Bone 70 (2015) 37–47

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Bone journal homepage: www.elsevier.com/locate/bone

Review

Characterization of bone marrow-derived mesenchymal stem cells in aging Natasha Baker a, Lisa B. Boyette b, Rocky S. Tuan a,c,⁎ a b c

Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA Thomas E. Starzl Transplantation Institute, Department of Surgery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA McGowan Institute for Regenerative Medicine, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA

a r t i c l e

i n f o

Article history: Received 18 July 2014 Revised 16 October 2014 Accepted 22 October 2014 Available online 28 October 2014 Edited by: Paolo Bianco Keywords: Bone marrow mesenchymal stem cells Aging

a b s t r a c t Adult mesenchymal stem cells are a resource for autologous and allogeneic cell therapies for immunemodulation and regenerative medicine. However, patients most in need of such therapies are often of advanced age. Therefore, the effects of the aged milieu on these cells and their intrinsic aging in vivo are important considerations. Furthermore, these cells may require expansion in vitro before use as well as for future research. Their aging in vitro is thus also an important consideration. Here, we focus on bone marrow mesenchymal stem cells (BMSCs), which are unique compared to other stem cells due to their support of hematopoietic cells in addition to contributing to bone formation. BMSCs may be sensitive to age-related diseases and could perpetuate degenerative diseases in which bone remodeling is a contributory factor. Here, we review (1) the characterization of BMSCs, (2) the characterization of in vivo-aged BMSCs, (3) the characterization of in vitro-aged BMSCs, and (4) potential approaches to optimize the performance of aged BMSCs. This article is part of a Special Issue entitled “Stem Cells and Bone”. © 2014 Elsevier Inc. All rights reserved.

Contents Introduction . . . . . . . . . . . . . . . . . Characterization of BMSCs . . . . . . . . . . BMSCs and aging in vivo . . . . . . . . . . . Characteristics of BMSCs aged in vivo . . . . . Aging and immunological properties of BMSCs . Characterization of in vitro-aged BMSCs . . . . Tools to monitor and prevent in vitro BMSC aging Conclusion . . . . . . . . . . . . . . . . . Acknowledgement . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . .

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Introduction Non-hematopoietic multipotent cells in the stromal compartment of the bone marrow were first identified by Friedenstein and colleagues [1]. These cells have high proliferative potential and the ability to differentiate into chondrocytes, osteoblasts, adipocytes [2], and stromal cells that support hematopoiesis [3]. Furthermore, they have ⁎ Corresponding author at: Center for Cellular and Molecular Engineering, Department of Orthopaedic Surgery, University of Pittsburgh School of Medicine, 450 Technology Drive, Room 221, Pittsburgh, PA 15219-3143, USA. Fax: +1 412 624 5544. E-mail address: [email protected] (R.S. Tuan).

http://dx.doi.org/10.1016/j.bone.2014.10.014 8756-3282/© 2014 Elsevier Inc. All rights reserved.

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37 38 38 39 41 44 44 45 45 46

immunomodulatory activity, which may contribute to immunesuppression and tissue healing [4]. Most commonly known as “mesenchymal stem cells” or “multipotent mesenchymal stromal cells” (MSCs), cells labeled as MSCs have been isolated from a variety of extramedullary tissues, are widely believed to serve as a reserve to replace damaged and aged cells, and are well recognized for their potential use in tissue-regenerative cell therapies [5–10]. Importantly, the regenerative function of tissue resident MSCs is said to decline after 30 years of age [6], and their exhaustion is recognized as a potentially important component of aging [11,12]. Our focus here is on the effects of aging on MSCs in the bone marrow, which we refer to hereon as BMSCs.

