Study of Telomere Length Reveals Rapid Aging of Human Marrow Stromal Cells following In Vitro Expansion
http://www.100md.com
《干细胞学杂志》
a Stem Cell Research Group, Giving for Living Postgraduate Centre, Royal Manchester Children’s Hospital, Manchester, United Kingdom;
b Laboratory Medicine, Stepping Hill Hospital, Poplar Grove, Stockport, United Kingdom;
c Willink Biochemical Genetics Unit, Royal Manchester Children’s Hospital, Manchester, United Kingdom;
d Cancer Research UK Gene Therapy Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, United Kingdom
Key Words. Mesenchymal stem cells ? Transplantation
Correspondence: Ilaria Bellantuono, Ph.D., Stem Cell Research Group, Giving for Living Postgraduate Centre, Royal Manchester Children’s Hospital, Manchester, United Kingdom, M27 4HA. Telephone: 44-161-7272385; Fax: 44-161-7272679; e-mail: Ilaria.Bellantuono@cmmc.nhs.uk
ABSTRACT
Marrow stromal cells (MSCs), or mesenchymal stem cells, are a promising therapeutic tool. They can be readily isolated by plastic adherence, differentiated into tissues such as bone and cartilage, and genetically modified by viral vectors . Clinically, MSCs may be used to enhance hematopoietic stem cell (HSC) engraftment post-transplantation, to correct inherited disorders of bone and cartilage, or as vehicles for gene therapy such as in osteogenesis imperfecta .
The use of MSCs to reconstitute bone marrow stroma or to sustain healthy osteogenesis relies on the long-lasting engraftment of MSCs with a residual replicative capacity to produce differentiated progeny, substitute damaged tissue, and sustain tissue turnover for life. To date, reinfusion of MSCs has resulted in poor engraftment and limited cell survival . Previous studies have shown that human MSCs cultured in vitro display a tendency to lose their proliferative potential, homing capability, and in vivo bone-forming efficiency over time . Whether even a limited expansion in vitro is sufficient to age the cells to a point where their successful clinical use is compromised has not yet been addressed.
Telomeres are specialized structures present at the ends of eukaryotic chromosomes. They have been associated with the molecular machinery critical for cell replicative lifespan, and their shortening is known to play an important part in the cell molecular aging process . In human cells, such as fibroblasts, telomeres shorten progressively during successive cell divisions, down to a threshold length at which they undergo replicative senescence . Consequently, mean telomere restriction fragment (mTRF) lengths can be used to extrapolate the replicative history and remaining replicative capacity of a cell population.
In the study reported here, we investigated the mTRFs of human MSCs during in vitro expansion and correlated this with mTRF changes with donor age. Our data show that, with present protocols, in vitro culture for just 7–10 population doublings (PDs) gives a reduction in mTRF length that, depending on the age of the MSC donor, is equivalent to the loss of more than half their lifespan by the time they are reinfused.
MATERIALS AND METHODS
In Vitro Expansion of MSCs
MSCs were derived from 10 donors of pediatric age (hMSC0–18) and five donors aged 59–75 years (hMSC59–75). After primary passage, fluorescence-activated cell sorter (FACS) analysis showed no expression of either CD34 or CD45 antigens in all MSC cultures, and all stained positive with SH2 antibody . MSC frequency in bone marrow samples, as determined by the CFU-F assay, showed a significant decrease in hMSC59–75 compared with hMSC0–18 (3.2 ± 1.7 versus 29.0 ± 4.7 per 106 mononuclear cells, p < .001).
The in vitro growth kinetics of MSCs were investigated from the primary passage until cells in culture ceased to replicate for at least 3 weeks. At this point, the cultures were considered senescent and were terminated. Most of hMSC0–18 had a faster expansion rate than hMSC59–75 (Table 1; Fig. 1A, 1B) while exhibiting the well-documented spindle-shape cell morphology (Fig. 2A) and low levels of alkaline phosphatase expression (Fig. 2B). After primary confluence, their rate of growth progressively declined. At growth arrest, hMSC0–18 lines had undergone on average 30.6 ± 2.2 PDs after 197.4 ± 25.3 days of culture.
