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Dia-Specific Induced
Pluripotent Stem Cells
In-Hyun Park,1,7Natasha Arora,1,7Hongguang Huo,1,7Nimet Maherali,2,3,7Tim Ahfeldt,2,5,7Akiko Shimamura,4
M.William Lensch,1,7,9Chad Cowan,2,6,7Konrad Hochedlinger,2,7and George Q.Daley1,7,8,9,*
1Department of Medicine,Division of Pediatric Hematology Oncology,Children’s Hospital Boston,and Dana-Farber Cancer Institute; Department of Biological Chemistry and Molecular Pharmacology,Harvard Medical School,Karp Family Rearch Building7214,
300Longwood Avenue,Boston,MA02115罗列
2Massachutts General Hospital Cancer Center and Center for Regenerative Medicine and Department of Stem Cell and Regenerative Biology,185Cambridge Street,Boston,MA02114,USAtigermap
3Department of Molecular and Cellular Biology,Harvard University,7Divinity Avenue,Cambridge,MA02138,USA
4Department of Pediatrics,Division of Hematology/Oncology,University of Washington and Fred Hutchinson Cancer Rearch Center, 1100Fairview Avenue N.,Seattle,WA98109,USA
5Department of Biochemistry and Molecular Biology II:Molecular Cell Biology,University Medical Center Hamburg-Eppendorf, Hamburg20246,Germany
6Stowers Medical Institute
7Harvard Stem Cell Institute
8Division of Hematology,Brigham and Women’s Hospital
kids怎么读
what是什么意思9Howard Hughes Medical Institute,Children’s Hospital Boston
Karp Family Rearch Building7214,300Longwood Avenue,Boston,MA02115
*Correspondence:george.daley@childrens.harvard.edu
DOI10.ll.2008.07.041
SUMMARY
Tissue culture of immortal cell strains from diad patients is an invaluable resource for medical re-arch but is largely limited to tumor cell lines or transformed derivatives of native tissues.Here we describe the generation of induced pluripotent stem(iPS)cells from patients with a variety of genetic dias with either Mendelian or complex inheri-tance;the dias include adenosine deamina deficiency-related vere combined immunodefi-ciency(ADA-SCID),Shwachman-Bodian-Diamond syndrome(SBDS),Gaucher dia(GD)type III, Duchenne(DMD)and Becker muscular dystrophy (BMD),Parkinson dia(PD),Huntington dia (HD),juvenile-ont,type1diabetes mellitus(JDM), Down syndrome(DS)/trisomy21,and the carrier state of Lesch-Nyhan syndrome.Such dia-spe-cific stem cells offer an unprecedented opportunity to recapitulate both normal and pathologic human tissue formation in vitro,thereby enabling dia investigation and drug development. INTRODUCTION
Cell culture has been the backbone of basic biomedical rearch for many decades,and countless insights into both normal and pathologic cellular process have been gleaned by studying human c
ells explanted in vitro.Most of the human cell lines in wide u today carry genetic and epigenetic artifacts of accom-modation to tissue culture and are derived either from malignant tissues or are genetically modified to drive immortal growth (Grimm,2004).Primary human cells have a limited life span in culture,a constraint that thwarts inquiry into the regulation of tis-sue formation,regeneration,and repair.Indeed,many human cell types have never faithfully been adapted for growth in vitro,and the lack of accessible models of normal and patho-logic tissue formation has rendered many important questions in human development and dia pathogenesis inaccessible. Human embryonic stem cells isolated from excess embryos from in vitro fertilization clinics reprent an immortal propaga-tion of pluripotent cells that theoretically can generate any cell type within the human body(Lerou et al.,2008;Murry and Keller, 2008).Human embryonic stem cells allow investigators to ex-plore early human development through in vitro differentiation, which recapitulates aspects of normal gastrulation and tissue formation.Embryos shown to carry genetic dias by virtue of preimplantation genetic diagnosis(PGD;genetic analysis of single blastomeres obtained by embryo biopsy)can yield stem cell lines that model single-gene disorders(Verlinsky et al., 2005),but the vast majority of dias that show more complex genetic patterns of inheritance are not reprented in this pool.
