| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
TISSUE-SPECIFIC STEM CELLS |
aExperimental Fetal Medicine Group, Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, London, United Kingdom;
bCentre for Fetal Care, Queen Charlotte's & Chelsea Hospital, London, United Kingdom
Key Words. Adult stem cells • Telomere • Telomerase • Real-time reverse transcription-polymerase chain reaction • Mesenchymal stem cell • Fetal stem cells
Correspondence: Pascale V. Guillot, Ph.D., Experimental Fetal Medicine Group, Institute of Reproductive and Developmental Biology, Imperial College London, Hammersmith Campus, Du Cane Road, London W12 0NN, U.K. Telephone: 44 (0)207-594-2121; Fax: 44 (0)207-594-2154; e-mail: Pascale.Guillot{at}imperial.ac.uk
Received on April 11, 2006;
accepted for publication on November 14, 2006.
First published online in STEM CELLS EXPRESS November 22, 2006.
 |
ABSTRACT |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
|---|
|
INTRODUCTION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
|---|
Pluripotency indicates the capacity to differentiate into cell types of all the three germ layers. Study of its relationship with gene expression led to identification of several markers of pluripotent stem cells, such as the transcription factor Oct-4 [14, 15], which prevents stem cells from differentiating and is expressed in ESC, germ cells, and early whole embryos [16, 17]. Despite the fact that adult MSC possess broader differentiation capacity than originally thought, no consistent data indicate that they express pluripotency markers. Contrary to the expectation that only stem cells from embryonic tissues express Oct-4, however, it has recently been suggested that Oct-4 may be expressed in ontologically immature postembryonic stem cells, such as in second-trimester amniotic fluid [15, 18]. In terms of other pluripotency markers, Nanog is a homeodomain protein present in pluripotent human cell lines and absent from differentiated cells, which directs pluripotency and differentiation of undifferentiated ESC [19]. Pluripotent human stem cells can also be characterized by expression of SSEA-3 and SSEA-4, the keratan sulfate-associated antigens Tra-1-60 and Tra-1-81 [20], and Rex-1, all expressed at high levels in undifferentiated ESC and absent in differentiated cells [21].
Other factors relevant to cell therapy are self-renewal and senescence. Replicative stability is conferred by telomeres, double-stranded DNA (TTAGGG)n repeat sequences up to 20 kb long, with a single strand of the same sequence acting as a protective cap for the chromosomal ends. Because DNA polymerase cannot fully duplicate these sequences, telomeres shorten with successive cell division and DNA replication to reach a critical length, which triggers cell division arrest [22]. This replicative senescence [23] occurs after a finite number of cell divisions known as the Hayflick limit, which depends on the telomere length of the starting population [24]. ESC escape telomere shortening, as after each cell division, telomerase enzyme extends telomeres to compensate for sequence loss during replication [25–27]. Telomerase activity is closely related to the expression of human telomerase reverse transcriptase (hTERT), which encodes the catalytic and a rate-limiting subunit of telomerase. Adult human MSC cultured in vitro lack telomerase activity [28] and hTERT expression [29], which results in telomere shortening with serial passaging [29]. However, when hTERT is ectopically overexpressed, telomerase activity is restored resulting in longer telomeres and increased cellular lifespan [30]. Telomerization of MSC by hTERT overexpression maintains stem cell characteristics of MSC during long-term culture and allows large-scale cell expansion [30]. Some but not all early human fetal tissues express telomerase [31], but telomere and hTERT activities have not been investigated in fetal MSC.
With a view to understanding the relative benefits of fetal versus adult sources for MSC therapy, our goal was to explore whether first-trimester fetal MSC from blood, liver, and bone marrow were phenotypic intermediates between ESC and adult MSC. We characterized pluripotency markers, growth kinetics, and telomere status in human first-trimester fetal blood, liver, and bone marrow MSC and compared them with adult bone marrow MSC.
|
MATERIALS AND METHODS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
|---|
Cell Sources
Human first-trimester fetal MSC from blood, liver, and bone marrow were harvested as described previously [12]. Adult MSC from bone marrow were obtained from Prof. F. Dazzi (Imperial College London, London, U.K.) and Tulane Center for Gene Therapy (Tulane University, New Orleans, LA). Endometrial cells were obtained from Prof. J. Brosens (Imperial College London). TERT-MSC DNA and proteins were from Prof. M. Kassem (University of Aarhus, Aarhus, Denmark). The immortalized human embryonic kidney cell line 293T [32] and the human immortalized osteoblast cell line Saos-2 cells were purchased from ATCC (CRL-11268 and CRL HTB-85; Teddington, U.K., http://www.atcc.org). Human fetal MSC were characterized by their adherence to plastic, their fibroblast-like spindle morphology, and their immunophenotype. Consistent with their nonhematopoietic, nonendothelial origin, cultured human fetal MSC were all CD45-, CD34-, CD14-, and CD31-negative. Furthermore, we confirmed their mesenchymal nature by immunophenotyping (SH2-, SH3-, and SH4-positive and expression of laminin, fibronectin, and vimentin) and ability under permissive conditions to differentiate down the osteogenic and adipogenic lineages [12, 33].