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BMSCs may be a particularly important MSC population to consider for three reasons. First, their activity supports that of another distinct population of progenitor cells that give rise to cells of the blood and immune system: hematopoietic progenitors (reviewed in [10]). Second, BMSCs could be used to encourage the repair of extramedullary tissues (reviewed in [13]). Third, BMSCs may influence bone growth and remodeling. They form the osteoblasts that deposit osteoclacin in mineralized bone tissue, and through paracrine stimuli can regulate osteoclasts, which orchestrate osteocalcin release and activation through bone resorption [14,15]. The central integration of bone marrow activity in the body places BMSCs in a position that may be particularly sensitive to aging and age-related diseases. Reciprocally, changes in BMSC activity could contribute to aging in both bone marrow and extramedullary tissues. Therefore, it is vital that we understand more about the biology of BMSCs in the context of aging. Here, we review (1) the characterization of BMSCs, (2) the characterization of in vivo-aged BMSCs, (3) the characterization of in vitro-aged BMSCs, and (4) potential approaches to optimize the performance of aged BMSCs. Characterization of BMSCs The International Society for Cellular Therapy (ISCT) suggested the following criteria for the identification of MSCs: (1) adherence to plastic; (2) differentiation into chondrocytes, osteoblasts, and adipocytes under standard in vitro differentiating conditions; and (3) expression of surface markers CD105, CD73, and CD90, in the absence of CD45, CD34, CD14, CD11b, CD79α, CD19, and HLA-DR [16]. However, these criteria, while convenient, have not been completely helpful to the research community because they are based on artificial in vitro observations. The immunological markers in particular have not been very helpful, especially for the research of BMSC behavior in vivo, as the MSC immunophenotype may be altered artificially by in vitro culture (reviewed in [19]). Meanwhile, other potential positive and negative antigenic markers have been suggested. With contrasting combinations of MSC markers described by different researchers, challenges remain in the field regarding BMSC characterization [10,17,18]. The confusion in the literature may be resolved if BMSCs represent a heterogeneous population of progenitors at different stages of differentiation. Indeed, oscillations in immunophenotype characteristics are known to occur during developmental osteogenesis in the chick tibia [5] and BMSCs may reside in three distinct niches within bone, namely, endosteal, stromal and perivascular niches, with different immunophenotypic features within each (reviewed in [19]). Conversely, some researchers have argued that MSCs are a homogeneous population [20]. Those who argue MSCs are a homogeneous population have found CD271 to be the most consistent marker of these cells [20]. However, these authors also admit that levels of CD146 expression are not uniform across the population. Sacchetti and colleagues have shown that at least one subpopulation of CD146+ CD45- cells can be enriched from human BMSC preparations. This CD146+ population is capable of self-renewal and reconstitution of bone and a hematopoiesissupporting stroma when transplanted into mice [3]. This is important because in vivo assay of cellular activity is the most reliable means of BMSC characterization. CD146+ could therefore be one characteristic marker of BMSCs or of a subpopulation of early BMSC progenitor cells. Other researchers have identified CD146 as a marker specific to BMSCs compared to hematopoietic stem cells (HSCs), and shown the in vitro chondrogenesis, osteogenesis and adipogenesis of CD146+ BMSCs [21]. Furthermore, an age-related decline in BMSCs expressing CD146 has been reported [22] (For further information on novel MSC markers, see reviews by [10,17,18]). It has also been suggested that a non-adherent multipotent BMSC/ HSC precursor may exist, although this is a more controversial idea. Dominici et al. showed that in mice, a subpopulation of non-plastic adherent bone marrow cells could reconstitute the hematopoietic system

