cell最新文章下载

Stem Cell Reports

Report

Direct Comparison of Autologous and Allogeneic T ransplantation of iPSC-Derived Neural Cells in the Brain of a Nonhuman Primate

Asuka Morizane,1Daisuke Doi,1Tetsuhiro Kikuchi,1Keisuke Okita,1Akitsu Hotta,1,2,3Toshiyuki Kawasaki,4 Takuya Hayashi,5Hirotaka Onoe,4Takashi Shiina,6Shinya Yamanaka,1and Jun Takahashi1,7,8,*

1Center for iPS Cell Research and Application(CiRA),Kyoto University,Kyoto606-8507,Japan

2PRESTO,Japan Science and Technology Agency,Kawaguchi332-0012,Japan

3Institute for Integrated Cell–Material Sciences(iCeMS),Kyoto University,Kyoto606-8501,Japan

4Bio-function Imaging Team,RIKEN Center for Life Science Technologies(RIKEN CLST),Kobe650-0047,Japan

5Functional Architecture Imaging Unit,RIKEN Center for Life Science Technologies(RIKEN CLST),Kobe650-0047,Japan

6Department of Basic Medical Science and Molecular Medicine,Tokai University School of Medicine,Isehara259-1143,Japan

7Department of Biological Repair,Institute for Frontier Medical Sciences,Kyoto University,Kyoto606-8507,Japan

8Department of Neurosurgery,Kyoto University Graduate School of Medicine,Kyoto606-8507,Japan

*Correspondence:jbtaka@cira.kyoto-u.ac.jp

https://www.wendangku.net/doc/522706380.html,/10.1016/j.stemcr.2013.08.007

This is an open-access article distributed under the terms of the Creative Commons Attribution License,which permits unrestricted use,distribution,and reproduction in any medium,provided the original author and source are credited.

SUMMARY

Induced pluripotent stem cells(iPSCs)provide the potential for autologous transplantation using cells derived from a patient’s own cells.How-ever,the immunogenicity of iPSCs or their derivatives has been a matter of controversy,and up to now there has been no direct comparison of autologous and allogeneic transplantation in the brains of humans or nonhuman primates.Here,using nonhuman primates,we found that the autologous transplantation of iPSC-derived neurons elicited only a minimal immune response in the brain.In contrast,the allografts caused an acquired immune response with the activation of microglia(IBA-1+/MHC class II+)and the in?ltration of leukocytes(CD45+/ CD3+).Consequently,a higher number of dopaminergic neurons survived in the autografts.Our results suggest that the autologous transplan-tation of iPSC-derived neural cells is advantageous for minimizing the immune response in the brain compared with allogeneic grafts.

INTRODUCTION

In recent studies,murine induced pluripotent stem cell (iPSC)-derived teratomas in the subcutaneous space induced an immune response in syngeneic mice(Zhao et al.,2011). In contrast,syngeneic transplantation of skin and bone marrow tissues(Araki et al.,2013)or endothelial,hepatic, and neuronal cells(Guha et al.,2013)derived from iPSCs showed a limited or no immune response,respectively. These rodent studies investigated the immunogenicity of teratomas,chimeric mouse-derived tissues,or ectopic grafts, but did not convincingly simulate the clinical situation. Parkinson’s disease is one of the most promising targets for cell therapy with pluripotent stem cells,in which differenti-ated dopaminergic(DA)neurons are transplanted into the putamen of a patient’s brain(Lindvall and Bjo¨rklund, 2011).In order to assess the immunogenicity of iPSC-derived neural cells in a primate brain,we generated iPSCs from four cynomolgus monkeys and directly compared the autologous and allogeneic transplantation of iPSC-derived neural cells. RESULTS

iPSCs Derived from Nonhuman Primates Differentiate into DA Neurons

For the?rst two animals(Nos.1and4),we established iPSCs from?broblasts derived from the oral mucosa using retroviral vectors(Okita et al.,2011).For the other two an-imals(Nos.6and8),we used peripheral blood mononu-clear cells(PBMCs)with nonintegrating episomal vectors (Okita et al.,2013).We selected the best clone from each animal according to the following criteria:a stable embry-onic stem cell(ESC)-like morphology of the colonies after passaging,expression of pluripotent markers,few or no in-tegrated transgenes(Figures1A–1F;Figure S1available online),and the potential for stable neural differentiation.

A PCR analysis revealed that all of the clones with retroviral vectors showed apparent expression of remaining trans-genes(Figures S1C and S1D),whereas the clones with episomal vectors never did(Figure S1F).To detect the iPSC-derived cells in a brain,we introduced GFP(Figures 1G and1H).The selected clones of iPSCs had the potential to generate teratomas in the testes of a severe combined im-munode?ciency(SCID)mouse within12weeks(Figures 1I–1M).

To ef?ciently generate DA neurons from monkey iPSCs, we modi?ed previously described protocols(Eiraku et al., 2008;Chambers et al.,2012;Morizane et al.,2011).Brie?y, dissociated iPSCs were incubated in ultralow-attachment 96-well plates in medium containing inhibitors of bone morphogenetic protein(BMP)and Activin/NODAL signaling to initiate neural induction.To induce differenti-ation of the cells toward midbrain DA neurons,purmorph-amine/FGF8and FGF2/FGF20were added sequentially

(Figure 1N).During differentiation,the expression of a pluripotent marker (OCT4)gradually decreased,whereas the levels of neural and DA markers increased (Figures 1O

and 1P).The differentiated neurons expressed markers characteristic of midbrain DA neurons (LMX1A,FOXA2,TH,and PITX3;Figures 1Q and 1R).Besides DA

neurons,

Figure 1.Characterization of Primate iPSCs and iPSC-Derived Neurons

(A–H)Phase-contrast images (A,B,and G)and immunostaining for pluripotent markers (C–F)of iPSCs (T7).GFP was detected during live imaging (G and H)in the same ?eld.