Tissue Culture
All cells were plated at 104 cells per cm2 and cultured in 10-cm2 dishes with expansion medium (i.e., Dulbecco's modified Eagle's medium; Sigma-Aldrich, St. Louis, http://www.sigmaaldrich.com) supplemented with 10% fetal bovine serum (Gibco-BRL, Gaithersburg, MD, http://www.gibcobrl.com) and 2 mmol/l L-glutamine, 50 IU/ml penicillin, 50 mg/ml streptomycin (Gibco-BRL) at 37°C in 5% CO [2]. Subconfluent (70%–80%) cells were detached with 0.05% trypsin-0.01% EDTA (Gibco-BRL), and only early passage cells (passages 2–7) were studied.
Immunofluorescence
Cells grown exponentially were fixed in 4% PFA in 125 mM HEPES (pH 7.6; 10 minutes, 4°C), 8% PFA in the same buffer (50 minutes, 4°C) and permeabilized in 0.5% Triton X-100 in phosphate-buffered saline (PBS) (30 minutes, gentle rocking). After fixation and permeabilization, cells were rinsed (3x) in PBS, incubated (30 minutes) with 20 µM glycine in PBS, blocked (1 hour) with PBS+ (PBS supplemented with 1% BSA, 0.2% fish skin gelatin, 0.1% casein; pH 7.6), incubated (2 hours) with primary antibodies in PBS+, washed (5x over 1.5 hours) in PBS+, incubated (1 hour) with secondary antibodies in PBS+, washed (overnight, 4°C) in PBS+, and rinsed (3x) in PBS, before being mounted in VectaShield labeled with 4,6-diamidino-2-phenylindole (Vector Laboratories, Burlingame, CA, http://www.vectorlabs.com) and visualized immediately. For fluorescence microscopy (Axioscope I microscope equipped with CCD camera and iPlab software; Carl Zeiss, Jena, Germany, http://www.zeiss.com), images were collected sequentially (TIFF files), transferred to Adobe Photoshop (Adobe Systems Inc., San Jose, CA, http://www.abode.com), and contrast-stretched without further processing. The following primary antibodies were used: mouse monoclonal IgG Oct-4 (Santa Cruz Biotechnology Inc., Santa Cruz, CA, http://www.scbt.com), IgG goat polyclonal Nanog (Santa Cruz Biotechnology), mouse monoclonal IgM SSEA-3, IgG SSSEA-4, IgM TRA-1–61, and IgM TRA-1–81 (from the ES Cell characterization kit; Chemicon, Temecula, CA, http://www.chemicon.com). All primary antibodies were used at a 1:50 dilution. Secondary antibodies for immunofluorescence were donkey anti-mouse or anti-goat IgG conjugated with fluorescein isothiocyanate (1:100 and 1:1000, respectively; multiple-labeling grade; Jackson Immunoresearch Laboratories, West Grove, PA, http://www.jacksonimmuno.com).
Reverse Transcription-Polymerase Chain Reaction
Total RNA was extracted from cells using the RNeasy Mini RNA isolation kit (Qiagen, Hilden, Germany, http://www1.qiagen.com). Total RNA was eluted from the mini columns with 50 µl of RNase-free water. The amount of total RNA isolated was quantified by optical density at 260 nm (OD260). Starting from 1 µg of total RNA, 20 µl of cDNA was synthetized using Pd(N)6 random hexamers (Amersham Biosciences, Piscataway, NJ, http://www.amersham.com) and 1 µl of 200 U M-MLV reverse transcriptase in the presence of dNTPs (Promega, Madison, WI, http://www.promega.com). The reaction was performed for 10 minutes at 25°C, for 60 minutes at 42°C, and for 10 minutes at 75°C. cDNA was stored at –20°C until use.
cDNA was amplified by 30 cycles of denaturation (60 seconds at 94°C), annealing (30 seconds, 60°C), and elongation (30 seconds, 72°C), followed by a final step at 72°C for 5 minutes, using primer sequences previously published [34] and blasted against expected published sequences to confirm specificity (data not shown).