Table 1. Growth properties of hMSC0–18 , hMSC0–18E, and hMSC59–75
Figure 1. Growth kinetics of hMSC cultures. The cumulative number of PDs were plotted against time in culture. (A): hMSC0–18 kinetics (filled circles), each line representing an hMSC0–18 culture (n = 8); hMSC0–18E kinetics (open circles), each line representing an hMSC0–18E culture (n = 2). (B): hMSC59–75 kinetics, each line representing an hMSC59–75 culture (n = 5). Abbreviations: hMSC, human marrow stromal cell; PD, population doubling.
Figure 2. Osteogenic differentiation of hMSCs. (A–C): hMSC0–18 culture at 14 PDs. (D–F): hMSC0–18E culture at 37 PDs. (A, D): May and Grunwald and Giesma staining of undifferentiated cultures. Alkaline phosphatase expression stain (pink) and Von Kossa stain (black) (B, E) of undifferentiated cultures and (C, F) following osteogenic differentiation. (G): Alkaline phosphatase expression (pink) and Von Kossa stain (black) of undifferentiated hMSC0–18 culture at 28 PD. (H): Alkaline phosphatase expression of undifferentiated hMSC59–75 culture at 16 PDs. (I): Alkaline phosphatase expression and Von Kossa stain of hMSC59–75 culture at 16 PDs following osteogenic differentiation. Abbreviations: hMSC, human marrow stromal cell; PD, population doubling.
Interestingly, two of the hMSC0–18 cultures (hMSC0–18E) proliferated for over 40 PDs (Fig. 1A). They followed a similar pattern of growth kinetics to hMSC0–18 in the first 25 PDs. Thereafter, the rate of growth was maintained at an average steady rate of 1 PD every 14.8 ± 0.6 days for over 40 PDs, with no growth arrest observed. They maintained the spindle-shape morphology even at later time points (Fig. 2D) and exhibited low levels of alkaline phosphatase expression (Fig. 2E). This was in contrast to all other hMSC0–18 lines where, even at earlier PDs, a progressive change in cell morphology was observed, with cells becoming wider and flatter and showing increased alkaline phosphatase expression and Von Kossa–stained mineralized deposits (Fig. 2G). hMSC0–18E retained good osteogenic capacity (Fig. 2F) but reduced adipogenic capacity (data not shown).
hMSC59–75 showed severely reduced proliferative capacity (Table 1; Fig. 1B) with a slower growth rate than that in hMSC0–18. No cells with the spindle-shape morphology were observed; only cells exhibiting a larger and flatter morphology were present. They formed a significantly thinner monolayer at first confluence (1.7 ± 0.9 x 104 cells/cm2 versus 6.2 ± 0.1 x 104 cells/cm2, p < .001) and exhibited increased alkaline phosphatase expression and Von Kossa–stained mineralized matrix formation (Fig. 2H). hMSC59–75 cultures showed reduced alkaline phosphatase upregulation and calcium deposition with osteogenic differentiation (Fig. 2I) when compared with hMSC0–18 cultures at similar PD (Fig. 2C).
Telomere Kinetics during In Vitro Expansion and In Vivo
Length of mTRF was measured on genomic DNA taken from hMSC0–18 cells and hMSC59–75 at each passage when cell numbers were sufficient. A significant decrease was found in hMSC0–18 between the mTRF lengths at primary passage and at senescence by an average total of 1.0 kb ± 0.2 (range 0.4–2.5 kb, p = .002, Fig. 3A). The kinetic study in hMSC59–75 was limited, but no significant difference in the rate of mTRF loss was observed (on average, 88 bp/PD ± 10 for hMSC0–18 and 78 bp/PD ± 34 for hMSC59–75, p = .82). A strong correlation was found between total mTRF loss and total number of PDs occurring between the two measurements (R = .93 Pearson coefficient; Fig. 3B).