(I–M)Teratoma formation at 3months after transplantation in the testes of SCID mice.H&E staining of the sections showed histological features of the neuroepithelium (J),cartilage (K),muscle (L)and gut-like epithelium (M).(N)The protocol used for neural differentiation.

(O)Expression analyses of neural markers and Oct4by ?ow cytometry.The negative control was a cell sample stained only by secondary antibody (PSA-NCAM)or the isotype control (TUB b III and OCT4).The positive control for OCT4was undifferentiated (day 0)iPSCs.(P)qPCR for the differentiation of donor cells.The data are shown as the means ±SD (n =4independent experiments).(Q)Immunostaining of primate iPSC-derived neurons on day 39.

(R)Quanti?cation of immunocytochemical analyses for each iPSC line.Data are shown as the means ±SD (n =3independent experiments).SER,serotonin,TUB b III,b -tubulin class III.Scale bars:200m m in (A)–(H),50m m in insets of (C)–(F),100m m in (J)–(M)and (Q).See also Figure S1and Tables S2and S3.

Stem Cell Reports

Immunogenicity of iPSC-Derived Neurons in Primates

other types of neurons,such as serotonergic and GABAergic neurons,as well as proliferating neural progen-itors positive for KI67,were also observed as minor popula-tions(Figures1Q and1R).OCT4was not detected by immunocytochemistry or?ow cytometry in the differenti-ation culture even from the retroviral iPSCs(Figure1O). Expression of Major Histocompatibility Complex

by Monkey iPSCs In Vitro

Next,we investigated the expression of major histocom-patibility complex class I(MHC-I)in iPSC-derived neural cells.Flow cytometry using antibodies against human leukocyte antigen(HLA)-A,HLA-B,and HLA-C revealed that mature neurons on days35and71expressed only a low level of MHC-I,and that the expression was enhanced in response to interferon gamma(IFN-g:25ng/ml for48hr; Figure2A).The expression level of the mRNAs was approx-imately1:100compared with peripheral blood cells in both ?broblast-and blood-cell-derived iPSCs,which was again increased by exposure to IFN-g(Figure2B).These results suggest that donor-derived neurons could express MHC-I when INF-g was secreted by the host brain under in?am-matory conditions.

To ensure that the MHC-Is of the host animal and donor cells were mismatched in the allotransplantation cases,we performed genotyping of the expressed MHCs from the monkeys,which were purpose-bred,second-generation (F2),captive-born animals.As shown in Figures2C and 2E,and Table S1,each monkey expressed different MHCs in terms of the A and B alleles.Based on these results,we chose the most mismatched combination for allotrans-plantation(Figures2D and2E).

Autografts Elicit Only a Minimal Immune Response

in the Primate Brain

We injected the iPSC-derived neural cells(day28)into the original monkey as an autograft,and into the MHC-mis-matched monkey as an allograft(Figure2D).Each animal received six separate injections( 8.03105cells in a4m l suspension per tract)in the left striatum,and was observed for 3.5–4months without immunosuppression.In the brain,both brain-resident microglia and circulating immune cells work as key players in immunological re-sponses.Once the microglia are activated,they develop an-tigen-presenting activity.PK11195selectively binds to the translocator protein that is expressed on activated micro-glia(Shah et al.,1994;Vowinckel et al.,1997).Therefore, positron emission tomography(PET)studies with[11C] PK11195have been used to visualize brain in?ammation in patients(Debruyne et al.,2003).

In sequential PET studies,we observed increased uptake of[11C]PK11195in one allograft(animal No.10)at 3months(Figures3A and3B).We could not detect any apparent uptake in the other animals or at any other time points(Figure S2).Intriguingly,the serum level of IFN-g temporarily increased at2months after the transplant in three animals(Figure3C).An immuno?uorescence study conducted at3.5–4months showed that MHC-II+cells were more frequently found in allografts than in auto-grafts,especially in the monkey with increased uptake of [11C]PK11195(Figure3D,No.10).The MHC-II staining never overlapped with that of GFP of the donor cells(Fig-ure3F),whereas it generally overlapped with that of IBA1 (Figure3G),indicating that MHC-II was expressed by host-derived microglia.Consistently,the number and den-sity of IBA1+cells were higher in allografts than in auto-grafts(Figures3E,3H,and S4C).An increase in the expres-sion of MHC might trigger the recruitment of circulating immune cells,including T cells.An immuno?uorescence study revealed that more CD45+cells(a marker for pan-leukocytes)accumulated in allografts compared with auto-grafts(Figures3I and3J).Most of the CD45+cells were CD3+T cells,and60%of them were CD8+killer T cells(Fig-ures3K and3L).These?ndings suggest that an acquired immune response was elicited only in the allografts in the primate brain.

DA Neurons Survive in Both Types of Grafts,but

a Higher Number Are Observed in Autografts

In order to evaluate the survival of the grafted cells,we per-formed MRI scanning and a histological analysis at3–4months posttransplantation(Figure4).Hematoxylin and eosin(H&E)staining and immunostaining for GFP of the brain slices demonstrated that the grafted cells survived in both auto-and allotransplantation without immuno-suppression(Figures4A–4F).Furthermore,there were no signi?cant differences in volume between the auto-and allografts(Figure4K).Immunostaining for tyrosine hy-droxylase(TH),a marker for DA neurons,revealed that a large number of DA neurons(4,428±1,130per tract,n= 22)survived in the autografts(Figures4G,4H,4L,and 4M).The surviving DA neurons coexpressed the markers of a mesencephalic phenotype,such as FOXA2,NURR1, and the dopamine transporter(DAT)(Figures4N–4P). Even in allografts without immunosuppression,the TH+ neurons survived well(2,247±641per tract,n=22),but the number and density were lower than in autografts(Fig-ures4I,4J,4L,and4M).We also found a small number of astrocytes(GFP+/GFAP+),as well as mature neurons (GFP+/NEUN+),in vivo(Figure S4B).

