Expression of Telomerase Extends the Lifespan and Enhances Osteogenic Differenti - 《stemcells》2004-2005文献干细胞之家



免疫细胞治疗专区	欢迎关注干细胞微信公众号

12 3 4 5 6 7 8 9 10 ... 23 下一页

返回列表

查看: 261516\|回复: 221	go Expression of Telomerase Extends the Lifespan and Enhances Osteogenic Differenti [复制链接]

江边孤钓

版主

Rank: 7 Rank: 7 Rank: 7

积分: 0
威望: 0
包包: 483

楼主

发表于 2009-3-5 10:35 |只看该作者 |倒序浏览 |打印

a Division of Gene Therapy and% h/ p( m& K# c: q& z

b Division of Veterinary Medicine, Tulane National Primate Center, Tulane University Health Sciences Center, Covington, Louisiana, USA;

c Center of Gene Therapy and
. f+ n  L+ n5 S! b' ~1 ]2 s6 l
d Department of Pharmacology, Tulane University Health Sciences Center, Tulane University, New Orleans, Louisiana, USA;* g3 c4 }& c: \  F* V# A

e Department of Physiology, College of Medicine, Busan National University, Busan, South Korea1 j7 X8 H, t' j3 W+ i$ z& a

Key Words. Telomerase ? Adipose tissue stromal cell ? Osteogenic differentiation ? Microarray

Correspondence: Bruce A. Bunnell, Ph.D., Center for Gene Therapy, Department of Pharmacology, Division of Gene Therapy, Tulane National Primate Research Center, Tulane University Health Sciences Center, 18703 Three Rivers Road, Covington, LA 70433, USA. Telephone: 985-871-6594; Fax: 985-871-6587; e-mail: bbunnell@tulane.edu

ABSTRACT
) V# d$ {0 [2 E9 f+ a* d5 m
The finite proliferation of mammalian cells is considered to be the result of a reduction of telomere length . The telomere contains repeated sequences of six nucleotide bases, TTAGGG, located at the termini of individual chromosomes, and has been shown to be shortened by 33–120 bp at each cell division in human fibroblastic cells and lymphocytes, thus causing aging and finite mitotic capability . Telomere length is maintained by telomerase, a ribonuclear protein complex consisting of an integral RNA (hTR), which serves as the telomeric template; a catalytic subunit (hTERT), which has reverse transcriptase activity; and associated protein components . In the absence of hTERT, telomeres shorten during cell division because the DNA replication complex cannot completely copy telomeric DNA. Cellular senescence and growth arrest are proposed to occur when telomere lengths in germ cells and most cancer cells are decreased. However, ectopic expression of hTERT leads to telomere elongation and extended lifespan in several cell types .* w: M* M- Z6 A: ^0 C& M
0 u( |* C; |, U! k
Possible mechanisms of age-dependent bone loss may be attributed, at least in part, to a deficiency of osteoblast function or a decrease in the number of osteogenic progenitor cells rather than to an increase in bone resorption by osteoclasts . It has been suggested that telomere-associated cellular senescence may contribute to various age-related disorders. Recent studies reported that the introduction of hTERT into osteoblasts isolated from human trabeculae induced telomerase activity and extended the lifespan of these cells . However, the role of telomerase in bone formation, particularly with respect to maintenance of the osteogenic precursor cell population, is largely unknown. Pluripotent human bone marrow stromal cells (BMSCs) were originally described as progenitors of osteoblasts because of their capacity to form normal bone in vivo . Mesenchymal stem cells, including BMSCs and adipose stromal cell lines (ATSCs), are being analyzed as new therapeutic agents for repairing large bone defects that cannot undergo spontaneous healing .  L0 l4 e+ `2 r3 [, g+ B

