Neurospheres From Human Adipo Tissue Transplanted Into Cultured Mou Embryos can Contribute to Craniofacial Morphogenesis:A Preliminary Report
Takashi Naga,MD,PhD,*Daisuke Matsumoto,MD,1Miki Naga,MD,PhD,2
Kotaro Yoshimura,MD,PhD,1Tomokuni Shigeura,MS,1Makoto Inoue,PhD,4
Mamoru Hagawa,PhD,4Masaaki Yamagishi,MD,PhD,*Masafumi Machida,MD,PhD*
Tokyo,Japan
全国一本大学一览表Adipo-derived stromal cells(ASCs)are one of the most promising stem cell populations that differ-entiate into the mesodermal as well as neural lineages in vitro.In this study,we examined the neural differentiating potential of human ASCs by a neurosphere culture method.Neurospheres derived from human ASCs expresd Nestin and Musashi-1genes,which are marker genes for neural stem cells.When the cells were labeled with green fluorescent protein gene transfection by Sendai virus vector and transplanted into the head region of mou embryos using a whole embryo culture system,the cells were incorporated into the craniofacial development.Some transplanted cells appeared to migrate along the cond bran-chial arches,implicating some similarity to the cranial neura
l crest cells.Although preliminary,our results support an idea that ASC-derived neuro-spheres have properties of neural progenitors in vitro and in vivo.Key Words:Adipo-derived stromal cells,neuro-sphere,neural stem cells,embryo,stem cells A dipo-derived stromal cells(ASCs)were
originally reported as a subtype of the
menchymal stem cells(MSCs)isolated
from liposuction aspirates differentiating into the mesodermal tissues such as bone,cartilage, and adipo tissue.1Characterization of ASCs has recently been studied world wide by many groups, including ours.2Y4ASCs are now regarded as one of the most promising adult stem cells for regenerative medicine becau they can be harvested safely by liposuction,and a good yield can be anticipated.
Advances in stem cell rearch have resulted in a novel concept of cellular plasticity of differentiation beyond the boundary of germ layers.MSCs and ASCs can differentiate into neuronal(and thus ectodermal)derivatives,although the cells are primarily mesodermal.5,6Recent reports further indicate that stem cells with neural characteristics can be isolated from the mesodermal tissues such as the dermis and the heart.7Y9In the cas,the cells were harvested by a neurosphere method,w
hich was originally developed as a culture method of isolating spheres of neural stem cells from the embryonic and adult brain.10Y12However,it is to be elucidated whether this method is also applicable for obtaining neural stem cells from the adipo tissue or ASCs.
In this study,the neurospheres expressing neural stem cell marker genes were obtained from human ASCs.We also transplanted the cells into mou embryos cultured in vitro to examine whether the cells behave similar to neuronal cells in vivo.
49头颅移植
From the*Clinical Rearch Center,National Hospital Organization Murayama Medical Center,Tokyo,Japan;1Depart-ment of Plastic and Reconstructive Surgery,2Department of Nephrology and Endocrinology,University of Tokyo Graduate School of Medicine,Tokyo,Japan;and4DNAVEC Corporation, Tsukuba,Ibaraki,Japan.
Address correspondence and reprint requests to Dr.Takashi Naga,Head,Division of Advanced Medical Rearch,Clinical Rearch Center,National Hospital Organization Murayama Medical Center,2-37-1Gakuen,Musashimurayama-City,Tokyo 208-0011,Japan;E-mail:tnaga@jp
The first two authors contributed equally to this work.
M ATERIALS
AND
M ETHODS
Isolation of Human ASCs and Neurosphere Cell Culture
赞美
A
SCs were isolated from the human liposuction aspirates as reported previously.3The suctioned fat was digested with 0.075%collagena in phosphate-buffered saline (PBS)for 30minutes on a shaker at 37-C.Mature adipocytes and connective tissues were eliminated by centrifugation.Blood cells were also eliminated by treating with erythrocyte lysis buffer,and resultant ASC pellets were obtained.Alternatively,ASCs could be isolated from the fluid portions of liposuction aspirates by treating with erythrocyte lysis buffer and density gradient centrifugation with Ficoll (GE Healthcare Bio-sciences,Piscataway,NJ).