in lethally irradiated mice, and also form osteoblasts [23]. They demonstrated that formation of the latter was not a result of cell fusion events by performing cell karyotype analysis. Meanwhile, other investigators have reported isolation of non-plastic adherent bone marrow cells with osteogenic potential [24,25]. In mice, cells with osteogenic potential may be mobilized from the bone marrow to the peripheral circulation to contribute to bone fracture repair in remote locations [23, 26–28]. Cells with osteoblastic potential have also been identified in the circulation of humans [29]. These cells may be relevant to aging and regenerative medicine, as they were reported to increase after bone fracture in humans and are more abundant in the peripheral blood of boys undergoing pubertal growth than in that of men. Others argue that if they exist, these cells are so rare that their relevance in tissue repair is questionable, especially if tissue specific MSCs are present [20]. In summary, there is still not a consensus view on the characterization and lineage markers of BMSCs, and it is possible that BMSCs represent a heterogeneous population with some lineage hierarchy (Fig. 1). If so, it is important to note that the current literature on BMSC aging may encompass age-related changes to subpopulations of BMSCs that are as yet uncharacterized from other populations. With this idea in mind, it is interesting to note that aging causes changes to the clonal composition of the HSC compartment, with a shift towards myeloid bias [30]. Also, immune function has been documented to decline across many different cell types with aging, suggesting that a more inflammatory cytokine milieu may exist in patients of advanced age. This may mean that BMSCs reside in a biologically more challenging environment in people of advanced age, who are most in need of cellular therapies. BMSCs and aging in vivo Experiments in mice suggest that the aged milieu can suppress the function of adult stem cells [31–36]. Serum isolated from old compared to young mice can suppress the in vitro proliferation of stem cells [33]. If expanded beforehand, human embryonic stem cells (hESCs) have at least some capacity to antagonize this effect, suggesting that they produce some factor(s) that can neutralize the anti-proliferative factors in aged serum. Conversely, adult satellite cells appear to lack this ability [33]. Meanwhile, muscle-derived stem progenitor cells (MDSPCs) from aged mice or a mouse progeria model have declined regenerative functions as measured by in vitro studies [37]. More importantly, in vivo, administration of MDSPCs from young (14- to 21-day-old) mice prevents tissue degeneration and extends the life span and health span of aged mice [37]. These studies suggest that there may be a causal relationship between stem cell aging and organismal aging. Further research is required to confirm and characterize such a relationship, and it remains to be determined if BMSCs are involved in the pathogenesis of organismal aging. It could be hypothesized that BMSCs are susceptible to the hostility of an aged in vivo environment, and reciprocally, that BMSC aging may contribute to organismal aging/age-related diseases. For example, it has been speculated that osteoporosis could in part be caused by a deficiency in BMSC osteogenesis [38,39]. This is supported by BMSC atrophy in mouse models of bone aging [15]. However, others have reported no difference between BMSC populations from osteoporosis patients and healthy donors [40]. Disruption of BMSC function may also cause accelerated aging associated with metabolic syndrome, a disorder of energy utilization (see review by [42]). It is speculated that stem cells may become exhausted by demands for adipogenesis in this syndrome, as metabolic syndrome is also observed in lipodystrophies, which are diseases of adipose tissue degeneration [42]. Meanwhile, type 2 diabetes and prediabetes, which are observed in metabolic syndrome, may also cause BMSC dysfunction through the generation of advanced glycation endproducts (AGEs). These may accumulate in bone matrix [43] and can suppress proliferation, induce apoptosis, increase intracellular reactive oxygen species (ROS) production, and suppress matrix mineralization

N. Baker et al. / Bone 70 (2015) 37–47

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Fig. 1. Origins of bone marrow mesenchymal stem cells. Multipotent mesenchymal stem cells (MSCs) reside in the bone marrow with hematopoietic stem cells (HSCs) and their progeny. The subpopulations in the hematopoietic lineage are numerous and well-defined (see review by [41]). This lineage gives rise to circulating blood and immune cells, and osteoclasts that resorb bone. It is likely that MSC populations are also comprised of subpopulations of progenitor cells not yet defined. A common progenitor for both the HSC lineage and the MSC lineage may exist, but this remains controversial. Non-adherent osteoprogenitor cells have also been reported. These may be mobilized for distal bone growth and/or fracture repair, and perhaps represent a subpopulation in the MSC lineage.

during osteogenesis in BMSCs in vitro [44]. Similar metabolic changes are also observed in Hutchinson–Gilford progeria syndrome (HGPS), which is a severe disease of accelerated aging [45]. HGPS is caused by mutation of LMNA, which encodes Lamin-A [45,46], a nuclear scaffolding protein that also has a role in substrate-directed BMSC differentiation [47]. Collectively, these observations suggest a working theory in which BMSC dysregulation and interaction with the aged ECM could participate in aging processes associated with metabolic syndrome (Fig. 2). Further work is indicated to conclusively determine which of these pathways are most relevant to the human aging process in vivo. Characteristics of BMSCs aged in vivo The number of clonal cell colonies produced by bone marrow aspirates can be used to determine BMSC frequency in bone marrow.