Figure 3. mTRF lengths of hMSCs during in vitro expansion. (A): hMSC0–18 (n = 8, filled circles) and hMSC0–18E (n = 2, open circles) mTRF kinetics over cumulative PDs. (B): Total loss of telomere length in kb, calculated as the difference between the first mTRF measurement (mTRF1) and mTRF at the end of the culture (mTRFend) versus number of PDs. hMSC0–18 cultures are represented by filled circles (n = 8), hMSC0–18E by open circles (n = 2), and hMSC59–75 by triangles (n = 5). (C): Normal karyotype of one of two hMSC0–18E cultures at 35 PDs. (D): Average mTRF of hMSC0–18 plus hMSC0–18E (black bar, n = 10) and hMSC59–75 (gray bar, n = 5) at 16 PDs. (E): mTRF of hMSC0–18 plus hMSC0–18E (black bar, n = 10) and hMSC59–75 (gray bar, n = 5) at growth arrest. Abbreviations: hMSC, human marrow stromal cell; mTRF, mean telomere restriction fragment; PD, population doubling.
Interestingly, the two cultures hMSC0–18E that maintained the spindle-shape morphology and showed longer proliferative capacity also displayed mTRF shortening in a similar range to other hMSC0–18 over the first 25 PDs, but thereafter losses were below detection (Fig. 3A). No telomerase activity was detected in those cultures at 35 PDs (data not shown), and karyotypic analysis carried out at the same time did not show any abnormality (Fig. 3C).
To ascertain whether in vitro aging of MSCs was a natural property of MSC and not due to suboptimal culture conditions, we compared the mTRF of hMSC0–18 (n = 10) with that of hMSC59–75 (n = 5) after an equal number of divisions in culture. All MSC cultures were expanded in vitro, and mTRF was calculated at 16 PDs. We found that mTRF in hMSC0–18 was significantly longer than in hMSC59–75 (11.5 PDs ± 0.2 versus 10.3 PDs ± 0.1, p = .021; Fig. 3D). As the average total mTRF loss was 1.2 kb and the mean age difference between the two groups was 63 years, an average loss of 17 bp per year could be estimated. To determine whether there was an mTRF threshold at which MSCs stop proliferating regardless of their starting mTRF, mTRF length from cells at growth arrest was determined. We found no significant difference in mTRF length at growth arrest between hMSC0–18 and hMSC59–75 (10.4 ± 0.1 kb, n = 10 versus 10.4 ± 0.1, n = 5; p = .795; Fig. 3E). Interestingly, the mTRF length of hMSC59–75 at primary passage was found to be close to the mTRF threshold at which cells stopped proliferating.
DISCUSSION
In this study we used telomere length as a marker of aging, to quantify the remaining replicative capacity following in vitro expansion. Cells with great telomere shortening are expected to have little remaining proliferative capacity. This study highlights the severe aging of MSCs by expansion using present protocols. From our data expansion of about 10 PDs (minimal expansion of MSCs used for transplantation in osteogenesis imperfecta ), leads to an average loss of 1 kb of telomere sequence. Taking into account that the total loss can be at most about 2.5–3.5 kb when cells are derived from young donors, this loss is significant. As we have shown that cells derived from an adult donor have already undergone some substantial telomere erosion in vivo (17 bp/year), the expansion may lead to reinfusion of cells that have short telomeres and are severely compromised in their remaining long-term proliferative, differentiative, and homing capacity at a time when proliferation should be at its best to be able to initiate regenerative processes. The presence of lines where expansion occurs with little or no telomere erosion is encouraging and requires further investigation into the mechanisms of telomere maintenance and on culture conditions where this can reliably be obtained.
ACKNOWLEDGMENTS
Prockop DJ. Marrow stromal cells as stem cells for non-haemopoietic tissues. Science 1997;276:71–74.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Baxter MA, Wynn RF, Deakin JA et al. Retrovirally mediated correction of bone marrow-derived stem cells from patients with mucopolysaccharidosis type I. Blood 2002;99:1857–1859.
Bartholomew A, Patil S, Mackay A et al. Baboon mesenchymal stem cells can be genetically modified to secrete human erythropoietin in vivo. Hum Gene Ther 2001;12:1527–1541.
Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with Osteogenesis imperfecta. Nat Med 1999;5:309–313.
Gao J, Dennis JE, Muzic RF et al. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs 2001;169:12–20.
Devine SM, Bartholomew AM, Mahmud N et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol 2001;29:244–255.
