group has the trend of improvement.

These results integrate and show that adherent cell in induced vascularization effect (such as blood flow) and improves posterior-limb ischemia Effect in mouse model limb function also indicates these cells (for example originating from the adherent cell of Placenta Hominis) in treatment ischemic limb Purposes in body disease.

Embodiment 7

For treating the PLX-C of apoplexy

Object of this investigation is to assess whole body (intravenous) Transplanted Human PLX-C (being derived from the adherent cell of Placenta Hominis) in treatment Therapeutic efficiency in apoplexy.

Material and experimental technique

Experimenter, perform the operation and transplant

Hypertension, hypercholesterolemia, diabetes and microangiopathic male spontaneous hypertensive rat are suffered from use.Will be dynamic Thing is raised under conditions of temperature, air humidity and illumination/dark cycle are constant.Experimenter is assigned randomly to experimental group (see Table 8 below).

Table 8: the rat process group in curing apoplexy

Group #	Process	Number of animals
			1	PLX-C, batch 1 single administration	N=8
2	PLX-C, batch 1 administered twice	N=7
			3	PLX-C, batch 2 single administration	N=8
4	PLX-C, batch 2 administered twice	N=7
			5	Comparison-carrier solution	N=12

Animal accepts the 1x10 of single or twice different batches⁶PLX-C dosage.The equal intravenous of all graft procedures is implemented. Double injection group is transplanted after cerebral ischemia 10 and 24 hours, and single is transplanted and within 24 hours, carried out after a stroke.All transplanted Cell fluorescent dye PKH26 preliminary making.

Experimental cerebral ischemia is implemented by the right arteriae cerebri of permanent occlusion.It should be noted that an animal is the most dead.

MR investigation (MRI)

The MRI of lesion development was carried out with 1.5T scanner (Philips) at the 1st, 8,29 and 60 days.Measure Infarction volume and Brain atrophy also uses crown T2-sequence to be calculated as the meansigma methods of three blind researcher institute values.

Behavioral value

By using two independent behavioral value array measurement changes of function.Walk crossbeam experiment for for quantitative sensory- The conventional detection of movement defect.Making rat run the rod of horizontal positioned, the cage of rat is at rod end.Measure five crossing times And it is recorded as meansigma methods in the daytime.Hovering over and be assessed as on crossbeam 20 seconds, dropping is 30 seconds.In first week, every day and every seven days enter Row is measured until the observation period terminates.

Second detection the most modified neurological severity score (mNSS) comprises additional sensation, moves and reflect item Mesh.The result of mNSS is expressed as the scoring between 1 to 18, and wherein the fraction representation between 1 to 6 is slight, and 7 to 12 is moderate and 13 It is major injury to 18.Within the 1st, 4,7,14,21,28,35,42,49 and 56 days after cerebral ischemia, carry out mNSS scoring assessment.

Histology

After experiment periods terminates, put to death all rats the formalin solution through heart perfusion 4%.The brain group that will extract Knit freezen protective and be cut into 30 μm slabs.For the assessment of neuroglia reaction, resist with the one of anti-GFAP and carry out immune group Weave chemistry is studied.The GFAP+ cell density in the region that the inspection (sxemiquantitative) 750 μm near infarction border are wide.For star god Through the detection that glial cell is reactive, including 15 regions of equispaced 0.6mm.

Statistics

Institute is related to body weight, MRI analyzes and the Gauss distribution of the data of histological inspection with ANOVA and accordingly Grade ANOVA analytical statistics significant difference.

Carry out detailed statistical analysis by walking the data collected in crossbeam and mNSS experiment, be considered as the repetition of experimenter Measure and the of short duration progress (chromatographic analysis) of each experimenter.In order to balance the interindividual variation about the severity of brain injury, use Random intercept model.Therefore walk the data collected in crossbeam experiment need to convert to categorizing system.Here, the time value less than 5 seconds It is considered classification (0), within 5 to 10 seconds, is classification (1), within 10 to 15 seconds, be classification (2), within 15 to 20 seconds, be classification (3), hover as classification And drop as classification (5) (4).

Experimental result

Body weight

Periodically weigh and allow to well estimate the holistic health state of experimenter.The initial stage is observed in all groups Lose weight, get involved (data do not show) owing to anesthesia and operation.Then, it was observed that the rapid normalization of body weight and the stable course of disease Until experiment in the 60th day terminates (data do not show).The body weight increase that experimental group display is corresponding.

Walk crossbeam experiment

What in process of the test, all of experimental group all showed to walk crossbeam classification significantly reduces (data do not show).With compare Group (respectively-0.01247 contrast-0.02931) is compared, and observes away horizontal stroke in test group 1 (PLX-C batch 1 single administration) The significantly lower reduction of beam classification.Do not have between experimental group 3 (PLX-C batch 2 single administration) and matched group (data do not show) The evidence of statistically-significant difference.