The regeneration of diseased or damaged tissue is the principle goal of the emerging discipline of tissue engineering. A key requirement in tissue regeneration is the availability of the constituent cells. Adipose tissue stromal cells have been defined as multipotential adult stem cells, capable of differentiating into a variety of cell types such as osteoblasts, chondrocytes, adipocytes, muscle cells, and neural cells . Recently, our group and others reported that human and nonhuman primate-derived ATSCs and BMSCs can propagate in vitro and contain detectable levels of telomerase activity. Forced division of ATSCs in vitro may cause excessive telomere shortening in the descendent lineages, although ATSCs themselves possess telomerase activity. Indeed, recent studies have demonstrated that the telomerase activity of mesenchymal stem cells is not sufficient to completely compensate for the reduction of telomere length during continuous in vitro subculture. To extend the proliferative lifespan of ATSCs, supplementation with transduced exogenous hTERT may be necessary, because the self-renewal and replicative potential of these cells may depend on sufficient telomerase activity to maintain stable telomeres. Reconstitution of telomerase activity through expression of exogenous hTERT enables normal human fibro-blasts, as well as retinal epithelial, myometrial, and endothelial cells, to avoid senescence . After ectopic expression of telomerase, the lifespan of BMSCs was significantly increased, and proliferative capacity was extended in vitro. The cells were demonstrated to have an enhanced capacity for bone formation in vitro and in vivo . The enhanced formation and normal morphology of the ectopically formed bone strongly suggest that ATSC-TERT cells represent a highly useful candidate cell source for bone tissue regeneration and engineering.' U( {- u  E0 E+ U# ?
  b2 l8 `! C& d+ h5 Z' p) Q9 k
MATERIALS AND METHODS
6 l+ `4 t- `9 `/ m, w" h
Growth Characteristics and Telomerase Expression in ATSC-TERT Cells1 n2 M& l& n: _. s) K4 B# z9 m& _# S
4 ]  u/ W5 I; ~5 y  u
During prolonged periods of culture, the population of control ATSCs isolated from nonhuman primate fat underwent a progressive decrease in proliferative potential, and finally cells underwent senescence after passage 20 (80–90 days in culture). At the end of their proliferative lifespan, the cells were flatter and larger in morphology in a monolayer similar to that described for senescent fibroblasts (data not shown). hTERT-transduced ATSCs grow continuously for more than 9 months (>50 passages) without diminished cell expansion or rate of proliferation (Fig. 1). Their rate of proliferation resembled that of control ATSCs, and ATSC-TERTs retained their inhibition of cellular proliferation by cell-to-cell contact. The results indicate that the immortalization of the stromal cells by telomerase expression does not alter cell growth. The TRAP assay and telomerase immunocytochemistry method were used to examine telomerase activity in primary cultures of ATSCs (passage 0) and hTERT retrovirus-infected cells (passages 5, 10, 15, and 20). Naive ATSCs have low levels of protein and enzymatic telomerase activity (Figs. 2A–2C). Whereas ATSCs transduced with the hTERT retrovirus had reconstituted telomerase activity, the ATSC-TERTs continuously expressed high levels of telomerase over time (Figs. 2B, 2C).

Figure 1. Ectopic expression of hTERT induces immortalization of ATSCs. (A): ATSCs overexpressing TERT were generated by transduction with a TERT-expressing oncoretrovirus vector followed by neomycin selection to obtain stable clones. Control ATSCs at low passage (5) and late passage (20) (left). Untransduced cells demonstrated markedly reduced self-renewal potentials after 12 passages in vitro. Cells expressing hTERT (right) at low passage (5) and higher passage (20) maintained their thin spindle fibroblast morphology and growth rate. (B): Growth kinetics of control and TERT-expressing ATSCs. ATSC control cells showed markedly reduced expansion after 50 days in vitro. hTERT-overexpressing cells underwent continuous expansion without a lag growth phase and have been in continuous culture for more than 9 months with no marked alterations in their growth characteristics. Abbreviation: ATSC, adipose stromal cell line.
9 A' Z" b$ g. ]( ]
Figure 2. Telomerase expression in control and hTERT-expressing ATSCs. (A): Immunocytochemistry of hTERT in ATSC-TERT clones and control cells. (B): Telomeric repeat amplification protocol assay for telomerase activity measurement in control and TERT-expressing ATSCs. The negative control assay was performed, omitting the TERT enzyme extract from the reaction mixture. (C): Quantification of telomerase activity in ATSC-TERT cells and ATSCs was performed by polymerase chain reaction enzyme-linked immunosorbent assay procedure. Abbreviation: ATSC, adipose stromal cell line., A4 x9 O& C- X
9 }. {+ B; [. F9 K8 T/ c5 }6 l
Telomere Length Analysis of ATSC-TERT Cells