DISCUSSION

In this study,we induced DA neurons from monkey iPSCs by directed differentiation in vitro,and demonstrated that

Stem Cell Reports

Immunogenicity of iPSC-Derived Neurons in Primates

Stem Cell Reports

Immunogenicity of iPSC-Derived Neurons in Primates

Figure2.Identi?cation of MHC Expression and Typing of Donor Cells

(A)Flow-cytometric analyses for MHC-I(HLA-A,HLA-B,and HLA-C).Incubation of the cells with IFN-g for48hr increased the MHC-I expression(green).

(legend continued on next page)

autologous transplantation of the iPSC-derived cells eli-cited only a minimal immune response in the nonhuman primate brain.Previous reports have suggested that either autologous grafts of iPSC-derived neural cells (Emborg et al.,2013;Maria et al.,2013)or allogeneic grafts of fetal ventral mesencephalic cells (Redmond et al.,2008)can sur-vive in a primate brain without immunosuppression.None of these studies,however,directly compared the immuno-genicity of autologous grafts with that of allogeneic ones.Our results clearly show differences in both immunoge-nicity and cell survival between these two types of grafts,and support the idea that immunosuppression is not neces-sary for autologous transplantation of iPSC-derived neural cells into the brain.The [11C]PK11195PET study was useful for real-time visualization of this phenomenon.Further-more,this technique can also be applied to patients in the clinical setting,which would help to determine when and if immunosuppressive drugs can be withdrawn.

Although we did not examine acute immune responses or in?ammation within 48hr,it is noteworthy that the re-sponses were observed 2or 3months posttransplantation.In the case of iPSC-based transplantation,there are four possible mechanisms that can cause in?ammatory and im-mune responses:(1)direct allorecognition of mismatched MHC or minor antigens of the donor cells,(2)indirect al-lorecognition through host-derived antigen-presenting cells,(3)expression of fetal antigens due to immature stem cells or remaining transgenes,and (4)mechanical damage rather than MHC mismatch.Because of the low expression level of MHC-I by the donor cells,direct allore-cognition is unlikely to be the main cause.However,donor-cell-derived astrocytes (Figure S4B),which can express both MHC-I and MHC-II to recruit T cells in response to IFN-g (Akesson et al.,2009;Chastain et al.,2011),could be observed in the grafts.Furthermore,it takes a longer time for astrocytes to differentiate than neurons.Therefore,although there was no apparent expression of either MHC-I or MHC-II by the grafted GFP +cells,donor-derived astrocytes may have contributed to the direct reaction in the late stage.Considering the high expression level of MHC-II by host-derived microglia in the allografts (Figures 3D–3G),indirect allorecognition seems to have played a major role in the present study.This requires the internal-ization and processing of the alloantigens,which are then recognized in peptidic form bound to recipient MHC-II molecules,possibly accounting for the late onset of the immune response.

Autografts derived from iPSCs generated by retroviral vectors resulted in the accumulation of larger numbers of IBA1+and CD45+cells compared with those generated us-ing episomal vectors,probably due to the residual expres-sion of the transgenes (Figure S3).This indicates that resid-ual transgenes can be immunogenic,and that it is therefore critical to use integration-free iPSCs.Mechanical damage caused by needle trauma can also activate host astrocytes and microglia to secrete proin?ammatory cytokines,which recruit leukocytes.Consistently,we found IBA1+cells along the needle tract in the animals that received control injec-tions (Figures 3E and S4C),but this was limited to a small area and not likely to play a major role.

Another important ?nding is that,in spite of the im-mune responses mounted by the host brain,a substantial number of TH +cells survived in the allografts.This is consistent with previous clinical reports of human fetal cell transplantation.Postmortem analyses of the patients revealed robust survival of DA neurons in spite of the fact that numerous immune cells were present around the graft (Kordower et al.,1997).In two double-blind clinical trials,immunosuppressive drugs were never used (Freed et al.,2001)or were withdrawn after 6months (Olanow et al.,2003).In these cases,the cells from multiple fetuses were used without HLA matching,but more than 50,000TH +cells had survived after several years.Our quantitative PCR (qPCR)study in vitro showed that the expression of MHC-I increased in response to IFN-g ,but the expression level was still 1/10that of untreated monkey peripheral blood cells (Figure 2B).The in vivo studies revealed that the serum level of IFN-g increased at 2months,and CD45+cells (including CD8+cells)accumulated in the allo-grafts 3.5–4months after the transplant.On the other hand,the levels of INF-g in the cerebrospinal ?uid (CSF)and the levels of tumor necrosis factor a (TNF-a )in both the serum and CSF were below the limit of detection by ELISA (data not shown).An immuno?uorescence study did not reveal any apparent expression of MHC-I by the grafted cells (Figure S4A).Therefore,it is possible that the immune response in the primate brain was not strong enough to reject all of the donor cells.These ?ndings

(B)Temporal MHC-I expression analysis of monkey iPSCs by qPCR.The data were obtained from three (n =3for ?bro iPSCs)or ?ve (n =5for blood iPSCs)different experiments.Data are shown as the means ±SD.

(C and E)Monkey MHC-I (Mafa)allele sequences detected by next-generation sequencing (C)and cluster analysis of the monkey MHCs (E).The colored letters indicate a comparatively high expression level of the MHC-I allele,comprising >10%of cDNA sequence reads.The gray background indicates the MHC-A allele,and the others indicate the MHC-B allele.

(D)Combinations of donor cells and recipient animals.Episomal v.,established with episomal vectors;P,passage number just before starting the differentiation of donor cells;Retro v.,established with retrovirus vectors.See also Figure S4D and Tables S1–S3.