Many studies have utilized this colony forming unit-fibroblast (CFU-F) assay to test for altered BMSC frequency with in vivo age. Studies have also compared the immunophenotypes, proliferation, differentiation, immune-modulation capacities, gene expression, migration, and adhesion of “young” versus “old” BMSCs. In 2006, Sethe et al. [48] published a comprehensive review of many of these studies, with an excellent commentary on the plethora of experimental variables that may be responsible for the conflicting results obtained from them. In general, most studies in both humans and rodents have reported a decline in the frequency of CFU-Fs with the biological age of the bone marrow [48]. Subsequent studies agree with this consensus [49–51] and suggest that this may be due to increased susceptibility to senescence [49,52]. However, one study reported that in mice this age-related decline in BMSC frequency is only marginal compared to other aging effects on BMSCs (discussed further below) [53], while others have documented

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N. Baker et al. / Bone 70 (2015) 37–47

Fig. 2. BMSCs, metabolism, and aging. (A) Some normal homeostatic mechanisms affecting BMSCs: The rigidity of the cell matrix, via effects on mechanosensory pathways involving nuclear Lamin-A, may help to determine BMSC differentiation into adipocytes and osteoblasts, in combination with soluble growth factors. Osteogenesis leads to bone formation and storage of osteocalcin in mineralized tissue, while adipogenesis leads to the storage of energy in fatty acids. In times of high calorie intake, insulin signals the storage of glucose in sugar and fat and encourages bone remodeling (and hence bone growth) by preventing osteoblast inhibition of osteoclasts by osteoprotegerin secretion. Osteoclasts release active osteocalcin from bone, and osteocalcin triggers the release of insulin in a positive feedback loop. Leptin is the regulator of this loop. In the brain, leptin causes the suppression of serotonin signaling, and this leads to appetite suppression and de-repression of osteoblast activity by the sympathetic nervous system. Thus, leptin favors the conservation of bone mineral matrix and utilization of energy, while insulin favors energy storage and bone remodeling. For more information see review by [14]. (B) In the case of insulin resistance, blood glucose levels rise and advanced glycation end products (AGEs) can accumulate in the bone matrix. This may disrupt BMSC activity and mechanosensing. Disruption of BMSC Lamin A signaling in lipodystrophies may have similar effects. In both cases, the formation of adipocytes may be compromised, leading to an over-demand for adipogenesis, BMSC exhaustion, and metabolic syndrome.

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Table 1 BMSC proliferation characteristics as a function of age. Speciesa

“Y”a

“O”a

Hu Hu

1–5 years 2–13 years

Sample size

Findings in association with aging

50–70 years 20–50 years

Decline in proliferation rate Significant decline in cumulative population doubling No differences in proliferation rates

80–92 years

n = 6/group “Y” = 8 “O” = 17 “Y” = 11 “M” = 26 “O” = 13 n = 4/group

Hu

b45 years

45–65 years

N65 years

Hu

21–25 years

44–55 years

Rh

b5 years

8–10 years

N12 years

n = at least 4 /group

M

6 days

6 weeks

12 months

n = 3/group

M

b1 month

1–3 months

N3 months

n = at least 3 /group

M M M Rt

3–4 months 3–6 months 3 months 3 weeks

23–24 months 24 months 18 months 12 months

unclear n = 6/group n = 5/group

S

b3 days

4–4.5 years

n = 3/group

a

“M”a

12–18 months

No difference in maximal number of population doublings Decline in proliferation rate in Y compared to M and O Decline in proliferation rate in Y compared to M and O No difference in proliferation capacity, as measured by cumulative growth index Decline in proliferation rate Increase in proliferation rate Decreased self-renewal No significant differences in proliferation rate up to 100 passages

Notes

Ref. [50] [56] [54].

All cells underwent replicative senescence regardless of age. Increased expression of β-galactosidase. Cells N12 years old reported to senescence after 2 passages in vitro.

[57] [58]

[59] [51] Increase in senescence

Cells reported to remain in culture for up to 100 passages in vitro without reaching senescence, regardless of in vivo age

Decline in proliferation rate

[60]. [55] [53] [52]

[61]

Species: Hu, Human; Rh, Rhesus macaque; M, Mouse; Rt, Rat; S, Sheep; “Y,” Young; “M,” Median Age; “O,” Old.