Horwitz EM, Prockop DJ, Gordon PL et al. Clinical responses to bone marrow transplantation in children with severe Osteogenesis imperfecta. Blood 2001;97:1227–1231.
Mets T, Verdonk G. In vitro aging of human bone marrow derived stromal cells. Mech Ageing Dev 1981;16:81–89.
D’Ippolito G, Schiller PC, Ricordi C et al. Age-related osteogenic potential of mesenchymal stromal cells from human vertebral bone marrow. J Bone Miner Res 1999;14:1115–1122.
Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997;64:278–294.
Digirolamo CM, Stokes D, Colter D. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999;107:275–281.
Banfi A, Muraglia A, Dozin B et al. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 2000;28:707–715.
Stenderup K, Justesen J, Clausen C et al. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow cells. Bone 2003;33:919–926.
Rombouts WJC, Ploemacher RE. Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia 2003;17:160–170.
Wright WE, Shay JW. Historical claims and current interpretations of replicative aging. Nat Biotechnol 2002;20:682–688.
Blackburn EH. Switching and signaling at the telomere. Cell 2001;106:661–673.
Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458–460.
Allsopp RC, Harley CB. Evidence for a critical telomere length in senescent human fibroblasts. Exp Cell Res 1995;219:130–136.
Reyes M, Lund T, Lenvik T et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615–2625.
Friedenstein AJ, Luria E, Molineux G. Assays of the haemopoietic microenvironment. In: Testa NG, Molineux G, eds. Haemopoiesis. New York: Oxford University Press, 1993:189–198.
Jaiswal N, Haynesworth SE, Caplan AI. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295–312.
Nuttall MSC, Patton AJ, Olivera DL. Human trabecular bone cells are able to express both osteoblast and adipocytic phenotype: implications for osteopenic disorders. J Bone Miner Res 1998;13:371–382.
Page K, Stevens A, Lowe J. Bone. In: Bancroft JD, Stevens A, eds. Theory and Practice of Histological Techniques. NewYork: Churchill Livingston, 1996;309–340.
Bayliss High OB, Lake B. Lipids. In: Bancroft JD, Stevens A, eds. Theory and Practice of Histological Techniques. NewYork: Churchill Livingston, 1996:213–242.
Wynn RF, Cross MA, Hatton C et al. Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants. Lancet 1998;351:178–181.
Barry FP, Boynton RE, Haynesworth S et al. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an eptitope on endoglin (CD105). Biochem Biophys Res Commun 1999;265:134–139.
Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–650.
Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol 2000;28:875–884.
Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sc 2000;113:1161–1166.
Colter DC, Sekiva I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A 2001;98:7841–7845.
Von Zglinicki T. Telomeres and replicative senescence: is it only length that counts? Cancer Lett 2001;168:111–116.
Lundblad V. Telomere maintenance without telomerase. Oncogene 2002;21:522–531.
Bianchi G, Banfi A, Mastrogiacomo M. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res 2003;287:98–105.
Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43:405–413.
Simonsen JL, Rosada C, Serakinci 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.
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.
Zimmermann S, Voss M, Kaiser S et al. Lack of telomerase activity in human mesenchymal stem cells. Leukemia 2003;17:1146–1149.
Masutomi K, Yu EY, Khurts S et al. Telomerase maintains telomere structure in normal human cells. Cell 2003;114:41–253.(Melissa A. Baxtera, Rober)
b Laboratory Medicine, Stepping Hill Hospital, Poplar Grove, Stockport, United Kingdom;
c Willink Biochemical Genetics Unit, Royal Manchester Children’s Hospital, Manchester, United Kingdom;
d Cancer Research UK Gene Therapy Group, Paterson Institute for Cancer Research, Christie Hospital, Manchester, United Kingdom
Key Words. Mesenchymal stem cells ? Transplantation
Correspondence: Ilaria Bellantuono, Ph.D., Stem Cell Research Group, Giving for Living Postgraduate Centre, Royal Manchester Children’s Hospital, Manchester, United Kingdom, M27 4HA. Telephone: 44-161-7272385; Fax: 44-161-7272679; e-mail: Ilaria.Bellantuono@cmmc.nhs.uk
ABSTRACT
Marrow stromal cells (MSCs), or mesenchymal stem cells, are a promising therapeutic tool. They can be readily isolated by plastic adherence, differentiated into tissues such as bone and cartilage, and genetically modified by viral vectors . Clinically, MSCs may be used to enhance hematopoietic stem cell (HSC) engraftment post-transplantation, to correct inherited disorders of bone and cartilage, or as vehicles for gene therapy such as in osteogenesis imperfecta .