Growth Kinetics
To investigate whether fetal MSC were more readily expandable than adult MSC, we compared their growth kinetics, estimated by the cumulative population doubling over 50 days. Fetal and adult MSC were plated in triplicate at a concentration of 104 cells per cm2 in 10-cm2 dishes and successively subcultured at the same density when subconfluent [35]. The cells were detached and counted in a hemocytometer in trypan blue to exclude dead cells. This replating procedure was serially repeated over 50 days, and the cumulative cell doublings of the populations were plotted against time in culture to determine the growth kinetics of fetal and adult MSC expansion. The number of population doublings was determined by counting the number of adherent cells at the start and end of each passage.
To compare the growth rate of fetal and adult MSC at different cell density and to determine the effect of basic fibroblast growth factor (bFGF), we seeded bone marrow-derived adult MSC and fetal MSC from the same source (bone marrow) in triplicate in 12-well plates at 10, 100, 1,000, and 10,000 cells per cm2 with or without bFGF (5 ng/ml). The number of cells was counted every 2 days over a 2 weeks period, and the growth rates were determined by plotting the number of cells against time for different conditions. The relative cell morphology and size of fetal and adult bone marrow-derived MSC was assessed under a microscope (magnification, x10 and x20) on adherent cells stained by crystal violet and in trypsinized cells visualized in a hemocytometer chamber.
Real-Time Polymerase Chain Reaction Quantification
We used SYBR Green dye fluorescence (Applied BioSystems, Foster City, CA, http://www.appliedbiosystems.com) and the ABI Prism 7700 Sequence Detection system (Applied Biosystems) based on the assumption that any increase in fluorescence signal is proportional to the amount of specific polymerase chain reaction (PCR) product. As the cycle threshold value (Ct) to reach fluorescence is directly proportional to the log of the initial amount of input cDNA, we estimated the amount of target sequence in the experimental sample by plotting the Ct of an unknown sample on a standard curve created with serial dilution of a reference sample. All samples were run at least in duplicate. Standard curves were generated with the ABI Prism 7700 SDS 1.7 software, and r2 values were consistently 
0.998.
Relative Telomere Length Using Quantitative Real-Time PCR
Genomic DNA from experimental samples (Table 1) and the reference 293T cells was obtained using standard salting-out extraction after Miller et al. [36] and stored at –20°C until analysis. Relative telomere lengths were measured by SYBR Green real-time quantitative PCR amplification of telomere repeats (T) and single-copy gene 36B4 (S). The 36B4 gene was used to normalize for sample variations in DNA amount. The primers and thermal cycling profiles were adapted from Cawthon et al. [37]. T and S standard curves (Ct vs. log quantity) were generated using serial dilution of DNA (200 to 12.5 ng) from the telomerase-positive cell line 293T. Ct values in experimental samples were determined from semi-log amplification plots, and the standard curve was used to determine the quantity of telomere repeats, which is equivalent to the level of dilution of the arbitrary reference 293T needed to give the identical number of PCR cycles during the exponential phase. Relative telomere length was thus estimated as the Tquantity/Squantity (T/S) ratio, which is proportional to the mean terminal restriction fragment telomere (TRF) length [37]. The telomere and 36B4 PCRs were run on separate plates, and the standard curve was included in each run to allow relative quantification between samples (50 ng per sample). The concentration of reagents was 25 µl of 2x SYBR Green PCR Master Mix (Applied Biosystems), with the following final primer concentrations: Tel 1, 270 nM; Tel 2, 900 nM; 36B4u, 300 nM; and 36B4d, 500 nM. The thermal cycling conditions started with 95°C for 10 minutes; this step was followed by 40 cycles of 95°C for 15 seconds and then 54°C for 2 minutes for telomere PCR, and with 40 cycles of 95°C for 15 seconds and then 58°C for 1 minute for 36B4 PCR.
|
CT for each experimental sample, with CT = CT(experimental sample) – CT(293T calibrator).
Real-Time PCR Quantification of hTERT mRNA
Levels of hTERT expression were estimated by SYBR Green real-time quantification using primers and methods described by Buttitta et al. [40] To normalize hTERT expression for intersample differences in RNA input, quality, and reverse transcriptase efficiency, we amplified the housekeeping gene β-actin. The ratio between copy numbers of hTERT mRNA and β-actin mRNA was used to normalize the amount of hTERT mRNA for each sample and allowed comparison between samples.