Stem Cell Reports

Immunogenicity of iPSC-Derived Neurons in Primates

Stem Cell Reports Immunogenicity of iPSC-Derived Neurons in Primates

(legend on next page)

closely correlate with the results of previous murine exper-iments (Hudson et al.,1994;Shinoda et al.,1995).To apply our ?ndings to a more clinically relevant setting,we inves-tigated the expression of HLA-I during neural differentia-tion of human ESCs (hESCs)and iPSCs by qPCR (Fig-ure S4D).The expression level was 1/100compared with that of human peripheral blood cells in both hESCs and iPSCs,and it was similarly elevated in response to IFN-g .It is dif?cult to precisely compare immunogenicity in monkeys with that in humans,but the low expression level of MHC-I by the donor cells may account for the mild rejection in both monkey and human neural transplantation.

Our results indicate that autologous transplantation is bene?cial in terms of the immune response and cell sur-vival.However,this strategy is associated with higher costs and labor.An alternative method is allogeneic trans-plantation using HLA-matched iPSC stocks (Okita et al.,2011;Nakatsuji et al.,2008;Deleidi et al.,2011).There-fore,as a next step,it is critical to determine whether autografts have advantages over HLA-matched allografts and HLA-mismatched allografts with immunosuppres-sion.To answer this question,it will be necessary to establish iPSCs from MHC-homozygous monkeys and transplant the iPSC-derived cells into monkeys with the identical MHC haplotype.The precise in?uence of HLA mismatch therefore needs to be explored in such future studies.

EXPERIMENTAL PROCEDURES

Nonhuman Primates

Eight purpose-bred male cynomolgus monkeys (Macaca fascicula-ris )were used for iPSC generation and transplantation.The animal experiments were performed in accordance with the Guidelines for Animal Experiments of Kyoto University,the Institutional Animal Care and Use Committee of Kobe Institute in RIKEN,and the Guide for the Care and Use of Laboratory Animals of the Institute of Laboratory Animal Resources (Washington,DC,USA).See also Supplemental Experimental Procedures .

Generation and Neural Differentiation of iPSCs

Fibroblasts from the oral mucosa were transfected retrovirally with ?ve transgenes (OCT3/4,SOX2,KLF4,L-MYC ,and LIN28)(Okita et al.,2011).PBMCs were transfected with a combination of plasmid vectors (OCT3/4,SOX2,KLF4,L-MYC ,LIN28,shRNA for TP53,and EBNA1)as described previously (Okita et al.,2013).The primate iPSCs were maintained on mouse embryonic ?bro-blast feeders treated with mitomycin-C (Sigma-Aldrich).They were differentiated into DA neurons through the SFEBq method (Eiraku et al.,2008)with dual SMAD inhibitors (Chambers et al.,2012;Morizane et al.,2011;Figure 1N).The cells were transplanted on day 28of differentiation.For in vitro analysis,the cells were dissociated with Accumax (Innovative Cell Technologies)on day 28and cultured on eight-well glass chamber slides coated with poly-l-ornithine and laminin for an additional 11days (for a total of 39days).

Genotyping of MHC

MHC genotypes were assigned by comparing the sequences with known MHC allele sequences released from the Immuno Polymor-phism Database (https://www.wendangku.net/doc/522706380.html,/ipd/index.html ).See also Supplemental Experimental Procedures .

Immunostaining and Histological Analyses

For in vivo studies,the ?xed frozen brains were sliced at 40m m thickness and immunologically stained via the free-?oating method.The primary antibodies used are listed in Table S3.See also Supplemental Experimental Procedures .

MRI and PET Studies

PET scans with [11C]PK11195were performed with the use of an animal PET scanner (microPET Focus220;Siemens Medical Solu-tions)to identify the activation of microglia.High-resolution T1-weighted and T2-weighted images were obtained using a 3T MRI scanner (MAGNETOM Verio;Siemens AG)to identify the injection site of grafts in the postero-dorsal striatum and to evaluate graft survival.See also Supplemental Experimental Procedures .

Transplantation

Floating aggregates (day 28)were harvested and dissociated into small clumps of 20–30cells with Accumax.The cells were sus-pended in the last culture medium (Figure 1N),which was

Figure 3.Immune Responses following Autologous or Allogeneic Transplantation

(A and B)[11C]PK11195PET study of the allografts in animal No.10,in which the highest immune response was observed histologically.The illustration in (A)shows the method used for cell injection.

(C)Temporal changes in the serum level of IFN-g .Bottom:a two-way ANOVA was performed with Bonferroni’s multiple-comparisons test;n =4animals,*p <0.05.Data are shown as the means ±SEM.

(D–H)Histological analyses of the host-resident microglia.The image of H&E staining of animal No.8is shown as an anatomical reference for coronal sections (D).The arrowheads in (A)and (D)indicate the direction of the cell injections.(I–L)Histological analyses of in?ltrating leukocytes.

Scale bars:1cm (A),2mm (D),100m m (E and I),and 20m m (F,G,K,and L).Quantitative data are presented as the means ±SEM (n =22tracts).Ratio paired t tests were performed for the auto-and allo-tracts.***p =0.0003(H,upper),**p =0.0015(H,lower),**p =0.0035(J,upper),*p =0.0122(J,lower).All of the PET,MRI,and histological images show coronal sections.See also Figures S2–S4and Table S3.

Stem Cell Reports

Immunogenicity of iPSC-Derived Neurons in Primates

Stem Cell Reports

Immunogenicity of iPSC-Derived Neurons in Primates

Figure4.Graft Survival in a Primate Brain

(A–P)Histological analyses of a brain section with H&E staining,and the immunohistochemical?ndings.

(K)Quantitative analyses of the graft volume.

(L and M)Survival of TH+neurons in the grafts.

(N and O)The TH+neurons expressed markers of the midbrain phenotype:Foxa2(N)and Nurr1(O).

(P)The DAT was also positively stained.