a larger difference in BMSC abundance in marrow with age [51]. Such discrepancies could be attributed to the methodology employed for the CFU-F assay, which must use a single cell suspension to ensure colonies are clonal. This can be achieved with dilution plating. One study reports using this [54]. Some studies have highlighted that species and gender are important variables determining CFU-F frequency. Siegel et al. (2013) examined 50 different human BMSC populations. In analysis of mixed gender populations, those from young donors (b 45 years) yielded significantly more CFU-Fs than those from middle-aged (45–65 years) and old (N65 years) donors [54]. However, when analysis was performed on cells of the same gender, only young female MSCs yielded more CFU-Fs than older cells [54]. In fact, regardless of age, female cell populations yielded more CFU-Fs than male populations [54]. Similarly, Katsara et al. [51] calculated the frequency of BMSCs to be 3.5 fold higher in the bone marrow of young (b1-month-old) mice than in the

bone marrow of old (N3months old) mice, but conversely, found that female mice yielded fewer CFU-Fs than males in each age group. Meanwhile, there is also disagreement regarding the effects of in vivo aging on the proliferative capacity of BMSCs. Up to 2006, the majority of studies reported a decrease in the proliferation capacity and proliferation rate with the biological age of BMSCs [48]. Table 1 summarizes the conclusions of studies since 2006. Some studies have found no difference in the in vitro proliferative capacity of “young” and “old” BMSCs. However, it is notable that those studies also compared groups that were closer in age than in other studies (see Table 1). Meanwhile, one study in mice noted an increase in proliferation rate with in vivo age [55]. Aging and immunological properties of BMSCs Other recent studies have compared the frequency of immunophenotypic antigens in chronologically young and old BMSCs, but

Table 2 Immunological marker frequency in BMSCs as a function of age. Speciesa

“Y”a

Hu

“O”a

Sample size

Findings in association with aging

1–5 years

50–70 years

n = 6/group

Hu

2–13 years

20–50 years

“Y”= 8 “O”= 17

Hu

21–25 years

44–55 years

80–92 years

n= 4/group

Hu

- 4–5 months

0–16 years

19–86 years

“Y”= 6 “M”= 27 “O”= 53

Hu

b45 years

45–65 years

N65 years

“Y”= 11 “M”= 26 “O”= 13

No significant differences in the expression of CD29, CD90, CD105, CD45, CD34, CD133 No differences in the expression of CD45, CD14, CD90, CD29, CD44, CD105, CD106, and CD166 No differences in the expression of CD13, CD29, CD44, CD73, CD90, CD105, CD146, CD166, CD31, CD34, CD45 Gain in CD271 in “M” and “O” Loss of CD146 in “O” Three distinct subpopulations noted: CD271– CD146+ (in Y only) CD271bright CD146+ (dominant in Y and M) CD271bright CD146– (dominant in O) Lower expression per cell: CD71, CD90, CD106, CD140b, CD146, CD166 and CD274

Rh

b5 years

8–10 years

N12 years

n= 3/group

M

b1 month

1–3 months

N3 months

n = at least 3/group

a

“M”a

Lower CD90 expression Higher CD44 expression than in “Y” Higher CD73 expression Than in “Y”

Species: Hu, Human; Rh, Rhesus macaque; M, Mouse; Rt, Rat; S, Sheep; “Y,” Young; “M,” Median Age; “O,” Old.

Notes

Ref. [50] [56]

[57]

“Y” = Fetal, 15–20 weeks gestation “M” = all had pediatric malignancies, 10 of which were hematological “O” =10 healthy, 43 undergoing cardiac surgery

[22]

Immunogenicity varied with gender, more cells from female BMSC harvests were CD119+ and CD130+ than cells in male BMSC harvests

[54].

[58] [51]