It has been proposed previously that critically shortened telomeres mediate massive genomic instability and contribute to M2 crisis. We assessed telomere lengths in cells at different stages of proliferation. Unexpectedly, in cells expressing only endogenous hTERT (control ATSCs), the TRFs continued to shorten for 30 cell doublings after entering crisis. In ATSC-TERT cells, telomere length was maintained at least 50 doublings beyond the expected crisis point, and the bulk of the TRFs increased slightly in length and became more clustered at approximately a mean length of 23 kb, which is the length of telomeres in passage-0 ATSCs (Fig. 3). Homeostasis of telomere length was essentially achieved in ATSC control cells, presumably by the balance between telomere synthesis by telomerase and the erosions of telo-meres during proliferation. In ATSC-TERTs, telomere length did not increase even after 50 doublings past the normal crisis point. Average telomere length was approximately 15 kb at the last time point analyzed. Finally, telomeres continued to shorten in hTERT-expressing cells as the cells proliferated beyond the expected crisis point, with the average telomere length in these late-passage hTERT-expressing cells considerably shorter than in control cells in crisis (data not shown).

Figure 3. Expression of vector-derived hTERT and telomere length analysis. (A): Detection of hTERT by reverse transcription–PCR. Total cellular RNA was isolated from TERT-expressing ATSCs at passage 5 or 15 (p5, p15). One microgram of RNA was then analyzed for TERT expression using PCR primers specific for human and endogenous rhesus TERT mRNA. The PCR product was separated on 1.5% agarose gel and visualized by ethidium bromide staining. (B): Mean telomere length assessed by telomeric restriction fragment analysis of ATSCs at passage 5 and ATSC-TERT cells at passage 15. Abbreviations: ATSC, adipose stromal cell line; PCR, polymerase chain reaction.' q: I' f5 i: ~1 i4 }

Ectopic Expression of hTERT in ATSCs Does Not Alter Functional Characteristics6 {8 z+ F% C+ |