The use of MSCs to reconstitute bone marrow stroma or to sustain healthy osteogenesis relies on the long-lasting engraftment of MSCs with a residual replicative capacity to produce differentiated progeny, substitute damaged tissue, and sustain tissue turnover for life. To date, reinfusion of MSCs has resulted in poor engraftment and limited cell survival . Previous studies have shown that human MSCs cultured in vitro display a tendency to lose their proliferative potential, homing capability, and in vivo bone-forming efficiency over time . Whether even a limited expansion in vitro is sufficient to age the cells to a point where their successful clinical use is compromised has not yet been addressed.
Telomeres are specialized structures present at the ends of eukaryotic chromosomes. They have been associated with the molecular machinery critical for cell replicative lifespan, and their shortening is known to play an important part in the cell molecular aging process . In human cells, such as fibroblasts, telomeres shorten progressively during successive cell divisions, down to a threshold length at which they undergo replicative senescence . Consequently, mean telomere restriction fragment (mTRF) lengths can be used to extrapolate the replicative history and remaining replicative capacity of a cell population.
In the study reported here, we investigated the mTRFs of human MSCs during in vitro expansion and correlated this with mTRF changes with donor age. Our data show that, with present protocols, in vitro culture for just 7–10 population doublings (PDs) gives a reduction in mTRF length that, depending on the age of the MSC donor, is equivalent to the loss of more than half their lifespan by the time they are reinfused.
MATERIALS AND METHODS
In Vitro Expansion of MSCs
MSCs were derived from 10 donors of pediatric age (hMSC0–18) and five donors aged 59–75 years (hMSC59–75). After primary passage, fluorescence-activated cell sorter (FACS) analysis showed no expression of either CD34 or CD45 antigens in all MSC cultures, and all stained positive with SH2 antibody . MSC frequency in bone marrow samples, as determined by the CFU-F assay, showed a significant decrease in hMSC59–75 compared with hMSC0–18 (3.2 ± 1.7 versus 29.0 ± 4.7 per 106 mononuclear cells, p < .001).
The in vitro growth kinetics of MSCs were investigated from the primary passage until cells in culture ceased to replicate for at least 3 weeks. At this point, the cultures were considered senescent and were terminated. Most of hMSC0–18 had a faster expansion rate than hMSC59–75 (Table 1; Fig. 1A, 1B) while exhibiting the well-documented spindle-shape cell morphology (Fig. 2A) and low levels of alkaline phosphatase expression (Fig. 2B). After primary confluence, their rate of growth progressively declined. At growth arrest, hMSC0–18 lines had undergone on average 30.6 ± 2.2 PDs after 197.4 ± 25.3 days of culture.
Table 1. Growth properties of hMSC0–18 , hMSC0–18E, and hMSC59–75
Figure 1. Growth kinetics of hMSC cultures. The cumulative number of PDs were plotted against time in culture. (A): hMSC0–18 kinetics (filled circles), each line representing an hMSC0–18 culture (n = 8); hMSC0–18E kinetics (open circles), each line representing an hMSC0–18E culture (n = 2). (B): hMSC59–75 kinetics, each line representing an hMSC59–75 culture (n = 5). Abbreviations: hMSC, human marrow stromal cell; PD, population doubling.
Figure 2. Osteogenic differentiation of hMSCs. (A–C): hMSC0–18 culture at 14 PDs. (D–F): hMSC0–18E culture at 37 PDs. (A, D): May and Grunwald and Giesma staining of undifferentiated cultures. Alkaline phosphatase expression stain (pink) and Von Kossa stain (black) (B, E) of undifferentiated cultures and (C, F) following osteogenic differentiation. (G): Alkaline phosphatase expression (pink) and Von Kossa stain (black) of undifferentiated hMSC0–18 culture at 28 PD. (H): Alkaline phosphatase expression of undifferentiated hMSC59–75 culture at 16 PDs. (I): Alkaline phosphatase expression and Von Kossa stain of hMSC59–75 culture at 16 PDs following osteogenic differentiation. Abbreviations: hMSC, human marrow stromal cell; PD, population doubling.