Neurosphere culture was performed as described previously with slight modification.12Freshly isolated ASCs were plated at a density of 2Â107cells in 10cm uncoated dishes and cultured in the neurosphere culture medium at 37-C in an atmosphere of 5%CO 2in humid air.The neurosphere medium was a Dulbecco’s Modified Eagle’s Medium/F12(1:1)-bad medium supplemented with human recombinant epidermal growth factor (EGF,20ng/mL,PeproTech,Rocky Hill,
NJ),human recombinant basic fibroblast growth factor (FGF,20ng/mL,Kaken Pharmaceutical,Tokyo,Japan),2%B27supplement (Gibco,Carlsbad,CA),100U/mL penicillin,and 100m g/mL streptomycin.Half of the medium was replaced with a fresh medium on the fourth to fifth day,and the passaging was performed on the eighth day.
Quantitative Real-Time Rever-Transcripta Polymera Chain Reaction
Total mRNA was extracted using RNeasy-mini kit (Qiagen,Hilden,Germany)from the neurosphere cells derived from passage one ASCs,which were precultured in the normal medium containing M199medium and 10%fetal bovine rum (FBS).The preculturing was necessary for reducing the contam-ination of blood cells.Control mRNA was also extracted from the passage one undifferentiated ASCs cultured in M199plus 10%FBS.
Expressions of undifferentiated neural stem cell marker genes Nestin and Musashi-113and adipogenic differentiation marker Leptin were analyzed by real-time quantitative rever-transcription polymera chain reaction (RT-PCR)using an ABI PRISM
7700
Fig 1Neurosphere formation of adipo-derived stromal cells cultured in neurosphere medium for 5days (A),6days (B),and 7days (C)(magnification Â
200).
Fig 2Quantitative real-time rever-transcription polymera chain reaction analysis of gene expressions of neural stem cell marker Nestin (A),Musashi-1(B),and adipogenic differentiation marker Leptin (C).Control =undifferentiated adipo-derived stromal cells;Sphere =neurospheres.Assays were performed in triplicate,and standard errors are indicated by error bars.
THE JOURNAL OF CRANIOFACIAL SURGERY /VOLUME 18,NUMBER 1January 2007
50
(Applied Biosystems,Foster City,CA),as reported previously.Gene expression of the target quence was normalized to that of the houkeeping gene b-actin.Transcript level in the control(undifferen-tiated ASC)group was arbitrarily expresd as1. TaqMan chemistry and assay by design primers and probe ts were ud for human Nestin,Musashi-1, Leptin,and b-actin.All the primers and probe ts were purchad from Applied Biosystems.
Mou Whole Embryo Culture and Transplantation of Neurosphere-Like Cells Neurospheres derived from human ASCs were transfected with green fluorescent protein(GFP) gene using the Sendai virus
vector(DNAVEC Corp.Tsukuba,Japan),as reported previously.14,15 The original vector SeV/D F lacks the F gene encoding fusion protein necessary for penetration of ribonucleoprotein complex into infected cells, and is thus nontransmissible and nonpatho-genic.14The modified SeV/D F vector has addi-tional mutations to reduce its cytotoxicity,15and we ud the modified vector in the prent study. Neurospheres were incubated for1hour in the medium with the modified SeV/D F carrying the GFP gene at a multiplicity of infection of250and rind with PBS.