Statistical Analysis
After confirming normal distribution on histograms, standard parametric descriptive and two-tailed comparative statistics were used, and correlations were sought by linear regression using the least squares method. A value of p < .05 was considered significant. We used one-way analysis of variance for continuous variables.
|
RESULTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
|---|
|
|
Telomere Length
Figure 3D shows that fetal MSC had longer telomeres than adult MSC (p < .001), whereas adult MSC and endometrial cells were similar in length (fetal blood, 74.6% of reference 293T value; fetal liver, 71.7%; fetal bone marrow, 75%; p < .001 compared with adult bone marrow and endometrial cells, 34%). Applying a conversion factor [37] to convert T/S ratios to their corresponding mean TRF length, fetal blood, liver, and bone marrow were 11, 10.8, and 11.2 kb, respectively, which corresponds to approximately 
of the length of hTERT-maximized MSC, whereas adult bone marrow MSC and endometrial cells were 7.2 and 7 kb, respectively.
|
CT relative to 293T cells) was higher in fetal blood, liver, and bone marrow than adult bone marrow (p < .01). Adult MSC had repressed telomerase activity, similar to control adult endometrial cells (Fig. 4E). As expected, telomere length positively linearly correlated (p < .01) (Fig. 5). To know whether higher telomerase activity in the fetal samples corresponded to a greater amount of telomerase or higher enzymatic activity, we determined the quantities of hTERT mRNA relative to adult MSC using real-time RT-PCR and the CT method (Fig. 6). hTERT mRNA was expressed in fetal blood, liver, and bone marrow 5.2, 4.8, and 5.7 times more, respectively, than in adult bone marrow samples.
|
|
|
|
DISCUSSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
|---|
Although pluripotency marker expression has been considered a hallmark of ESC, this study shows that all three fetal sources of MSC express pluripotency markers. Although no previous study has tested this in fetal MSC, our finding is not entirely unexpected, given their earlier developmental origins and the other differences we have shown in fetal versus adult MSC. A few reports have suggested that early human MSC may express some pluripotency markers. Some MSC from second-trimester amniotic fluid express Oct-4 [9, 18], whereas Karlmark et al. [42] recently showed that second-trimester human amniotic fluid contains cell populations capable of activating Oct-4 and Rex-1 promoters. In addition, both hemopoietic and nonhemopoietic stem cell populations from umbilical cord blood have been shown to express some pluripotency markers, although these cells were not considered mesenchymal in origin [43–45].
We have previously shown multilineage potential of fetal MSC cultures, in particular that fetal but not adult MSC readily undergo myogenic differentiation [33]. Compared with adult bone marrow MSC, we recently found that the osteogenic efficiency of human fetal MSC was greater than adult MSC both in vitro and in vivo (unpublished data). Similarly in the rhesus monkey, third-trimester fetal MSC differentiated more readily down the osteogenic, chondrogenic, and adipogenic lineages than adult bone marrow-derived MSC [11]. Banfi et al. [35] showed that adult bone marrow MSC gradually lost their multiple differentiation potential during in vitro expansion, with their bone-forming efficiency in vivo reduced manyfold at first confluence compared with fresh bone marrow. Therefore, we speculate that lack of expression of early pluripotency markers in adult MSC may reflect a degree of multilineage lineage restriction.
Our results are comparable to previous reports documenting the slow replication and rapid senescence of adult MSC in vitro. The maximal population doublings we achieved in vitro were 70–80 for human fetal MSC [12], considerably greater than the 30–40 for adult MSC [35]. Adult MSC are only found at low frequency in bone marrow in vivo, and their low proliferative capacity is accompanied by progressive replicative aging and decrease in osteogenic potential when expanded in vitro, with no detectable telomerase activity [46]. Fetal MSC are smaller than adult MSC. Interestingly, cell morphology is affected by senescence, cell size increasing with in vitro ageing [24], whereas cells transduced with telomerase become smaller than their nontransduced counterparts [47].
Telomere length is relevant to cell therapy, especially in utero transplantation, when the recipient is younger, and stem cells will need to persist for a similar ontological lifespan. Telomeres protect chromosomal ends, and when protection is lost, certain genes can be activated to trigger senescence-related dysfunction and pathology, as in dyskeratosis congenital when DKC1 activation induces premature ageing and skin cancer [48]. Telomere status and cell proliferation are closely linked in MSC, with human adult MSC telomerized by overexpressing hTERT exhibiting extensive proliferation and acquiring an extended cell lifespan [49].