(legend continued on next page)

neurobasal medium with B27with added ascorbic acid,dibutyryl-cAMP ,glial-cell-line-derived neurotrophic factor,and brain-derived neurotrophic factor.We also added a ROCK inhibitor,Y27632,to increase the survival of the donor cells.The suspension was prepared at a concentration of 23105cells/m l,and 4m l of the suspension was injected through a 22-gauge needle with a Hamil-ton syringe.We made six (two coronal 3three sagittal)tracts of injection in one side of the putamen.In total,4.83106cells per animal (8.03105cells/tract 36tracts)were injected into one side of the putamen according to the coordinate decided by the MRI image of each monkey.The same volume of the culture me-dium was injected to the contralateral side as control.No immuno-suppressant was used.Under deep anesthesia,the animals were sacri?ced and perfused transcardially with 4%paraformaldehyde after 3.5–4months of observation.

Statistics

The data were expressed as the mean ±SD or mean ±SEM,and dif-ferences were tested by commercially available software Prism 6(GraphPad).With regard to the histological data for the auto-and allo-tracts,after con?rming normal distribution,we proceeded to perform statistical analyses with ratio paired t tests.Some tracts were omitted from the assessment because of technical problems;p values <0.05were considered to be signi?cant.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,four ?gures,and three tables and can be found with this article online at https://www.wendangku.net/doc/522706380.html,/10.1016/j.stemcr.2013.08.007.

ACKNOWLEDGMENTS

We thank Ms.E.Yamasaki and Mr.K.Kubota for their technical assistance,Drs.K.Tanaka and T.Sato for the genetic analyses,Dr.Y.Yamada for his valuable advice on the histological studies,Dr.Y.Ono for providing antibodies,and Dr.H.Kawamoto for his critical advice on the manuscript.This study was supported by grants from the Highway Project for Realization of Regenerative Medicine (Ministry of Education,Culture,Sports,Science and Technology),the Funding Program for World-Leading Innovative R&D on Science and Technology (FIRST Program,Japan Society for the Promotion of Science),and the Shimizu Foundation for Immunology and Neuroscience Grant for 2012.Received:June 23,2013Revised:August 29,2013Accepted:August 30,2013Published:September 26,2013

REFERENCES

Akesson,E.,Wolmer-Solberg,N.,Cederarv,M.,Falci,S.,and Ode-berg,J.(2009).Human neural stem cells and astrocytes,but not neurons,suppress an allogeneic lymphocyte response.Stem Cell Res.(Amst.)2,56–67.

Araki,R.,Uda,M.,Hoki,Y.,Sunayama,M.,Nakamura,M.,Ando,S.,Sugiura,M.,Ideno,H.,Shimada,A.,Nifuji,A.,and Abe,M.(2013).Negligible immunogenicity of terminally differentiated cells derived from induced pluripotent or embryonic stem cells.Nature 494,100–104.

Chambers,S.M.,Qi,Y.,Mica,Y.,Lee,G.,Zhang,X.J.,Niu,L.,Bils-land,J.,Cao,L.,Stevens,E.,Whiting,P .,et al.(2012).Combined small-molecule inhibition accelerates developmental timing and converts human pluripotent stem cells into nociceptors.Nat.Biotechnol.30,715–720.

Chastain, E.M.,Duncan, D.S.,Rodgers,J.M.,and Miller,S.D.(2011).The role of antigen presenting cells in multiple sclerosis.Biochim.Biophys.Acta 1812,265–274.

Debruyne,J.C.,Versijpt,J.,Van Laere,K.J.,De Vos,F.,Keppens,J.,Strijckmans,K.,Achten,E.,Slegers,G.,Dierckx,R.A.,Korf,J.,and De Reuck,J.L.(2003).PET visualization of microglia in multiple sclerosis patients using [11C]PK11195.Eur.J.Neurol.10,257–264.Deleidi,M.,Hargus,G.,Hallett,P .,Osborn,T.,and Isacson,O.(2011).Development of histocompatible primate-induced plurip-otent stem cells for neural transplantation.Stem Cells 29,1052–1063.

Eiraku,M.,Watanabe,K.,Matsuo-Takasaki,M.,Kawada,M.,Yone-mura,S.,Matsumura,M.,Wataya,T.,Nishiyama,A.,Muguruma,K.,and Sasai,Y.(2008).Self-organized formation of polarized cortical tissues from ESCs and its active manipulation by extrinsic signals.Cell Stem Cell 3,519–532.

Emborg,M.E.,Liu,Y.,Xi,J.,Zhang,X.,Yin,Y.,Lu,J.,Joers,V .,Swanson,C.,Holden,J.E.,and Zhang,S.C.(2013).Induced plurip-otent stem cell-derived neural cells survive and mature in the nonhuman primate brain.Cell Rep.3,646–650.

Freed,C.R.,Greene,P .E.,Breeze,R.E.,Tsai,W.Y.,DuMouchel,W.,Kao,R.,Dillon,S.,Win?eld,H.,Culver,S.,Trojanowski,J.Q.,et al.(2001).Transplantation of embryonic dopamine neurons for severe Parkinson’s disease.N.Engl.J.Med.344,710–719.Guha,P .,Morgan,J.W.,Mostoslavsky,G.,Rodrigues,N.P .,and Boyd,A.S.(2013).Lack of immune response to differentiated cells derived from syngeneic induced pluripotent stem cells.Cell Stem Cell 12,407–412.

Hudson,J.L.,Hoffman,A.,Stro

¨mberg,I.,Hoffer,B.J.,and Moor-head,J.W.(1994).Allogeneic grafts of fetal dopamine neurons:behavioral indices of immunological interactions.Neurosci.Lett.171,32–36.

(Q–S)Magnetic resonance images of a representative animal (No.6,autograft)at 3months after the transplant.The arrowheads indicate the directions of the cell injections.(Q)coronal,(R)axial,and (S)sagittal.The letter L indicates the left side.

Scale bars:2mm (A,B,D,F,H,and J),50m m (C,E,G,I,N,O,and P),and 1cm (Q–S).Quantitative data are presented as the mean ±SEM (n =22tracts).Ratio paired t tests were performed for the auto-and allo-tracts:**p =0.0021(L),**p =0.0088(M).See also Figure S4and Table S3.