The neurological severity score (mNss) modified

What all experimental grouies showed neurological score point significantly reduces (data do not show).Relatively with PLX-2 (PLX-C Secondary is administered) the mNSS result of experimenter treated discloses the superiority of the statistically significant compared with matched group.Batch 2 Twice repeated embryo transfer of (group 3) show and significantly improves in mNSS test compared with the simple injection of same batch.

Infarction volume is measured

Nuclear magnetic resonance is a kind of method fine for the height estimating internal brain injury and tissue forfeiture.Consider individuality Between fluctuation, the progress of Infarction volume is each expressed as the percent of the 1st day Infarction volume.The stalk of the 1st day between experimental group Dead volume is not significantly different from.Between 1st day and the 8th day, the overall development of Infarction volume shows the minimizing of about 50%.This is main Owing to initial stage cerebral edema regression.Damage internal progress with MRI detection and show that organizing 4 (PLX-C batch 2 administered twice) experimenters exists Within 60th day, show significantly reduced infarction coefficient (respectively 0.48 ± 0.02 contrast 0.60 ± 0.03, result does not shows).

These results integrate and show that Ink vessel transfusing gives PLX-C and causes in curing apoplexy function in two behavioral value extensive Multiple significantly improves.And, observe the quite big of twice transplanting of PLX-C and statistically significant relative to similar single injection Advantage.

The confirmation treating the behavior improvement measured through MRI in the experimenter of twice with PLX-C is obvious.Additionally, in examination Significantly reducing of Infarction volume and brain atrophy is observed at the end of testing.And, in two Function detection with compare and single note Not verified effect is compared after penetrating, it was observed that two doses transplants the stable improvement after PLX-C to functional rehabilitation.

Embodiment 8

Treatment needs the pathology of connective tissue regeneration and/or reparation

Adherent cell of the present invention treatment is used to need the pathology of osteanagenesis and/or reparation

(it is derived from Placenta Hominis or fat group to detect adherent cell of the present invention with animal model (such as, ripe New Zealand white rabbit) Knit and available from 3D cultivate, such as PLX-C cell) femur critical size segmental defect heal in effect.Animal is divided at random One group be fitted in three groups.1-10x10 is injected to A treated animal defect⁶Individual adherent cell (PLX-C cell).B treated animal is noted Penetrate PBS.In C treated animal, do not process defect.Post operation is immediately and interval prepares radiograph in one week.At the 12nd week, put to death dynamic Thing, the femur involved by removal, preparation is from defect and the non-decalcification tissue slice of adjacent bone.Carry out machinery, histological Test in the healing with detection defect and defect with histomorphometricall and the bone formation of surrounding.Additionally, reverse Record-polymerase chain reaction (RT-PCR) detection I type and the mRNA of II Collagen Type VI.

Tendon regeneration and/or the pathology repaired is needed with adherent cell of the present invention treatment

(such as PLX-C is thin to detect adherent cell of the present invention with animal model (such as, the New Zealand white rabbit of skeletal maturation) Born of the same parents) to tendon healing effect.Long for big toe tendon is transferred in the calcaneus tunnel of 2.5-mm diameter.Use or do not use PLX-C Treatment osseous tunnel.One group animal is assigned randomly in three groups.1-10x10 is injected to the injection of A treated animal defect or IV⁶ Individual PLX-C cell.B treated animal injection PBS.In C treated animal, do not process defect.After surgery the 2nd, 4 and 6 weeks results from Three samples of each group by using conventional histology and immunohistochemical localization I, II and type III collagen to heal Tendon to the assessment of the morphological characteristic of bone interface.

The pathology of regenerating bone or cartilage and/or reparation is needed with adherent cell of the present invention treatment

(such as PLX-C is thin to detect adherent cell of the present invention with animal model (such as, the New Zealand white rabbit of skeletal maturation) Born of the same parents) effect to Cartilage healing.Implement the full-thickness defects of fl far-end patellar groove articular cartilage.From covering quricipital fascia Remove the flap of about 6mm and with the edge of 6-0 catgut suture to artificial defect.One group animal is assigned randomly in three groups. 1-10x10 is injected to the injection of A treated animal defect or IV⁶Individual PLX-C cell.B treated animal injection PBS.In C treated animal, Do not process defect.Put to death animal.Transplanting PLX-C cell to after ten surroundings on osteochondral defect, excision distal femur is also carried out Histological assessment, advantage character based on repair tissue, color substrates, the systematicness on surface, structural intergrity, reparation thickness, Repair in involutory, the repair tissue between cartilage and surrounding normal cartilage without degeneration sign and surrounding normal cartilage without degenerative Change is to sample sxemiquantitative classification.