The potential therapeutic advantages of fetal MSC over adult MSC are not restricted to differentiation potential, growth kinetics, and telomere status. When transplanted into the fetus of immunodeficient SCID mice, donor cells from fetal liver cells showed a 10-fold engraftment advantage over those from adult bone marrow [50]. In preimmune fetal sheep, fetal MSC, like adult MSC, engrafted in multiple tissues and showed multilineage differentiation like their adult counterparts, but unlike adult bone marrow MSC, they appeared to contribute hemopoiesis [51]. In complementary work, we recently found that fetal MSC express a unique pattern of adhesion molecule expression, with higher levels of 
2 and 4β1 and binding to their extracellular matrix ligands compared with adult MSC, which would facilitate homing and engraftment [52]. Fetal MSC also present some immunological advantages. Their lack of intracellular HLA class II and slower response to stimulation by 
-interferon [53] might confer fetal donor cells with an immune tolerance advantage.
Although early fetal MSC have been isolated from other fetal organs [54], human fetal MSC from blood liver and bone marrow are the best characterized. Allogeneic use raises the ethical issues associated with tissues derived from termination of pregnancy. Autologous use for ex vivo gene therapy, however, would allow collection of fetal blood and possibly liver in continuing pregnancies, and recent reports of MSC in first-trimester placenta [55] and mid-trimester amniotic fluid raise the possibility of using more accessible sources of fetal MSC.
|
CONCLUSION |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
|---|
|
DISCLOSURES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
|---|
|
ACKNOWLEDGMENTS |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
|---|
|
REFERENCES |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionConclusionDisclosuresAcknowledgmentsReferences |
|---|
This article has been cited by other articles:
|
|
 |
|
  B. L. Yen, C. J. Chang, K.-J. Liu, Y. C. Chen, H.-I Hu, C.-H. Bai, and M.-L. Yen Brief Report--Human Embryonic Stem Cell-Derived Mesenchymal Progenitors Possess Strong Immunosuppressive Effects Toward Natural Killer Cells as Well as T Lymphocytes Stem Cells, February 1, 2009; 27(2): 451 - 456. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
Z.-Y. Zhang, S.-H. Teoh, M. S.K. Chong, J. T. Schantz, N. M. Fisk, M. A. Choolani, and J. Chan Superior Osteogenic Capacity for Bone Tissue Engineering of Fetal Compared with Perinatal and Adult Mesenchymal Stem Cells Stem Cells, January 1, 2009; 27(1): 126 - 137. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. T. Durkin, K. A. Jones, D. Rajesh, and A. F. Shaaban Early chimerism threshold predicts sustained engraftment and NK-cell tolerance in prenatal allogeneic chimeras Blood, December 15, 2008; 112(13): 5245 - 5253. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. A. Rider, C. Dombrowski, A. A. Sawyer, G. H. B. Ng, D. Leong, D. W. Hutmacher, V. Nurcombe, and S. M. Cool Autocrine Fibroblast Growth Factor 2 Increases the Multipotentiality of Human Adipose-Derived Mesenchymal Stem Cells Stem Cells, June 1, 2008; 26(6): 1598 - 1608. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. L. Troyer and M. L. Weiss Concise Review: Wharton's Jelly-Derived Cells Are a Primitive Stromal Cell Population Stem Cells, March 1, 2008; 26(3): 591 - 599. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
X.-Y. Wang, Y. Lan, W.-Y. He, L. Zhang, H.-Y. Yao, C.-M. Hou, Y. Tong, Y.-L. Liu, G. Yang, X.-D. Liu, et al. Identification of mesenchymal stem cells in aorta-gonad-mesonephros and yolk sac of human embryos Blood, February 15, 2008; 111(4): 2436 - 2443. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
P. V. Guillot, O. Abass, J. H. D. Bassett, S. J. Shefelbine, G. Bou-Gharios, J. Chan, H. Kurata, G. R. Williams, J. Polak, and N. M. Fisk Intrauterine transplantation of human fetal mesenchymal stem cells from first-trimester blood repairs bone and reduces fractures in osteogenesis imperfecta mice Blood, February 1, 2008; 111(3): 1717 - 1725. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Naujokat and T. Saric Concise Review: Role and Function of the Ubiquitin-Proteasome System in Mammalian Stem and Progenitor Cells Stem Cells, October 1, 2007; 25(10): 2408 - 2418. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| STEM CELLS | THE ONCOLOGIST | CME | ALPHAMED PRESS JOURNALS |