We examined whether ectopic expression of hTERT affected the multipotent characteristics of ATSCs. No marked differences between ATSC control and ATSC-TERT cells were detected. ATSC-TERT cells retained the ability to accumulate lipid droplets typical for the adipocyte phenotype, and they maintained osteogenic and chondrogenic differentiation potential (Fig. 4, adipogenesis , osteogenic , and chondrogenesis ). Also, ATSC-TERTs were induced toward the neurogenic lineage through neurosphere formation and final differentiation on PDL-laminin–coated substrate in NB media supplemented with B27, bFGF, and EGF. During neurogenic induction in NB media, both cell populations undergo a marked morphologic change from elongated fibroblast morphology to compact, spheroid bodies, which expand to larger spheroid bodies as the total cell number expands (Fig. 4, neurospheres). After detachment of the spheroid bodies from substrate, we performed neural induction for 4 days through suspension culture in Petri dishes, and then the intact neurospheres or dissociated neurospheres were layered on PDL-laminin–coated chamber slide and cultured for an additional 10 days. As soon as the cells were layered on the laminin-coated surface, the spheroid cell mass began to adhere and spread across the growth surface, forming long chains of cellular processes and, finally, the cell processes began to exhibit secondary branching with multiple extensions (Fig. 4, Tuj/ DAPI).$ k+ @" g. H% L8 t! Y  ?  }, R, p8 J
& e( {' }6 p$ P( Z: n, X! V, N% e2 z
Figure 4. Confirmation of multipotential differentiation of TERT-expressing ATSCs. Cells (passage 5) were subjected to differentiation along adipogenic, osteogenic, chondrogenic, and neurogenic lineages in vitro. Adipogenic differentiation was induced, and accumulation of lipid vacuoles was visualized under the microscope after Oil red O staining (adipogenic). Mineralization of the extracellular matrix was visualized by staining of the cultures with Alizarin red S and von Kossa reagents (osteogenic and chondrogenic). To assess neural differentiation, the ATSC-TERT cells were tested for their ability to form neurospheres after plating density (neurospheres). The neurospheres were collected and underwent extensive neural differentiation when cultured on PDL-laminin for 10 days and immunostained using neuronal lineage-specific antibody (TuJ1/DAPI). Abbreviations: ATSC, adipose stromal cell line; DAPI, 4',6'-diamidino-2-phenylindole.: u  Y- f4 S: R
4 B& r8 J7 z4 U- j. z
Enhanced In Vitro Osteogenesis and Chondrogenesis by ATSC-TERT
6 R- m  z# @. J- Z9 R, |
ATSCs are known progenitors of skeletal tissues and differentiate into osteoblast-like cells in culture supplemented with ascorbic acid and a source of glucocorticoid. However, ATSCs lose their osteogenic capacity during continuous subculture in vitro. This may limit their therapeutic use because of effective treatment of extensive bone defects that require the transplantation of large numbers of ex vivo–expanded ATSCs. To determine the functional role of telomerase during osteogenesis, we examined the osteogenic differentiation potential of ATSC-TERT cells in vitro. ATSCs typically begin to accumulate at calcium after 2–4 weeks of induction in osteogenic differentiation medium. However, ATSC-TERT cells were found to accumulate significant amounts of calcium after only 1 week of osteogenic induction in vitro (Fig. 5). We quantified the differences in the efficiency of nodule formation between the naive and TERT-expressing ATSCs by determining the number of stained nodules in 25 random fields. As shown in Figure 5C, there are more than three times more nodules in the TERT-expressing ATSCs compared with control ATSCs. After differentiation, an extensive number of calcium deposits derived from ATSC-TERT cells accumulated on the cell surface and ultimately were released into culture supernatant. We identified and quantified calcium deposits from culture supernatant after calcium-specific Fluo-3 staining and flow cytometry analysis (data not shown).. O) G8 ~9 M8 C+ e1 B3 B
! h: z" c6 a4 W8 c- k" F
Figure 5. Telomerase expression enhances osteoblast differentiation in vitro. Osteoblast differentiation of ATSC-TERT cells (A) and controlATSCs (B), both at passage 6. Osteoblast differentiation was induced with L-ascorbate-2-phosphate, dexamethasone, and inorganic phosphate for 7–14 days. Cells were stained with Alzarin red S and von Kossa for detection of calcified deposits in both cell populations after 7 days of osteoinduction. (C): The numbers of stained nodules in each population were counted in 25 random fields for quantification of differences in osteogenic differentiation potential. Abbreviation: ATSC, adipose stromal cell line.( ~: @' p0 N3 w; i: [) a

Also, after culture of ATSCs-TERT cells (passage 5) in the pellet culture system for chondrogenic differentiation, we stained von Kossa for calcium deposit and Toluidine blue O for proteoglycan, a chondrocyte marker. ATSCs-TERT had a highly formed calcium deposit and proteoglycan matrix (Fig. 6). Immunohistochemistry results showed that ATSC-TERT cells highly expressed collagen I and II in the matrix (Fig. 6). In contrast to ATSC-TERT, we failed to detect collagen-positive cells in control ATSCs.# |- f$ e/ c) V# E5 A' ^
" {  R- a9 @8 q6 q4 z0 \, V
Figure 6. Synthesis of bone and cartilage by ATSC-TERT cells and control ATSCs by immunohistochemistry for collagen I and II. (A): For chondrocyte differentiation, a pellet culture system was used. Approximately 3 x 106 ATSC-TERT cells (passage 6) and control ATSCs (passage 6) were cultured in medium containing insulin, ascorbic acid, ?-glycerophosphate, dexamethasone, and transforming growth factor ?1 for 14–21 days. For microscopic analysis, the pellets were embedded in paraffin, cut into 5-μm sections, and stained with HE, von Kossa (calcium), and Toluidine blue (purple color is proteoglycan and blue color is background). (B): Paraffin-embedded sections were stained for collagen synthesis using anti-collagen human type I and II antibodies, which were detected using fluorescein isothiocyanate–conjugated anti-mouse secondary antibody. The nucleus was counterstained with TO-PRO 3. Abbreviations: ATSC, adipose stromal cell line; HE, hematoxylin-eosin.  B3 v  l2 k( f, I! b