Interestingly, two of the hMSC0–18 cultures (hMSC0–18E) proliferated for over 40 PDs (Fig. 1A). They followed a similar pattern of growth kinetics to hMSC0–18 in the first 25 PDs. Thereafter, the rate of growth was maintained at an average steady rate of 1 PD every 14.8 ± 0.6 days for over 40 PDs, with no growth arrest observed. They maintained the spindle-shape morphology even at later time points (Fig. 2D) and exhibited low levels of alkaline phosphatase expression (Fig. 2E). This was in contrast to all other hMSC0–18 lines where, even at earlier PDs, a progressive change in cell morphology was observed, with cells becoming wider and flatter and showing increased alkaline phosphatase expression and Von Kossa–stained mineralized deposits (Fig. 2G). hMSC0–18E retained good osteogenic capacity (Fig. 2F) but reduced adipogenic capacity (data not shown).
hMSC59–75 showed severely reduced proliferative capacity (Table 1; Fig. 1B) with a slower growth rate than that in hMSC0–18. No cells with the spindle-shape morphology were observed; only cells exhibiting a larger and flatter morphology were present. They formed a significantly thinner monolayer at first confluence (1.7 ± 0.9 x 104 cells/cm2 versus 6.2 ± 0.1 x 104 cells/cm2, p < .001) and exhibited increased alkaline phosphatase expression and Von Kossa–stained mineralized matrix formation (Fig. 2H). hMSC59–75 cultures showed reduced alkaline phosphatase upregulation and calcium deposition with osteogenic differentiation (Fig. 2I) when compared with hMSC0–18 cultures at similar PD (Fig. 2C).
Telomere Kinetics during In Vitro Expansion and In Vivo
Length of mTRF was measured on genomic DNA taken from hMSC0–18 cells and hMSC59–75 at each passage when cell numbers were sufficient. A significant decrease was found in hMSC0–18 between the mTRF lengths at primary passage and at senescence by an average total of 1.0 kb ± 0.2 (range 0.4–2.5 kb, p = .002, Fig. 3A). The kinetic study in hMSC59–75 was limited, but no significant difference in the rate of mTRF loss was observed (on average, 88 bp/PD ± 10 for hMSC0–18 and 78 bp/PD ± 34 for hMSC59–75, p = .82). A strong correlation was found between total mTRF loss and total number of PDs occurring between the two measurements (R = .93 Pearson coefficient; Fig. 3B).
Figure 3. mTRF lengths of hMSCs during in vitro expansion. (A): hMSC0–18 (n = 8, filled circles) and hMSC0–18E (n = 2, open circles) mTRF kinetics over cumulative PDs. (B): Total loss of telomere length in kb, calculated as the difference between the first mTRF measurement (mTRF1) and mTRF at the end of the culture (mTRFend) versus number of PDs. hMSC0–18 cultures are represented by filled circles (n = 8), hMSC0–18E by open circles (n = 2), and hMSC59–75 by triangles (n = 5). (C): Normal karyotype of one of two hMSC0–18E cultures at 35 PDs. (D): Average mTRF of hMSC0–18 plus hMSC0–18E (black bar, n = 10) and hMSC59–75 (gray bar, n = 5) at 16 PDs. (E): mTRF of hMSC0–18 plus hMSC0–18E (black bar, n = 10) and hMSC59–75 (gray bar, n = 5) at growth arrest. Abbreviations: hMSC, human marrow stromal cell; mTRF, mean telomere restriction fragment; PD, population doubling.
Interestingly, the two cultures hMSC0–18E that maintained the spindle-shape morphology and showed longer proliferative capacity also displayed mTRF shortening in a similar range to other hMSC0–18 over the first 25 PDs, but thereafter losses were below detection (Fig. 3A). No telomerase activity was detected in those cultures at 35 PDs (data not shown), and karyotypic analysis carried out at the same time did not show any abnormality (Fig. 3C).