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there is not an obvious pattern in their results (Table 2). Furthermore, most of these studies were performed on cells that had undergone multiple passages in culture up to ten times, and in vitro culture alters the expression of cell surface antigens. Only two studies listed in Table 2 performed immunophenotypic analysis on early passage (P1–P2) cells, but both noted a decrease in the expression of CD90 with increasing age of the primate (human and non-human) subjects [54,58]. Therefore, loss of CD90 is one possible candidate marker for BMSC aging, but much further work is required in this area since BMSC characterization is in itself still a challenge. In particular, more characterization needs to be performed on BMSCs in vivo within their native environment. Another immunological marker to explore may be loss of CD146 expression. For example, although their results did not show ageassociated differences in human BMSC proliferation rates, Siegel et al. observed that within human BMSC isolates, a subpopulation of smaller and more proliferative cells exists, which is positive for CD146, a known perivascular marker, and may occur with higher frequency in young female MSC donor populations [54]. Perhaps this is the same subpopulation of BMSCs isolated by Sacchetti and colleagues [3], and/or one of the BMSC CD146+ subpopulations found to decline with human development/age by Maijenburg and colleagues [22]. Aging also appears to alter BMSC differentiation potential measured in vitro, but whether this occurs in vivo is still debatable. Most studies of the relationship between BMSC age and differentiation potential have only assayed in vitro differentiation. Previously, it was thought that with age, BMSCs lost osteogenic potential and gained adipogenic potential. However, data have been produced both to support and argue against this hypothesis. In 2006, Sethe et al. [48] could not make a conclusive comment regarding age-related effects on BMSC differentiation. Furthermore, Table 3 summarizes the findings from studies since 2006. One of these studies compared in vivo bone formation between “old” and “young” murine BMSCs and found that old BMSCs had significantly reduced in vivo ectopic ossification potential [53]. However, whether this occurs in humans and the relevance of this in relation to agerelated loss of bone mineral density remains to be determined. With the exception of one study in rat BMSCs, those studies listed in Table 3 that did not find any differences in the in vitro differentiation capacities between “young” and “old” BMSCs compared groups that were closer in age than in other studies. Two studies in different species documented a progressive decline in BMSC in vitro differentiation capacity

with age, with chondrogenic and osteogenic differentiation capacities declining earlier than adipogenesis. This may explain the previous notion that osteogenic potential is lost with age in favor for adipogenesis and may agree with the age-related decline in in vivo osteogenesis of murine BMSCs. Finally, it is important to note that age-related disease could potentially skew data regarding the characteristics of in vivo aged BMSCs, especially as many human BMSCs are isolated from the tissue discarded after total joint replacement and hence are probably derived from individuals with age-related degenerative disease. While studies comparing the proliferation and differentiation potentials of BMSCs of different in vivo ages are numerous and somewhat conflicting, those comparing gene expression are fewer and so far have yielded no common findings at all. In a comparative study of human BMSCs from 4 young donor sources (21–25 years old), 4 median-aged donor sources (44–55 years old), and 4 old donor sources (80– 92 years old) all at in vitro passage 2, 67 genes were found to be upregulated with age [57]. Most of these genes were related to regulation of the extracellular matrix, mesoderm development, synaptic vesicle endocytosis and chemotaxis, and included mesenchyme homeobox 2 (MEOX2), which represses the proliferation of mesodermal tissues and is affected in the fibroblasts of those with HGPS, and short stature homeobox 2 (SHOX2), which is thought to cause idiopathic short stature [55]. Meanwhile, 60 genes were found to be down-regulated with age. These genes were mostly in functional categories associated with DNA repair, mitosis, and transcriptional regulation, including various homeobox genes and peroxisome proliferator-activated receptor γ (PPARG) [57]. In a different study of human BMSCs isolated from cadaveric vertebral bones, BMSCs from a 60-year-old donor expressed lower levels of the tumor necrosis factor α receptor 1 (TNFR1), IL-6 receptor (IL6R), and interferon γ receptor 1 (IFNGR1) than BMSCs from a 23-year-old human donor [62]. However, this was only a comparison between two donor cell populations, and there is recognized donorto-donor variability in human BMSC populations and even within sampling of the same donor (reviewed in [19]). These results must therefore be viewed with caution. Meanwhile, in a study of BMSCs from young(b5 years old), middle- (8–10 years old), and old- (N12 years old) aged rhesus macaques, expression of many heat shock proteins (HSPs) was found to decline progressively with age, and the expression of various micro RNAs (miRNAs) was also altered with age [58]. Twenty-five percent of miRNAs probed were up-regulated with age, while