In Vivo Osteogenesis after Engraftment of ATSC-TERT Cells' M  E; b# z  o; q, R8 \( `2 v

We studied in vivo osteogenesis effects after implantation of ATSC-TERT subcutaneously with Matrigel and hydroxyapatite scaffolds in immunedeficient mice. Five weeks after transplantation of ATSC-TERT cells, we analyzed paraffin-embedded tissue using hematoxylin-eosin, Toluidine blue O for proteoglycan, and von Kossa for calcium deposition. The results of hematoxilin-eosin and von Kossa staining of implant tissue section revealed highly enhanced bone formation by ATSC-TERT cells compared with control ATSCs. Control ATSC-implanted tissue section failed to show any hematoxylin-eosin and von Kossa–positive staining (data not shown). Toluidine blue O staining showed that both implants did not express proteoglycan (Fig. 7).% B# M% J- `9 ?7 o" T% v
( s1 c- Z5 H: S' Y. L  c
Figure 7. In vivo bone formation by ATSC-TERT transplants. Cross-section of transplanted calcium phosphate scaffold or Matrigel scaffolds seeded with TERT-expressing ATSCs after 6 weeks. Left: Sections were stained with stained with HE. ATSC-TERT cells generated higher amounts of bone formation at 6 weeks after transplantation. Right: Toluidine blue O staining for detection of chondro-cyte differentiation in a section from a ATSC-TERT implant. We were not able to detect newly formed bone in any sections from transplanted control ATSCs. Abbreviations: ATSC, adipose stromal cell line; B, bone; HE, hematoxylin-eosin; M, marrow.( B3 w# E9 e& w* K" I5 Z- G' o, z3 d
' a$ m( ]# `' O5 {- x! I) Z
cDNA Expression Profile of ATSC-TERT# w& P# O) ?3 N) ~- t& Z& P
* D' H$ M8 `3 H+ x. _: M$ t
To analyze the gene expression pattern, we performed oligo-nucleotide microarray analysis. The gene expression profile in ATSC-TERT cells was compared with ATSC controls. Total RNA was harvested from both cultures, and gene expression profiles were compared using Affymetrix HG-U95a microarray (22,000 genes and ESTs). Affymetrix Microarray Suite 5.0 was used to scan and analyze the relative abundance of each gene. The signal output from each gene from the ATSC control profile was plotted against the ATSC-TERT profile (data not shown), and the correlation coefficient (r) was calculated for each comparison. The analysis of the gene expression levels demonstrated that fewer than 1% of the total genes were expressed at greater than 2.2-fold different levels in ATSCs and ATSC-TERT, as indicated by the r value (0.8). Tables 1 and 2 give a partial list assembled into gene function of upregulated (total number of genes = 288) or downregulated (total number of genes = 580) genes expressed in ATSC-TERT compared with naive ATSCs. Relative expression of telomerase, AP2, BDNF, and MAP2 were examined by real-time RT-PCR. Comparing expression of those genes in ATSCs and ATSC-TERT revealed that some neural lineage-related genes are highly upregulated in ATSC-TERT, and that was consistent with our Affymetrix Microarray result (data not shown).$ H5 S3 A7 L! w: W

Table 1. Expressed gene profile of ATSCs and ATSC-TERT cells and partial list of genes that were upregulated in ATSC-TERT cells' ?4 u" t' L2 v6 i8 ]) H: k
$ J3 v5 C+ b  q" D) }% Y
Table 2. Expressed gene profile of ATSCs and ATSC-TERT cells and partial list of genes that were downregulated in ATSC-TERT cells# W  m4 ~. e$ N
( q& y' h: \0 \) N9 }7 @1 p
DISCUSSION
% u6 u/ V/ ?3 F2 T6 C* m/ u4 \6 x2 F
We are grateful to Cynthia Trygg for supporting basic experiments. The work was supported by grant RR00164 from the National Center for Research Resources, National Institutes of Health, and a grant from the State of Louisiana Millennium Health Excellence Fund and the Louisiana Gene Therapy Research Consortium.