To ascertain whether in vitro aging of MSCs was a natural property of MSC and not due to suboptimal culture conditions, we compared the mTRF of hMSC0–18 (n = 10) with that of hMSC59–75 (n = 5) after an equal number of divisions in culture. All MSC cultures were expanded in vitro, and mTRF was calculated at 16 PDs. We found that mTRF in hMSC0–18 was significantly longer than in hMSC59–75 (11.5 PDs ± 0.2 versus 10.3 PDs ± 0.1, p = .021; Fig. 3D). As the average total mTRF loss was 1.2 kb and the mean age difference between the two groups was 63 years, an average loss of 17 bp per year could be estimated. To determine whether there was an mTRF threshold at which MSCs stop proliferating regardless of their starting mTRF, mTRF length from cells at growth arrest was determined. We found no significant difference in mTRF length at growth arrest between hMSC0–18 and hMSC59–75 (10.4 ± 0.1 kb, n = 10 versus 10.4 ± 0.1, n = 5; p = .795; Fig. 3E). Interestingly, the mTRF length of hMSC59–75 at primary passage was found to be close to the mTRF threshold at which cells stopped proliferating.
DISCUSSION
In this study we used telomere length as a marker of aging, to quantify the remaining replicative capacity following in vitro expansion. Cells with great telomere shortening are expected to have little remaining proliferative capacity. This study highlights the severe aging of MSCs by expansion using present protocols. From our data expansion of about 10 PDs (minimal expansion of MSCs used for transplantation in osteogenesis imperfecta ), leads to an average loss of 1 kb of telomere sequence. Taking into account that the total loss can be at most about 2.5–3.5 kb when cells are derived from young donors, this loss is significant. As we have shown that cells derived from an adult donor have already undergone some substantial telomere erosion in vivo (17 bp/year), the expansion may lead to reinfusion of cells that have short telomeres and are severely compromised in their remaining long-term proliferative, differentiative, and homing capacity at a time when proliferation should be at its best to be able to initiate regenerative processes. The presence of lines where expansion occurs with little or no telomere erosion is encouraging and requires further investigation into the mechanisms of telomere maintenance and on culture conditions where this can reliably be obtained.
ACKNOWLEDGMENTS
Prockop DJ. Marrow stromal cells as stem cells for non-haemopoietic tissues. Science 1997;276:71–74.
Pittenger MF, Mackay AM, Beck SC et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143–147.
Baxter MA, Wynn RF, Deakin JA et al. Retrovirally mediated correction of bone marrow-derived stem cells from patients with mucopolysaccharidosis type I. Blood 2002;99:1857–1859.
Bartholomew A, Patil S, Mackay A et al. Baboon mesenchymal stem cells can be genetically modified to secrete human erythropoietin in vivo. Hum Gene Ther 2001;12:1527–1541.
Horwitz EM, Prockop DJ, Fitzpatrick LA et al. Transplantability and therapeutic effects of bone marrow-derived mesenchymal cells in children with Osteogenesis imperfecta. Nat Med 1999;5:309–313.
Gao J, Dennis JE, Muzic RF et al. The dynamic in vivo distribution of bone marrow-derived mesenchymal stem cells after infusion. Cells Tissues Organs 2001;169:12–20.
Devine SM, Bartholomew AM, Mahmud N et al. Mesenchymal stem cells are capable of homing to the bone marrow of non-human primates following systemic infusion. Exp Hematol 2001;29:244–255.
Horwitz EM, Prockop DJ, Gordon PL et al. Clinical responses to bone marrow transplantation in children with severe Osteogenesis imperfecta. Blood 2001;97:1227–1231.
Mets T, Verdonk G. In vitro aging of human bone marrow derived stromal cells. Mech Ageing Dev 1981;16:81–89.
D’Ippolito G, Schiller PC, Ricordi C et al. Age-related osteogenic potential of mesenchymal stromal cells from human vertebral bone marrow. J Bone Miner Res 1999;14:1115–1122.
Bruder SP, Jaiswal N, Haynesworth SE. Growth kinetics, self-renewal and the osteogenic potential of purified human mesenchymal stem cells during extensive subcultivation and following cryopreservation. J Cell Biochem 1997;64:278–294.