Stem Cell Reports

Immunogenicity of iPSC-Derived Neurons in Primates

Kordower,J.H.,Styren,S.,Clarke,M.,DeKosky,S.T.,Olanow,C.W., and Freeman,T.B.(1997).Fetal grafting for Parkinson’s disease: expression of immune markers in two patients with functional fetal nigral implants.Cell Transplant.6,213–219.

Lindvall,O.,and Bjo¨rklund,A.(2011).Cell therapeutics in Parkin-son’s disease.Neurotherapeutics8,539–548.

Maria,S.,Helle,B.,Tristan,L.,Gaynor,S.,Arnar,A.,Michele,M., Teresia,O.,Oliver,C.,Roger,S.,et al.(2013).Improved cell therapy protocols for Parkinson’s disease based on differentiation ef?-ciency and safety of hESC-,hiPSC-,and non-human primate iPSC-derived dopaminergic neurons.Stem Cells31,1548–1562. Morizane,A.,Doi,D.,Kikuchi,T.,Nishimura,K.,and Takahashi,J. (2011).Small-molecule inhibitors of bone morphogenic protein and activin/nodal signals promote highly ef?cient neural induc-tion from human pluripotent stem cells.J.Neurosci.Res.89, 117–126.

Nakatsuji,N.,Nakajima,F.,and Tokunaga,K.(2008).HLA-haplo-type banking and iPS cells.Nat.Biotechnol.26,739–740. Okita,K.,Matsumura,Y.,Sato,Y.,Okada,A.,Morizane,A.,Oka-moto,S.,Hong,H.,Nakagawa,M.,Tanabe,K.,Tezuka,K.,et al. (2011).A more ef?cient method to generate integration-free hu-man iPS cells.Nat.Methods8,409–412.

Okita,K.,Yamakawa,T.,Matsumura,Y.,Sato,Y.,Amano,N.,Wata-nabe,A.,Goshima,N.,and Yamanaka,S.(2013).An ef?cient nonviral method to generate integration-free human human-induced pluripotent cells from cord blood and peripheral blood cells.Stem Cells31,458–466.Olanow,C.W.,Goetz,C.G.,Kordower,J.H.,Stoessl,A.J.,Sossi,V., Brin,M.F.,Shannon,K.M.,Nauert,G.M.,Perl,D.P.,Godbold,J., and Freeman,T.B.(2003).A double-blind controlled trial of bilat-eral fetal nigral transplantation in Parkinson’s disease.Ann.Neu-rol.54,403–414.

Redmond,D.E.,Jr.,Vinuela,A.,Kordower,J.H.,and Isacson,O. (2008).In?uence of cell preparation and target location on the behavioral recovery after striatal transplantation of fetal dopami-nergic neurons in a primate model of Parkinson’s disease.Neuro-biol.Dis.29,103–116.

Shah,F.,Hume,S.P.,Pike,V.W.,Ashworth,S.,and McDermott,J. (1994).Synthesis of the enantiomers of[N-methyl-11C]PK11195 and comparison of their behaviours as radioligands for PK binding sites in rats.Nucl.Med.Biol.21,573–581.

Shinoda,M.,Hudson,J.L.,Stro¨mberg,I.,Hoffer,B.J.,Moorhead, J.W.,and Olson,L.(1995).Allogeneic grafts of fetal dopamine neu-rons:immunological reactions following active and adoptive im-munizations.Brain Res.680,180–195.

Vowinckel,E.,Reutens,D.,Becher,B.,Verge,G.,Evans,A.,Owens, T.,and Antel,J.P.(1997).PK11195binding to the peripheral benzo-diazepine receptor as a marker of microglia activation in multiple sclerosis and experimental autoimmune encephalomyelitis. J.Neurosci.Res.50,345–353.

Zhao,T.,Zhang,Z.N.,Rong,Z.,and Xu,Y.(2011).Immunogenicity of induced pluripotent stem cells.Nature474,212–215.

Stem Cell Reports Immunogenicity of iPSC-Derived Neurons in Primates

生产线布局说明

制动器生产线平面图如下： 1-磨床；2-助力臂覆盖区域；3-小车；4-零件放置台；5-工人工作位置；6-液压机；7-工作平台1；8-辊子滑台；9-工作平台2；10-工作平台3；11-油桶； 12-成品存放板

生产线所完成的工序包括装配制动底板、磨摩擦片外圆、装配凸轮、装配防护罩及后臂总成、收集成品及注油。以上工序中，工件的搬运、翻转依靠助力臂，工件在辊子滑台上依靠手推移动，装配组件放置在可提升的小车内，小零件放在工作平台上的零件盒内。 一、图示各部分功能介绍： 1-磨床，磨摩擦片外圆，工件由助力臂夹持搬运。 2-助力臂覆盖区域，助力臂采用气压夹持，人工操作，能实现工件的圆周移动、上下移动和翻转等动作，覆盖区域保证工序间夹持工件的要求，安装位置方便工人操作。 3-小车，放置装配的组件，采用液压提升装置，便于拿取。小车的尺寸大小为1000*500*1200（mm）。 4-零件放置台，放置些许制动底板和制动蹄备用。台的尺寸大小为2500*800*750（mm）。 5-工人工作位置，预留出足够空间，保证操作和移动的方便。 6-液压机，装配制动底板和制动蹄，用液压力代替人力压支撑销。 7-工作平台1，安装螺钉和钢丝锁线，平台上有专门区域放置螺钉和钢丝锁线等零件。工作平台的尺寸大小为1000*800*750（mm）。 8-辊子滑台，放置各工序完成后的工件，方便移动。滑台的尺寸大小为1500*800*750（mm），为两工序之间留出2~3个缓冲件，协调工作节拍。 9-工作平台2，安装凸轮和回位弹簧，，平台上有专门区域放置回位弹簧等零件。