Mou whole embryo culture was performed as reported previously.16Y19Nine mou embryos at embryonic day(E)8were discted out without damaging yolk sacs,and the GFP-transfected neuro-sphere cells were transplanted using micropipettes into the head region of the embryos.The embryos were cultured for approximately40hours,and prence or abnce of the GFP-positive transplanted cells was investigated under a fluorescent discting microscope.All experimental procedures were per-formed at the University of Tokyo under approval of the ethical committee.
proposal是什么意思R ESULTS
W e first cultured human ASCs in the neuro-sphere culture medium containing EGF and basic FGF without rum.On the third day of culture of freshly prepared ASCs,the floating ASCs started to form
small mass(data not shown).The neuro-sphere-like cellular aggregates were clearly obrved on the fifth day(Fig1A).The number and the size of the spheres became increasingly larger within the next2days(Fig1,B and C).The passaging was performed on the eighth day when the spheres were dissociated and resuspended in the new medium.The spheroids were newly formed after culturing again for veral days(data not shown),suggesting lf renewal of the neurosphere cells.
To characterize the neurosphere cells,we next examined expressions of neural stem cell marker Nestin and Musashi-1genes and adipocyte marker Leptin by quantitative real-time RT-PCR.Expressions of Nestin and Musashi-1genes were remarkably up-regulated in the neurosphere cells compared with the control ASCs without culturing in the neurosphere medium(Fig2,A and B),suggesting characteristics of neural progenitor.Converly,Leptin
expression Fig3Neural crest-like migrations of green fluorescent protein(GFP)-transfected,adipo-derived stromal cell-derived neurospheres grafted into mou embryo cultured in vitro.(A)Appearances of mou embryos cultured for40 hours from embryonic day8.(B and C)Fluorescent views of embryos.GFP-positive neurosphere cells were arranged in a row(arrows),suggesting their migration along cond branchial arch.Bars=500m m.
upgradeable
ADIPOSE-DERIVED NEUROSPHERE/Naga et al
51
was dramatically reduced in the neurospheres (Fig2C),indicating loss of adipogenic potential.
To investigate functions of the neurosphere cells in vivo,we labeled the cells by the modified Sendai virus vector carrying the GFP gene and transplanted them into the head region of the E8mou embryos. After the embryos were cultured for approximately 40hours in vitro,the transplanted GFP-positive cells were clearly obrved and appeared viable in only two embryos of the nine cultured embryos.The GFP-positive cells were incorporated into the cranio-facial region as well as the heart and the trunk in the two embryos(Fig3).Notably,the transplanted cells were arranged in a row along the cond branchial arch(arrows in Fig3,B and C)in a quite similar pattern t
o the neural crest cells migrating within the cond branchial arch.Although not confirmatory,this result suggests a intriguing possi-bility that neurosphere cells derived from ASCs have neural crest-like properties.
D ISCUSSION
A SCs are probably one of the most well-known
2020高考语文试题stem cells among plastic surgeons.ASCs were originally reported by Zuk et al1from the clinical samples of liposuction aspirates.According to their broad spectrum of differentiation potential,ASCs have been ud in a number of preclinical animal studies of in vivo regeneration of a various tissues such as bone,20,21cartilage,22vesls,4,23,24soft tissue,4bone marrow,25and so on.Even a clinical ca was reported,in which a calvarial defect was repaired by ASCs combined with scaffold.26Several groups reported neural differentiation of ASCs in vitro,5,6,27and Kang et al28reported functional recovery of the rat model with cerebral infarction after ASC transplantation in vivo.
The neurosphere method was originally reported by Reynolds et al10,11and is one of the most frequently ud methods for isolating neural stem cells from the embryo or from the adult central ner
vous systems.However,this method has not yet been applied for obtaining neural stem cells from adipo tissue or the ASC population.In this preliminary study,we obtained neurospheres from the ASCs in human liposuction aspirates.Prolifera-tion of the cells was quite rapid,possibly faster than other neurospheres from various tissue origins such as the dermis and the heart,7Y9suggesting advantages of ASCs as a origin of neuronal progeni-tors for regenerative medicine.The neurosphere cells expresd Nestin and Musashi-1,marker genes for neural stem cells,probably reflecting their tendency of differentiating into neuronal progeni-tors.This view is further supported by inhibition of their expression of Leptin,a marker for adipogenic differentiation and maturation.