REFERENCES2 J! B7 [' E; u( ^8 O% ?0 P  L  }

Zhu J, Wang H, Bishop JM et al. Telomerase extends the lifespan of virus-transformed human cells without net telomere lengthening. Proc Natl Acad Sci U S A 1999;96: 3723–3728." G  g7 J" \. M: l

Blackburn EH. Structure and function of telomerase. Nature 1991;350:569–573.
" s' b- t6 K; g/ E
Yamada O, Akiyama M, Kawauchi K et al. Overexpression of telomerase confers a survival advantage through suppression of TRF1 gene expression while maintaining differentiation characteristics in K562 cells. Cell Transplant 2003;12: 365–377.
4 @: x8 ^4 u' d
Moyzis RK, Buckingham JM, Cram LS et al. A highly conserved repetitive DNA sequence, (TTAGGG)n, present at the telomeres of human chromosomes. Proc Natl Acad Sci U S A 1988;85:6623–6626.! V" @# M9 I7 Z! n: H! |

Shi S, Gronthos S, Chen S et al. Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression. Nat Biotechnol 2002;20:587–591." b% W0 U5 j# X: o  G

Simonsen JL, Rosada C, Serakini N et al. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol 2002;20:592–596.+ `) p, v( D2 L8 R9 N3 [- C% M3 P

Saldanha SN, Andrews LG, Tollefsbol TO et al. Analysis of telomerase activity and detection of its catalytic subunit, hTERT.Anal Biochem 2003;315:1–21.
0 M# T! @0 j* s5 s- _1 M6 _
Harley CD, Futcher AB, Greider CW et al. Telomeres shorten during aging of human fibroblasts. Nature 1990; 345:458–460.

Vaziri H, Schachter F, Uchida I et al. Loss of telomeric DNA during aging of normal and trisomy 21 human lymphocytes. Am J Hum Genet 1993;52:661–667.7 V5 X: q" a+ n5 G7 I* x
. p/ T% z8 y0 E
Prescott JC, Blackburn EH. Telomerase: Dr. Jekyll or Mr. Hyde? Curr Opin Genet Dev 1999;9:368–373.

Meyerson M, Counter CM, Eaton EN et al. hEST2, the putative human telomerase catalytic subunit gene, is up-regulated in tumor cells and during immortalization. Cell 1997;90:785–795.+ |; l9 `( U8 A

Urqidi V, Tarin D, Goodison S et al. Role of telomerase in cell senescence and oncogenesis. Annu Rev Med 2000;51: 65–79.. p9 ^$ B" B# O3 d

Vaziri H, Benchimol S. Reconstitution of telomerase activity in normal human cells leads to elongation of telomeres and extended replicative life span. Curr Biol 1998;8:279–282.
0 S% E6 z" y0 S
Yang J, Chang E, Cherry AM et al. Human endothelial cell life extension by telomerase expression. J Biol Chem 1999;274:26141–26148.: u: ^, }8 M  l+ o; c) K+ q* i: _
, l3 \" J# `. Q! ]3 p# i& S; _
Kassemm M, Ankersen L, Eriksen EF et al. Demonstration of cellular aging and senescence in serially passaged long-term cultures of human trabecular osteoblasts. Osteoporos Int 1997;7:514–524.# E% v% k7 U4 w! k0 g
7 k; t* i' S) W
Caplan AI, Bruder SP. Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med 2001;7:259–264.1 j: H$ {. e! D3 X

Krebsbach PH, Kuznetsov SA, Satomura K et al. Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts. Transplantation 1997;63:1059–1069.
& L. f+ z2 p* i# g
Reddi AH. Morphogenesis and tissue engineering of bone and cartilage: inductive signals, stem cells, and biomimetic biomaterials. Tissue Eng 2000;6:351–359.
; D! r1 }4 A1 K3 ^6 N" R* A
Kang SK, Lee DH, Bae YC et al. Improvement of neurological deficits by intracerebral transplantation of human adipose tissue-derived stromal cells after cerebral ischemia in rats. Exp Neurol 2003;183:355–366.
( d1 U# ~2 p# s2 N& L$ j
Safford KM, Hicok KC, Safford SD et al. Neurogenic differentiation of murine and human adipose-derived stromal cells. Biochem Biophys Res Commun 2002;294:371–379.
$ I  Y  [' T2 U# v/ y
Parfitt AM. Bone forming cells in clinical conditions. In: Hall BK, ed. The Osteoblast and Osteocyte. London: Telford, 1990:351–426.' w/ G+ Q% J  v# S7 X' q, n5 I