A tractable method for establishing immortal cultures of plu-ripotent stem cells from diad individuals would not only fa-cilitate dia rearch but also lay a foundation for producing autologous cell therapies that would avoid immune rejection and enable correction of gene defects prior to tissue reconstitu-tion.One strategy for producing autologous,patient-derived pluripotent stem cells is somatic cell nuclear transfer(NT).In a proof of principle experiment,NT-embryonic stem(ES)cells generated from mice with genetic immunodeficiency were ud to combine gene and cell therapy to repair the genetic de-fect(Rideout et al.,2002).To date,NT has not proven success-ful in the human and,given the paucity of human oocytes,is Cell134,877–886,September5,2008ª2008Elvier Inc.877
destined to have limited utility.In contrast,introducing a t of transcription factors linked to pluripotency can directly repro-gram human somatic cells to produce induced pluripotent stem(iPS)cells,a method that has been achieved by veral groups worldwide(Lowry et al.,2008;Park et al.,2008b;Taka-hashi et al.,2007;Yu et al.,2007).Given the robustness of the approach,direct reprogramming promis to be a facile source of patient-derived cell lines.Such lines would be immediately valuable for medical rearch,but current methods for reprog-ramming require infecting the somatic cells with multiple viral vectors,thereby precluding consideration of their u in trans-plantation medicine at this time.
Human cell culture is an esntial complement to rearch with animal models of dia.Murine models of human congenital and acquired dias are invaluable but provide a limited repre-ntation of human pathophysiology.Murine models do not al-ways faithfully mimic human dias,especially for human con-tiguous gene syndromes such as trisomy21(Down syndrome or DS).A mou model for the DS critical region on distal human chromosome21fails to recapitulate the human cranial abnormal-ities commonly associated with trisomy21(Olson et al.,2004). Orthologous gments to human chromosome21are prent on mou chromosomes10and17,and distal human chromo-some21corresponds to mou chromosome16where trisomy 16in the mou is lethal(Nelson and Gibbs,2004).Thus,a true murine equivalent of human trisomy21does not exist.Murine strains carrying the same genetic deficiencies as the human bone marrow failure dia Fanconi anemia demonstrate DNA repair defects consistent with the human ,Chen et al.,1996),yet none develop the spontaneous bone marrow failure that is the hallmark of the human dia.
For cas where murine and human physiology differ,dis-ea-specific pluripotent cells capable of differentiation into the various tissues affected in each condition could undoubtedly provide new insights into dia pathophysiology by permitting analysis in a human system,under controlled con
ditions in vitro, using a large number of genetically modifiable cells,and in a manner specific to the genetic lesions in each whether known or unknown.Here,we report the derivation of human iPS cell lines from patients with a range of human genetic dias.RESULTS AND DISCUSSION
Dermalfibroblasts or bone marrow-derived menchymal cells were obtained from patients with a prior diagnosis of a specific dia and ud to establish dia-specific lines of human iPS cells(Table1).This initial cohort of cell lines was derived from patients with Mendelian or complex genetic disorders,in-cluding Down syndrome(DS;trisomy21);adenosine deami-na deficiency-related vere combined immunodeficiency (ADA-SCID);Shwachman-Bodian-Diamond syndrome(SBDS); Gaucher dia(GD)type III;Duchenne type(DMD)and Becker type(BMD)muscular dystrophy;Huntington chorea (Huntington dia;HD);Parkinson dia(PD);juvenile-on-t,type1diabetes mellitus(JDM);and Lesch-Nyhan syn-drome(LNSc;carrier state).
Patient-derived somatic cells were transduced with either four (OCT4,SOX2,KLF4,and c-MYC)or three reprogramming fac-tors(lacking c-MYC).Following2to3weeks of culture in hES cell-supporting conditions,compact refractile ES-like colonies emerged among a background offibroblasts,as previously de-scribed(Park et al.,2008a,2008b).Although our previous report ud additional factors(
h TERT and SV40LT)to achieve reprog-ramming of adult somatic cells,we have found the four-factor cocktail to be sufficient as long as we employ a higher multiplicity of retroviral infection.Additionally,we generated a single line from a carrier of Lesch-Nyhan syndrome usingfive doxycy-cline-inducible lentiviral vectors(OCT4,SOX2,KLF4,c-MYC, and NANOG),a strategy that has been ud to isolate murine iPS cells(Brambrink et al.,2008;Stadtfeld et al.,2008)but previously had not been attempted with human somatic cells. Characterization of the iPS cell lines is prented below. Mutation Analysis in iPS Cell Lines
The iPS cell lines were evaluated to confirm,where possible,the dia-specific genotype of their parental somatic cells.Analy-sis of the karyotype of iPS cell lines derived from two individuals with Down syndrome showed the characteristic trisomy21 anomaly(Figure1A).Aneuploidies such as that occurring in DS are unambiguously associated with advanced maternal age(re-viewed in Antonarakis et al.,2004)and,as such,are
occasionally Table1.iPS Cells Derived from Somatic Cells of Patients with Genetic Dia
878Cell134,877–886,September5,2008ª2008Elvier Inc.