Do the ASC-derived neurosphere cells behave as neuronal progenitors in vivo?Our attempt of grafting the cells into the cultured mou embryo revealed that some of the cells migrate along the cond branchial arch and contribute to craniofacial mor-phogenesis.Their migratory pattern is quite similar to that of cranial neural crest cells,as we reported previously.16The neural crest cells are an embryonic cellular population characterized by extensive migra-tion and a unique repertoire of differentiation.29The neural crest cells are often regarded as stem or progenitor cells for peripheral neurons and Schwann cells,and the craniofacial skeletal menchyme is also neural-crest derived.17,19,29,30Recent studies indicate that the neural crest stem cells can be harvested from th
e emingly‘‘mesodermal’’tissues of adult ani-mals,such as the dermis,7the hair follicular dermal papilla,8or the heart,9by means of the neurosphere method,implicating that it is also the ca in the adipo tissue.Becau our data are preliminary and we have a small sample size,further studies such as tho with detailed expression analysis of neural/ neural crest marker genes and large-scale in vivo grafting are necessary to confirm this interesting idea. R EFERENCES
1.Zuk PA,Zhu M,Mizuno H,et al.Multilineage cells from
human adipo tissue:implications for cell-bad therapies.
Tissue Eng2001;7:211Y228
2.Zuk PA,Zhu M,Ashjian P,et al.Human adipo tissue is a
source of multipotent stem cells.Mol Biol Cell2002;13: 4279Y4295
3.Yoshimura K,Shigeura T,Matsumoto D,et al.Characteriza-
tion of freshly isolated and cultured cells derived from the fatty and fluid portions of liposuction aspirates.J Cell Physiol 2006;208:64Y76
4.Matsumoto D,Sato K,Gonda K,et al.Cell-assisted lipotransfer
(CAL):supportive u of human adipo-derived cells for soft tissue augmentation with lipoinjection.Tissue Eng.In Press.
5.Ashjian PH,Elbarbary AS,Edmonds B,et al.In vitro differ-
entiation of human procesd lipoaspirate cells into early neural progenitors.Plast Reconstr Surg2003;111:1922Y1931
6.Kokai LE,Rubin JP,Marra KG.The potential of adipo-
derived adult stem cells as a source of neuronal progenitor cells.Plast Reconstr Surg2005;116:1453Y1460
7.Toma JG,Akhavan M,Fernandes KJ,et al.Isolation of
multipotent adult stem cells from the dermis of mammalian skin.Nat Cell Biol2001;3:778Y784
8.Fernandes KJ,McKenzie IA,Mill P,et al.A dermal niche for
clazziquai
multipotent adult skin-derived precursor cells.Nat Cell Biol 2004;6:1082Y1093
9.Tomita Y,Matsumura K,Wakamatsu Y,et al.Cardiac neural
crest cells contribute to the dormant multipotent stem cell in the mammalian heart.J Cell Biol2005;170:1135Y1146
10.Reynolds BA,Tetzlaff W,Weiss S.A multipotent EGF-responsive
THE JOURNAL OF CRANIOFACIAL SURGERY/VOLUME18,NUMBER1January2007 52
striatal embryonic progenitor cell produces neurons and astro-cytes.J Neurosci1992;12:4565Y4574
11.Reynolds BA,Weiss S.Generation of neurons and astrocytes
from isolated cells of the adult mammalian central nervous system.Science1992;255:1707Y1710
12.Kanemura Y,Mori H,Kobayashi S,et al.Evaluation of in vitro
proliferative activity of human fetal neural stem/progenitor cells using indirect measurements of viable cells bad on cellular metabolic activity.J Neurosci Res2002;69:869Y879 13.Kaneko Y,Sakak
ibara S,Imai T,et al.Musashi-1:an
evolutionally conrved marker for CNS progenitor cells including neural stem cells.Dev Neurosci2000;22:139Y153 14.Li HO,Zhu YF,Asakawa M,et al.A cytoplasmic RNA vector
derived from nontransmissible ndai virus with efficient gene transfer and expression.J Virol2000;74:6564Y6569
15.Inoue M,Tokusumi Y,Ban H,et al.Nontransmissible virus-
like particle formation by F-deficient ndai virus is tempera-ture nsitive and reduced by mutations in M and HN proteins.J Virol2003;77:3238Y3246
16.Naga T,Sanai Y,Nakamura S,et al.Roles of HNK-1
carbohydrate epitope and its synthetic glucuronyltransfera genes on migration of rat neural crest cells.J Anat2003;203: 77Y88
17.Naga T,Naga M,Osumi N,et al.Craniofacial anomalies of
the cultured mou embryo induced by inhibition of sonic hedgehog signaling:an animal model of holoproncephaly.