Ligament regeneration and/or the pathology repaired is needed with adherent cell of the present invention treatment

(such as PLX-C is thin to detect adherent cell of the present invention with animal model (such as, the New Zealand white rabbit of skeletal maturation) Born of the same parents) effect to Ligament healing.Implement the mono cortex round defect of diameter 8mm.One group animal is assigned randomly in three groups. 1-10x10 is injected to the injection of A treated animal defect or IV⁶Individual PLX-C cell.B treated animal injection PBS.In C treated animal, Do not process defect.Transplant PLX-C cell and put to death animal to ten surroundings in ligament defect.Carry out Histological assessment and according to reparation The dominance confrontation sample sxemiquantitative classification of tissue.

It should be appreciated that for clarity sake, some feature of the present invention illustrated in the context of independent embodiment Offer also can be provided in single embodiment.Otherwise, for the sake of brief, the basis illustrated in single embodiment context The various features of invention, it is possible to separately or with any suitable sub-combination provide.

Be elaborated although the present invention combines its specific embodiments, it is apparent that the most alternative, modifications and changes for Will be apparent to for those skilled in the art.Accordingly, it is intended to embrace fall in the spirit and broad range of accessory claim All such alternative, modifications and variations.The all publications, patents and patent applications that this specification is previously mentioned and GenBank accession number is incorporated at this entirely through introducing this specification with it, and its degree is as clearly and point out often individually As one independent publication, patent or patent application or GeneBank accession number are incorporated herein by reference.It addition, in the application The quoting or identify and be not construed as recognizing the prior art that such reference can be used as the present invention of any reference.

REFERENCES

(Additional References are cited in text)

Bauer, Thomas W., Muschler, George F., Bone Graft Materials:An Overview Of theBasic Science.Clinical Orthopaedics&Related Research.371:10-27, February2000.

Carstanjen B, Desbois C., Hekmati M, and Behr L.Successful engraftment of culturedautologous mesenchymal stem cells in a surgically repaired soft palate defect in anadult horse.Can J Vet Res.2006 April；70 (2): 143-147.

Bruder SP, et al.1998 The effect of implants loaded with autologous mesenchymal stemcells on the healing of canine segmental bone defects.J Bone Joint Surg Am.80 (7): 985-96

Chao Wan, Qiling He, Gang Li, 2006.Allogenic peripheral blood derived mesenchymalstem cells(MSCs)enhance bone regeneration in rabbit ulna critical- sized bonedefect model.Journal of Orthopaedic Research 24(4)610-618.

Herthel D.J.2001, Enhanced Suspensory Ligament Healing in 100 Horses by StemCells and Other Bone Marrow Components.AAEP PROCEEDINGS/Vol.47.

Gordon et al, Tendon Regeneration Using Mesenchymal Stem Cells.p313- 320 in TendonInjuries.Springer London.2005.

Horwitz et al., 1999.Transplantability and therapeutic effects of bone marrow derivedmesenchymal cells in children with osteogenesis Imperfecta.Nat.Med.5:309-313.

Horwitz et al., 2002.Isolated allogeneic bone marrow-derived mesenchymal cellsengraft and stimulate growth in children with osteogenesis Imperfecta:Implications for cell therapy of bone.PNAS 99 (13) 8932-8937.

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Claims (11)

1. the adhering substrate cell cultivated purposes in the medicine of preparation treatment ischemia, wherein said stromal cell derives from tire Dish, and make it adhere on adhesion material, thereby separate adherent cell, and be subsequently transferred to three-dimensional (3D) condition, in three-dimensional (3D) breed in cultivating.

2. the purposes of claim 1, the adhering substrate cell of wherein said cultivation can suppress the immunoreation in experimenter.

3. the purposes of claim 1, the adhering substrate cell of the described cultivation of at least a part of which 10% is in proliferation period.

4. the purposes of claim 1, wherein said dimensional culture includes three-dimensional bioreactor.

5. the purposes of claim 1 or 4, the cultivation in described dimensional culture of the adhering substrate cell of wherein said cultivation is filling Pour down realization.

6. the purposes any one of claim 1-4, the condition of culture of wherein said dimensional culture includes selected from polyester and poly-third The adhesion material of alkene.

7. the purposes any one of claim 1-4, the adhering substrate cell of wherein said cultivation comprise selected from CD73, CD90, The positive mark of CD29 and CD105 expresses.

8. the purposes any one of claim 1-4, the adhering substrate cell of wherein said cultivation comprise selected from CD3, CD4, The negative marker of CD45, CD80, HLA-DR, CD11b, CD14, CD19, CD34 and CD79 is expressed.

9. claim 1 purposes, the adhering substrate cell of wherein said cultivation comprises stromal stem cell phenotype.

10. the purposes of claim 1, wherein said ischemia is peripheral arterial disease (PAD).

The purposes of 11. claim 1, wherein said ischemia is central nervous system (CNS) ischemia.