Figure1.Genotypic Analysis of Dia-Specific iPS Cell Lines
(A)Two different,primaryfibroblast specimens,DS1and DS2from male patients with Down syndrome(trisomy21),were ud to derive DS1-iPS4and DS2-iPS10.Each has a47,XY+21karyotype over veral passages(G-banding analysis).
(B)Fibroblast(ADA and GBA)or bone marrow menchymal cells(SBDS)were ud to generate iPS cell lines.Mutated alleles identical to the original specimens were verified by DNA quencing.Adenosine deamina deficiency line ADA-iPS2is a compound heterozygote:GGG to GAA double transition in exon7of one allele(G216R substitution);the cond allele is an exon10frameshift deletion(-GAAGA)(Hirschhorn et al.,1993).Shwachman-Bodian-Diamond syndrome line SBDS-iPS8is also a compound heterozygote:point mutations at the IV2+2T>C intron2splice donor site and an IVS3À1G>A mutation of the SBDS gene (Austin et al.,2005).GD-iPS3(Gaucher dia type III):a1226A>G point mutation(N370S substitution)and a guanine inrtion at nucleotide84of the cDNA (84GG)(Beutler et al.,1991).
(C)Fibroblasts from patients diagnod with either Duchenne(DMD)or Becker type muscular dystrophy(BMD):DMD-iPS1has a deletion over exons45–52(mul-tiplex PCR for the dystrophin gene).
We could not determine a deletion in BMD-iPS1using two different multiplex PCR ts though the assays do not cover the entire coding region.DMD2is a patient control(exon4deletion).The control is genomic DNA from a healthy volunteer.Huntington dia(HD)is caud by a trinucleotide repeat expansion within the huntingtin locus.DNA quencing shows that HD-iPS1has one normal(<35repeats)and one expanded allele(72 repeats).HD2is a positive control from a cond Huntington patient with one normal and one expanded allele(54repeats).The control is genomic DNA from a healthy volunteer.
Cell134,877–886,September5,2008ª2008Elvier Inc.879
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880Cell134,877–886,September5,2008ª2008Elvier Inc.
detected in the preimplantation embryo when in vitro fertilization (IVF)is coupled with PGD.While it is possible that a discarded IVF embryo found to have trisomy21could be donated to at-tempt hES cell derivation,it is important to point out that many gestating DS embryos do not survive the prenatal period. Some studies place the frequency of spontaneous fetal demi (miscarriage)in DS to be above40%(Bittles et al.,2007). Thus,the derivation of a human iPS cell line with trisomy21 from an existing individual may be preferable,as such a line is most likely to harbor the complex genetic and epigenetic modi-fiers that favor full-term gestation and,by virtue of the often lengthy medical history,will be a more informative resource for correlative clinical rearch.
Creation of iPS cell lines from patients with single-gene disor-ders allows experiments on dia phenotypes in vitro,and an opportunity to repair gene defects ex vivo.The resulting cells,by virtue of their immortal growth in culture,can be extensively characterized to ensure that gene repair is preci and specific, thereby reducing the safety concerns of random,viral-mediated gene therapy.Repair of gene defects in pluripotent cells pro-vides a common platform for combined gene repair and cell re-placement therapy for a variety of genetic disorders,as long as the pluripotent cells can be differentiated into relevant somatic stem cell or tissue populations.
Three dias in our cohort of iPS cells are inherited in a clas-sical Mendelian manner as autosomal recessive congenital dis-orders and are caud by point mutations in genes esntial for normal immunologic and hematopoietic function:adenosine de-amina deficiency,which caus vere combined immune deficiency(ADA-SCID)due to the abnce of T cells,B cells, and NK cells;Shwachman-Bodian-Diamond syndrome,a con-genital disorder characterized by exocrine pancreas insuffi-ciency,skeletal abnormalities,and bone marrow failure;and Gaucher dia type III,an autosomal recessive lysosomal storage dia characterized by pancytopenia and progressive neurological deterioration due to mutations in the acid beta-glu-cosida(GBA)gene.Sequence analysis of the ADA gene in the dia-associated ADA-iPS2cell line revealed a compound heterozygote:a GGG to GAA transition mutation at exon7,caus-ing a G216R amino acid substitution(Figure1B);the other allele is known to have a frameshift deletion(-GAAGA)in exon10 (Hirschhorn et al.,1993).The SBDS-iPS8cell line harbors point mutations at the IV2+2T>C intron2splice donor site (Figure1B)and IVS3À1G>A mutation(Austin et al.,2005).Mo-lecular analysis of the GBA gene in the Gaucher dia line re-vealed a1226A>G point mutation,causing a N370S amino acid substitution(Figure1B);the cond allele is known to have a fra-meshifting inrtion of a single guanine at cDNA nucleotide84 (84GG)(Beutler et al.,1991).The Lesch-Nyhan syndrome carrier line harbors heterozygous deficiency of the HPRT gene(Nuss-baum et al.,1983).