J Craniofac Surg2005;16:80Y88
18.Naga T,Naga M,Yoshimura K,et al.Angiogenesis within
the developing mou neural tube is dependent on sonic hedgehog signaling:possible roles of motor neurons.Genes Cells2005;10:595Y604
19.Yamada Y,Naga T,Naga M,et al.Gene expression
changes of sonic hedgehog signaling cascade in a mou model of fetal alcohol syndrome.J Craniofac Surg2005;16:1055Y1061 20.Cowan CM,Shi YY,Aalami OO,et al.Adipo-derived adult
stromal cells heal critical-size mou calvarial defects.Nat Biotechnol2004;22:560Y567
21.Dragoo JL,Lieberman JR,Lee RS,et al.Tissue-engineered bone
from bmp-2-transduced stem cells derived from human fat.
Plast Reconstr Surg2005;115:1665Y1673
22.Dragoo JL,Samimi B,Zhu M,et al.Tissue-engineered cartilage
and bone using stem cells from human infrapatellar fat pads.
J Bone Joint Surg Br2003;85:740Y747
感叹号英文23.Planat-Benard V,Silvestre JS,Cousin B,et al.Plasticity of
human adipo lineage cells toward endothelial cells:physio-logical and therapeutic perspectives.Circulation2004;109: 656Y663
24.Miranville A,Heeschen C,Sengenes C,et al.Improvement of
postnatal neovascularization by human adipo tissue-derived stem cells.Circulation2004;110:349Y355
25.Cousin B,Andre M,Arnaud E,et al.Reconstitution of lethally
irradiated mice by cells isolated from adipo tissue.Biochem Biophys Res Commun2003;301:1016
Y1022coats
26.Lendeckel S,Jodicke A,Christophis P,et al.Autologous stem
cells(adipo)and fibrin glue ud to treat widespread traumatic calvarial defects:ca report.J Craniomaxillofac Surg2004;32:370Y373
27.Fujimura J,Ogawa R,Mizuno H,et al.Neural differentiation of
adipo-derived stem cells isolated from GFP transgenic mice.
Biochem Biophys Res Commun2005;333:116Y121
六级英语成绩查询28.Kang SK,Lee DH,Bae YC,et al.Improvement of neurological
deficits by intracerebral transplantation of human adipo tissue-derived stromal cells after cerebral ischemia in rats.Exp Neurol2003;183:355Y366
29.Le Douarin N,Kalcheim C.The Neural Crest,ed2.Cambridge:
Cambridge University Press,1999
30.Naga T,Nakamura S,Harii K,et al.Ectopically localized
HNK-1epitope perturbs migration of the midbrain neural crest cells in pax6mutant rat.Dev Growth Differ2001;43: 683Y692
ADIPOSE-DERIVED NEUROSPHERE/Naga et al
53