10-工作平台3，安装防护罩和后臂总成，平台上专门区域放置螺栓、垫圈等零件。 11-油桶，对装配成品的关键部位注油以润滑、防锈。 12-成品存放板，存放装配好的成品，成品的搬运依靠助力臂。 二、各工序工人任务： 1）工人1，主要完成制动底板的装配，包括压支撑销、装螺钉和钢丝锁线。 2）工人2，主要完成磨摩擦片外圆和装凸轮、回位弹簧，包括工件的翻转。工件的搬运依靠助力臂。 3）工人3，主要完成防护罩和后臂总成的装配。 4）工人4，主要完成收集成品和润滑成品。工件的搬运依靠助力臂。

生产线改造之布局

生产线改造方案布局 一、设施布置设计的原则： （1）符合工艺过程的要求 （2）有效利用空间 （3）物料搬运费用最少 （4）保持生产和安排的柔性 （5）适应组织结构的合理化和管理的方便 （6）为职工提供方便、安全、舒适的作业环境 二、设施布置基本流动模式： 选择车间内部流动模式的一个重要因素是车间入口和出口的位置。 流动模式还受生产工艺流程、生产线长度、场地、建筑物外形、物料搬运方式与设备、储存要求等方面的影响 基本流动模式有如下图的五种。 三、布局形式： （a）直线形（b）L形（c）U形（d）环形 1、工艺原则布置（Process Layout） 一种将相似设备或功能集中布置在一个地方的布置形式，适用于多品种小批量的生产方式。 2、产品原则布置（Product Layout） 也称装配线布置，是一种根据产品制造的步骤来安排设备或工作过程的方式。适用于少品种、大批量的生产方式。 下面举例说明产品原则布置在装配线平衡中的应用过程 例1J型手推车要在一个传送带上组装，每天需生产500辆，每天的生产时间为420分钟。表4－1列出了手推车的组装步骤及其时间，请根据周期时间和作业次序 的限制，求使工作地点数量最少的平衡方式。

设计流程如下： （1）绘制双代号网络图，如下图所示。

（5）分配各工作地点的作业，分配结果如表4－3所示

3、定位布置（Fixed Layout） 产品（由于体积或重量庞大）停留在一个位置上，设备、人员、材料都围绕着产品而转。具有相对较少的产品数量。 4、成组技术布置（Group Layout） 将不同的机器组成加工中心（工作单元）来对形状和工艺相似的零件进行加工。 适应于中小批量生产。 好处： （1）改善人际关系 （2）提高操作技能 （3）减少在制品和物料搬运 （4）缩短生产准备时间 步骤： （1）将零件分类，建立零件分类编码系统。 （2）识别零件组的物流类型，以此作为工艺布置和再布置的基础。 （3）将机器和工艺分组，组成工作单元。

生产单元布局

工艺专业化布局和产品专业化布局 生产设备的布置通常有两种形式，工艺专业化和产品专业化 设备 类型 工种 工艺 方法 加工 对象 生产类型 工艺布局 相同 相同 相同 不同 单件小批 产品布局 不同 不同 不同 相同 大量 1. 工艺专业化布置形式 机群式布置，这个概念与厂区布置的工艺专业化是相同的，只是工艺的概念更 小一些，指把同种类型的设备和人员集中布置在一个地方。 这种布置方式常常用于用同样的 设备来制造和装配各种不同的部件。比较是用于品种多产量小的生产类型。 其特点是：同类设备集中，加工技术单一，生产系统柔性大；加工对象多，工 艺路线差别大，物料搬运有交叉，难以使工件搬动自动化；在各工序之间成批搬运， 加工周期长；周转环节多，不易管理。 2. 产品专业化布置形式 流水线布置，按产品的加工过程顺序配置设施，布置成一条专门的加工生产线。这 种形式适合于品种少产量达的生产类型。 其特点是：生产效率高；生产流程连续性好，可缩短生产周期；降低搬运费用；计 划管理简单，生产易控制；但加工线应变能力差，缺乏柔性。 车间门 车间门

需要注意的是在生产车间内部的布置也应该遵循工艺性、经济性和安全性原则， 具体有以下要求： 1.尽可能保持生产过程的连续性； 2.工件加工中的运送路线要短； 3.车间内要留出足够的通到面积，通道要直； 4.充分保证生产用面积，提高利用率； 5.设备布置要保证安全。 传统生产线的弊端 直线型生产线的生产方式下，人员在一个周期内的来回时间及行走距离较多，如图2所示。某个单元生产周期不能够得到合理限制造成整个系统的周期过长，产品成本增加，如局部环节出现问题会影响整个生产线的连续性，形成在制品堆积的严重现象。 面向产品族的“制造单元”？ 制造单元是以柔性设备为核心的若干台设备的组合，将这些设备按照每一个产品“族”相类同的工艺顺序排列，在制造单元内完成制造这些产品/零件族的全部过程。制造单元不是针对每一个零件的，这样就不是精益制造了。制造单元起码有以下几个特征和功能：？ a）面向一个“产品/零件族”。“族”是一组形状和制造过程相同或相似的零件。因而精益的制造单元是柔性的，大大地减少了由于换型造成的生产停滞。？？ b）制造单元内的设备顺序是按工艺流程排列的，考虑到零件进出单元方向的一致性，经常排列成U形，所以又称其为U形单元，从而保证了＞物流的流程距离最短，而消除了多 种形式的浪费。？？ c）工件在制造单元中按流程顺序自然流转，从而简化了管理，节省了信息的传递和信息流距离。？？ d）制造单元内的工人是多能的，单元团队的成员之间是相互支持和相互替代的，使更加发挥工人和团队的积极性，？？ e）所以建立制造单兀是实行精益生产的关键步骤之一。 单元生产模式是一种基于追求无浪费理念，以工作单元(机械设备、生产人员和在制品的物流系统)为基本组成，对生产线合理布置，进行单一或多品种的生产方式。单元式生产