Zuk PA, Zuh M, Mizuno H et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng 2001;7:211–228.9 \5 b$ p8 h" d& d2 _$ f

Zuk PA, Zhu M, Ashjian P et al. Human adipose tissue is a source of multipotent stem cells. Mol Biol Cell 2002;13: 4279–4295.' ]) f5 n& t- v- W+ Z6 c

Bodnar AG, Ouellette M, Frolkis M et al. Extension of lifespan by introduction of telomerase into normal human cells. Science 1998;279:349–352." c. t. ?6 X) i8 j6 X

Dickson MA, Hahn WC, Ino Y et al. Human keratinocytes that express hTERT and also bypass a p16(INK4a)-enforced mechanism that limits life span become immortal yet retain normal growth and differentiation characteristics. Mol Cell Biol 2000;20:1436–1447.0 u+ y, O4 `! `
5 c' W  I5 C* M
Yang J, Nagavarapu U, Relloma K et al. Telomerized human microvasculature is functional in vivo. Nat Biotechnol 2001;19:219–224.8 M8 O0 o8 G2 E, F% u

Ilsopp RC, Morin GB, Horner JW et al. Effect of TERT over-expression on the long-term transplantation capacity of hematopoietic stem cells. Nat Med 2003;9:369–371.

Condon J, Yin S, Mayhew B et al. Telomerase immortalization of human myometrial cells. Biol Reprod 2002;67: 506–514.5 u, L' q7 Z0 u; Z' h. ?
5 R9 M$ Q6 F& V0 ~; J
Di Donna S, Mamchaoui K, Cooper RN et al. Telomerase can extend the proliferative capacity of human myoblasts, but does not lead to their immortalization. Mol Cancer Res 2003;1:643–653.5 L+ |. J1 M' T& P  `+ Y, L

Funk WD, Wang CK, Shelton DN et al. Telomerase expression restores dermal integrity to in vitro-aged fibroblasts in a reconstituted skin model. Exp Cell Res 2000;258:270–278.

Gronthos S, Chen S, Wang CY et al. Telomerase accelerates osteogenesis of bone marrow stromal stem cells by upregulation of CBFA1, Osterix, and Osteocalcin. J Bone Miner Res 2003;18:716–722.
; t( j- p. {3 W
Gronthos S, Mankani M, Brahim J et al. Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. Proc Natl Acad Sci U S A 2000;97:13625–13630.

Harada S, Rodan GA. Control of osteoblast function and regulation of bone mass. Nature 2003;423:349–355.; G* m( |: C5 E/ O$ m
6 R% R: Q$ e( k1 ~( q" ]9 U; I- C9 S' s
Jiang XR, Jimenez G, Chang E et al. Telomerase expression in human somatic cells does not induce changes associated with a transformed phenotype. Nat Genet 1999;21:111–114.
1 u' r4 ^, M0 K9 I' \
Morales CP, Holt SE, Ouellette M et al. Absence of cancer-associated changes in human fibroblasts immortalized with telomerase. Nat Genet 1999;21:115–118." f6 }* W) p" P+ K  x$ t

Shay JW, Wright WE. The use of telomerized cells for tissue engineering. Nat Biotechnol 2000;18:22–23.
& S4 e6 q4 Z% p$ m
Tuan R. Boning up on telomerase. Nat Biotechnol 2002;20: 560–561.

Zheng Q, Zhou G, Morello R et al. Type X collagen gene regulation by Runx2 contributes directly to its hypertrophic chondrocytes-specific expression in vivo. J Cell Biol 2003;162:833–842.(Soo Kyung Kanga,c, Lorna )

收藏0 分享0 顶0 踩0