Table 3 Differentiation potential of BMSCs as a function of age. Speciesa “Y”a

“M”a

“O”a

Sample size

Findings in association with aging

45–65 years

N65 years

“Y” =11 “M” =26 “O” =13 n = 4/group “Y” = up to 9 “M” = up to 4 “O” = up to 9

No difference in differentiation capacities

Hu

b45 years

Hu Rh

21–25 years 44–55 years b5 years 8–10 years

80–92 years N12 years

M

6 days

6 weeks

12 months

M

b1 months

1–3 months

N3 months

M M

3 months 3–6 months

18 months 12–18 months 24 months

Rt

4 months

15 months

Rt

3 weeks

12 months

a

No difference in differentiation capacities Progressive decline in osteogenic and adipogenic potential, with osteogenic potential reaching a significant reduction at an earlier age than adipogenic potential n = 3/group 6 weeks: Diminished chondrogenic and (6 for adipogenesis) osteogenic differentiation 12 months: Diminished chondrogenic, osteogenic, and adipogenic differentiation n = at least 3/group No gross differences in adipogenesis and osteogenesis observed Decreased osteogenic capacity n = 6/group 12–18 months: Increased osteogenic differentiation 24 months: Decreased osteogenic differentiation 2 male, 2 female/group Dramatic reduction in osteo-, adipo-, and chondro-genesis n = 5/group No difference in osteogenic and adipogenic potential

Species: Hu, Human; Rh, Rhesus macaque; M, Mouse; Rt, Rat; S, Sheep; “Y,” Young; “M,” Median Age; “O,” Old.

Notes

Ref. [54].

[57] [58]

[59]

Gender differences noted

[51] [53] [55]

[63] Cells reported to remain in culture for up [52] to 100 passages in vitro without reaching senescence, regardless of in vivo age

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38% were down-regulated [58]. It is also interesting to note that out of five core circadian clock genes assayed, the mRNA for one, Rev-erb α, was significantly different in old and young cells over the duration of 2 days, with younger cells expressing significantly lower basal levels [58]. Global gene expression analysis of young versus old BMSCs in a mouse model revealed changes in differentiation, cell cycle, and growth factor genes. Specifically, there was a decline in expression of regulators of adipogenesis, tumor suppressors p53 and p21, and hepatocyte growth factor (HGF) and VEGF [64]. In a study of passage, 0 BMSCs from 3-month-old and 24-month-old C57BL/6 mice, increased age was associated with a general decrease in gene expression (with about 90% of 927 genes altered with age down-regulated). This ageassociated down-regulation affected genes involved in migration and osteogenic differentiation, with Col1a1 expression down-regulated 83-fold, and particularly genes involved in inflammatory responses (e.g., cytokine receptors) [62]. An age-related decline in expression of inflammatory response genes may indicate that BMSCs undergo an age-related decline in their immunomodulatory activity. However, Siegel et al. [54] found that biological age did not affect the ability of human BMSCs to suppress proliferation of activated allogeneic T-cells in vitro. Similarly, in vivo age has not been found to affect suppression of in vitro activated T-cell proliferation by murine BALB/c BMSCs [51]. Interestingly, older BMSCs cocultured with activated T-cells were found to secrete more IL-6 than younger cells co-cultured with activated T-cells [54]. This may have in vivo significance because IL-6 is an osteoclastogenic stimulus [65], and BMSCs from a mouse model of early aging secrete higher levels of IL-6 and have higher osteoclastogenesis-inducing activity [15]. There are age-associated changes in BMSC activity that do seem to agree in different species. For example, in vivo age is associated with a significant reduction in the antioxidant capacity of rat BMSCs [52]. This is in agreement with an increase in ROS accumulation seen with rhesus macaque BMSC aging, which is concomitant with the decline in expression of many HSPs with age [58]. Furthermore, in vivo aging of rat BMSCs is associated with a significant reduction in basal migration capacity [52]. This agrees with a reduction in in vitro wound healing of murine BMSCs from 23- to 24-month-old C57BL/6 mice compared to those from 3- to 4-month-old mice [60]. Finally, BMSCs from 6-dayold and 6-week-old mice are significantly more adhesive than those from 1-year-old mice [59], while BMSCs from 3-month-old mice secrete an extracellular matrix (ECM) that is more proliferation- and osteogenesis-promoting than that from older mice [53]. Collectively, these data suggest that general age associated changes may occur in BMSC adhesion, migration, resistance to oxidative stress, and cytokine and ECM secretion. These changes may have important consequences on tissue healing mediated by BMSCs, through their homing, migration, and paracrine stimulation of other cells. These are important considerations for use of in vivo-aged BMSCs for cellular therapies. While the possible uses of BMSCs as cellular therapies for different clinical problems are still under investigation, it does appear that aged BMSCs generally perform less well than their younger counterparts in various disease models [50,62,66–70]. There is a great deal of interest in the clinical application of stem cell-based therapies, with no less than twenty ongoing clinical trials exploring the role of these cells in treating cardiovascular disorders alone [71]. The disease models in which most comparisons have been made between the therapeutic efficacy of old and young BMSCs are myocardial infarction (MI) models. After surgical induction of MI by coronary artery ligation, Fan et al. (2010) found that cardiac function recovered better in immunosuppressed rats that 15 minutes later received injection of human BMSCs from young donors (1–5 years old) as compared to rats that received BMSCs from old donors (50–70 years old) or control medium alone [50]. The same group repeated these results in a separate study, except with BMSC injection 30 minutes after coronary artery ligation, and found that old BMSCs did not suppress TNF-α production in the post-