01第一章 U型生产线布局汇总

第一章 U型生产线布局 【本章重点】 好的生产线布局将为高效率的作业从根本上打下良好的基础，因此设计一个良好的生产线布局至关重要，U型布局目前被公认为是最高效率的生产线布局方法。使用U型布局可以使标准作业顺利进行，使作业管理变得一目了然，使制造现场变得井然有序。 本章主要内容 U型生产线布局的定义 U型生产线布局的优势 使用适合于U型线布局的设备 第一节U型生产线布局的定义 一、改变传统的设备布局思想 在大量制造的工厂中，设备按照种类排布在一起，各工作的几层组织按照加工的内容进行分布，这样的生产线布局要求每两个工程之间必须使用搬运来连接，这导致了工程间在制品大量增加，制造的连续性被破坏。 在进行重新安排生产流程按照U型布局时要充分考虑设备布局的合理性和先进性，必须保证如下原则顺利实施，以确保目标的完成。 1、一个流的生产 在整个生产线布局中必须设法保证在生产过程中的中间在库制品数量最少，即消除中间在库的停滞，让工件像河水 2、所有的零件及完成品使用同样的节拍（Takt Time） Takt来源德语单词，意思是音乐的节拍，在生产制造过程中力求使所有的零件在同一期间使用相同的速度——“节拍”进行制造，不断地使全部零件流动起来，使制造（含组装）以一个流的方法向河水一样的流动，避免过度制造情况发生。 按照节拍进行生产除了考虑适应客户需要的速度外，还充分的考虑了人性化的需要。人喜欢有节奏的事物，例如人对自己喜欢的歌曲听几遍就会记住，但是如果背同样长短的古文或英文单词你会觉得困难的多，因为歌曲有节拍。如果让

精益布置 批量布置 人在生产线作业时，和唱歌一样的富有节奏，那么，人的作业将会轻松很多，这也是更多的考虑了人性化管理的内容后所产生的结果。 3、柔性生产系统 由于售出的速度会在不同时期发生变化。因此，必须使生产线布局能够适应不同生产节拍的要求，利用一人多序的方法，调整人的作业范围达到适应不同生产速度之目标。在作业中，以人的动作为中心，不考虑设备的能力，使人的作业达到最高效率化，要明确设备停止并不能造成成本提高，而人的停止是最大的浪费。 工序/设备 A B C D 1 ☆ ☆ ☆ 2 ☆ ☆ ☆ 3 ☆ ☆ ☆ ☆ 4 ☆ ☆ ☆ ☆ 在这里，需要充分理解一人多机和一人多序的不同 二、建立流程型生产线布局 将设备布局的形式从集群式改造为流程式之后，随着生产制造过程中的物流距离快速缩短，连续流制造已经可以成为现实。在这里我们必须按照工艺流程的顺序排布设备，而不是按照设备的种类，以解决工程间产生的批量流动问题。 从工厂设计开始就必须充分考虑到U 型生产线布局的思路，按照如下原则考虑： 1、联合厂房设计：字制造型工厂中，为了加强信息的沟通和缩短物流距离，尽可能打破传统的按车间建设厂房的方法，使用大型联合厂房可以将相关的工程紧密的连接在一起，以达到减少成本之目标。 2、原动力合理排布：厂房建设好后，原动力尽可能在工厂内均匀分布，在合理的距离上留出接口，因为精益化的改造设备布局可能会经常进行调整，如果由于原动力的问题而不能进行设备移动将是十分遗憾的事情。原动力不仅指电力，同时也包括诸如水、压缩空气、煤气等等。 随着客户对产品需求发生的变化，大量制造的方法生产出来的产品已经远远不能适应客户的需求，因此小批量、多品种制造产品的柔性制造生产线越来越得到大家的重视。 U 型线布局是典型的柔性制造布局，他打破了传统的按照加工种类排布设备

生产线布局情况

生产线布局情况 Company number：【WTUT-WT88Y-W8BBGB-BWYTT-19998】

固体制剂车间生产线的布局情况 固体制剂车间片剂、硬胶囊剂、颗粒剂、锭剂及纯化水系统工艺流程图。 江苏黄河药业股份有限公司固体制剂车间由山东省医药工业设计院根据98版GMP要求设计，其主体为单层结构，固体制剂车间按照产品生产工艺流程及GMP生产要求，主要布置为生产一般区、30万级洁净区、工艺用水制备系统、空调净化系统及压缩机系统等；一般区主要为外包装工序及办公区域；30万级洁净区布置配料、压片、包衣、充填、内包装工序。车间年生产能力为片剂12亿

片、硬胶囊剂3亿粒。车间工艺布局合理，建筑面积共计3742㎡。在洁净区设计上采用人流道、物流道分开，有效避免了交叉污染的可能，物流通道上采用传递窗将物料进入洁净区，保证物料不对洁净区产生污染，确保生产区域符合洁净要求。洁净区空气洁净度，经盐城市药品检验所静态测试，符合30万级要求。车间生产剂型主要为片剂、硬胶囊剂、颗粒剂、锭剂四个个剂型共计116个品种（常年生产37个品种）。经对生产片剂、硬胶囊剂、颗粒剂、锭剂各品种工艺、设备验证及再验证表明，生产设备符合生产需求，生产工艺稳定。空气净化、压缩空气、纯化水等经过验证及再验证，符合药品生产要求。各项标准健全，各有关规程已经建立，运转以来，能够保障生产质量管理要求。有效运行，持续改进。 固体制剂车间现有员工xx人，均严格按GMP要求进行培训，经考核合格后持证上岗。 固体制剂车间主要生产工艺流程：原辅料---前处理（粉碎） ---称量配料---制粒---干燥---总混---充填或压片 ---内包装（瓶装或铝塑包装）---外包装。 固体制剂车间使用的主要生产及生产能力：