Digirolamo CM, Stokes D, Colter D. Propagation and senescence of human marrow stromal cells in culture: a simple colony-forming assay identifies samples with the greatest potential to propagate and differentiate. Br J Haematol 1999;107:275–281.
Banfi A, Muraglia A, Dozin B et al. Proliferation kinetics and differentiation potential of ex vivo expanded human bone marrow stromal cells: implications for their use in cell therapy. Exp Hematol 2000;28:707–715.
Stenderup K, Justesen J, Clausen C et al. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow cells. Bone 2003;33:919–926.
Rombouts WJC, Ploemacher RE. Primary murine MSC show highly efficient homing to the bone marrow but lose homing ability following culture. Leukemia 2003;17:160–170.
Wright WE, Shay JW. Historical claims and current interpretations of replicative aging. Nat Biotechnol 2002;20:682–688.
Blackburn EH. Switching and signaling at the telomere. Cell 2001;106:661–673.
Harley CB, Futcher AB, Greider CW. Telomeres shorten during ageing of human fibroblasts. Nature 1990;345:458–460.
Allsopp RC, Harley CB. Evidence for a critical telomere length in senescent human fibroblasts. Exp Cell Res 1995;219:130–136.
Reyes M, Lund T, Lenvik T et al. Purification and ex vivo expansion of postnatal human marrow mesodermal progenitor cells. Blood 2001;98:2615–2625.
Friedenstein AJ, Luria E, Molineux G. Assays of the haemopoietic microenvironment. In: Testa NG, Molineux G, eds. Haemopoiesis. New York: Oxford University Press, 1993:189–198.
Jaiswal N, Haynesworth SE, Caplan AI. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295–312.
Nuttall MSC, Patton AJ, Olivera DL. Human trabecular bone cells are able to express both osteoblast and adipocytic phenotype: implications for osteopenic disorders. J Bone Miner Res 1998;13:371–382.
Page K, Stevens A, Lowe J. Bone. In: Bancroft JD, Stevens A, eds. Theory and Practice of Histological Techniques. NewYork: Churchill Livingston, 1996;309–340.
Bayliss High OB, Lake B. Lipids. In: Bancroft JD, Stevens A, eds. Theory and Practice of Histological Techniques. NewYork: Churchill Livingston, 1996:213–242.
Wynn RF, Cross MA, Hatton C et al. Accelerated telomere shortening in young recipients of allogeneic bone-marrow transplants. Lancet 1998;351:178–181.
Barry FP, Boynton RE, Haynesworth S et al. The monoclonal antibody SH-2, raised against human mesenchymal stem cells, recognizes an eptitope on endoglin (CD105). Biochem Biophys Res Commun 1999;265:134–139.
Caplan AI. Mesenchymal stem cells. J Orthop Res 1991;9:641–650.
Deans RJ, Moseley AB. Mesenchymal stem cells: biology and potential clinical uses. Exp Hematol 2000;28:875–884.
Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sc 2000;113:1161–1166.
Colter DC, Sekiva I, Prockop DJ. Identification of a subpopulation of rapidly self-renewing and multipotential adult stem cells in colonies of human marrow stromal cells. Proc Natl Acad Sci U S A 2001;98:7841–7845.
Von Zglinicki T. Telomeres and replicative senescence: is it only length that counts? Cancer Lett 2001;168:111–116.
Lundblad V. Telomere maintenance without telomerase. Oncogene 2002;21:522–531.
Bianchi G, Banfi A, Mastrogiacomo M. Ex vivo enrichment of mesenchymal cell progenitors by fibroblast growth factor 2. Exp Cell Res 2003;287:98–105.
Greider CW, Blackburn EH. Identification of a specific telomere terminal transferase activity in Tetrahymena extracts. Cell 1985;43:405–413.
Simonsen JL, Rosada C, Serakinci 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.
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.
Zimmermann S, Voss M, Kaiser S et al. Lack of telomerase activity in human mesenchymal stem cells. Leukemia 2003;17:1146–1149.
Masutomi K, Yu EY, Khurts S et al. Telomerase maintains telomere structure in normal human cells. Cell 2003;114:41–253.(Melissa A. Baxtera, Rober)