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Two lines were derived from dermalfibroblasts cultured from patients with muscular dystrophy.Multiplex PCR analysis with primer ts amplifying veral(but not all)intragenic intervals of the dystrophin gene(Beggs et al.,1990;Chamberlain et al., 1988)revealed the deletion of exons45–52in the iPS cells de-rived from a patient with Duchenne muscular dystrophy(DMD; Figure1C).Despite analysis for gross genomic defects by multi-plex PCR,a deletion was not detected in iPS cells derived from a patient with Becker type muscular dystrophy(BMD;Figure1C). As BMD is a milder form of dia,and the dystrophin gene one of the largest in the human genome,definition of the genetic le-sion responsible for this condition is sometimes elusive(Prior and Bridgeman,2005).
Given that numerous groups have pioneered the directed dif-ferentiation of neuronal subtypes,and that genetically defined ES cells from animal models of amyotrophic lateral sclerosis have revealed important insights into the pathophysiology of mo-tor neuron deterioration(Di Giorgio et al.,2007),there is consid-erable interest in generating iPS cell lines from patients afflicted with neurodegenerative dia.We generated iPS cell lines from a patient with Huntington chorea(Huntington dia; HD)and verified the prence of expanded(CAG)n polyglut-amine triplet repeat quences(72)in the proximal portion of the huntingtin gene(Figure1C;Riess et al.,1993)in one allele and19repeats in the other(where the normal range is35or less;Chong et al.,1997).
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Pluripotent cell lines will likewi be valuable for studying neu-rodegenerative conditions with more complex genetic predispo-sition,as well as metabolic dias known to have familial pre-dispositions but for which the genetic contribution remains unexplained.We have generated lines from a patient diagnod with Parkinson dia(PD)and another from a patient with ju-venile-ont(type I)diabetes mellitus(Table1).Given that the conditions lack a defined genetic basis,genotypic verification is impossible at this time.
The Lesch-Nyhan syndrome is caud by mutations in hypo-xanthine-guanine phosphoribosyltransfera(HPRT),an X-linked enzyme in purine metabolism that when deficient leads to abnormal accumulation of uric acid and a neurologic disorder characterized by cognitive deficits and lf-mutilating behavior. Cells carrying either intact or deficient HPRT enzyme function can be lectively cultured in media containing hypoxanthine-Aminopterin-Thymidine(HAT)or6-thioguanine(6-TG),respec-tively.Strategies for inducing specific mutation or gene repair by homologous recombination werefirst established for the HPRT locus(Doetschman et al.,1987,1988;Thomas and Ca-pecchi,1987).We have generated an iPS cell line from a female carrier(LNSc-iPS2)that will be a valuable resource for studies of homologous recombination in iPS cells and for analysis of X chromosome reactivation during reprogramming and random inactivation with differentiation.
Figure2.Dia-Specific iPS Cell Lines Exhibit Markers of Pluripotency
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ADA-iPS2,GD-iPS1,DMD-iPS1,BMD-iPS1,DS1-iPS4,DS2-iPS10,PD-iPS1,JDM-iPS1,SBDS-iPS1,HD-iPS4,and LNSc-iPS2were established fromfibroblast or menchymal cells(Table1).Dia-specific iPS cell lines maintain a morphology similar to hES cells when grown in coculture with mou embryonic feeder fibroblasts(MEFs).Dia-specific iPS cells express alkaline phosphata(AP).Also,as shown here via immunohistochemistry,dia-specific cells express pluripotency markers including Tra-1–81,NANOG,OCT4,Tra-1-60,SSEA3,and SSEA4.4,6-Diamidino-2-phenylindole(DAPI)staining is shown at right and indicates the total cell content per image.
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