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MI myocardial tissue to the same extent as young BMSCs [66]. In a similar model, Kang et al. (2012) measured the effectiveness of human BMSCs seeded on collagen patches for surgical ventricular restoration 2 weeks after MI [67]. They found that BMSCs from 4 human donors over 66 years old were less effective at restoring cardiac function than BMSCs from 4 human donors under 57 years old. Also in a similar model, Zhang et al. (2005) reported that older (2-year-old) rat BMSCs do not improve cardiac function better than medium injection alone when introduced 3 weeks after injury [68]. These authors reported that this was in contradiction to the results they had previously found with younger cells, although they did not directly compare young and old rat BMSCs in the same study. Meanwhile, cardiac recovery was also reported to be better in C57BL/6 mice that received BMSCs from young (2-month-old) versus old (18-month-old) donor mice after induced MI [69]. Myocardial repair is not the only domain in which young BMSCs have outperformed old BMSCs in disease models. In female mice in which acute respiratory distress syndrome (ARDS) is induced through intraperitoneal injection of Escherichia coli lipopolysaccharides (LPS), intravenous administration of young (3-month-old) murine BMSCs significantly reduces lung inflammation as compared to when old (24-month-old) cells are administered [62]. Furthermore, when ARDS is induced in wild-type mice sharing a parabiotic circulation with GFP+ mice, it is evident that fewer cells are recruited from old GFP+ mice to the injured lungs of wild type mice than are from young GFP+ mice [62]. The reduced recruitment of GFP+ cells from older animals is also associated with increased lung inflammation, and injured animals with old parabionts have more severe injury scores compared to injured animals with young parabionts [62]. Administration of old (60-year-old) and young (23-year-old) human BMSCs in LPS-induced ARDS mice followed a similar trend to these results, with significantly more neutrophil and lymphocyte infiltration in the lungs of animals that received older human BMSCs than in the lungs of animals that received younger human BMSCs [62]. However, only one young donor population and one old donor population were used for the human BMSCs in this study [62]. Despite the results discussed above in cardiovascular disease models, the age of BMSCs appears not to be a significant variable for all applications. Another area where there is much clinical interest in using BMSCs for tissue repair is in joint tissue regeneration. For example, clinical trials are ongoing around the world to evaluate the role of these cells in aiding cartilage regeneration, primarily in the knee [72]. Dressler et al., examined the differences between 1-year-old and 4-year-old autogenic BMSCs seeded in collagen type I gels in the repair of defects induced in the contralateral patellar tendons within 4-yearold rabbits [70]. No significant differences in tissue repair were measured between defects treated with gel implants containing 1-year-old BMSCs and those containing 4-year-old BMSCs. However, it is important to note that the 1- year-old BMSCs underwent a 3-year period of cryopreservation, and there was a trend toward decreased width, thickness, and mechanical properties including stiffness, grip-to-grip strain energy, and maximal force, in repair tissue from the defects treated with 4-year-old BMSCs, although the differences were not significant. The authors also acknowledged that such trends could have been observed due to surgical variability. Nevertheless, this study was important because it did allow the comparison of BMSCs from the same animal at different in vivo ages and eliminated variables associated with allogeneic studies. Interestingly, the authors also reported that the quality of repair in general in the 4-year-old rabbits may have been inferior to that seen previously in studies with 1-year-old rabbits. Although this could be due to experimental variables, it is possible that the aged environment may have more of a negative effect on repair. Further investigations on BMSCs from donors of advanced age (N60), i.e., individuals who will be most frequently in need of BMSCbased regenerative therapies, are required to determine whether ageassociated changes in the function of BMSCs can be overcome by

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pampering them with optimized culture conditions post-harvest. It is important to consider that differences in donor age may also be translated to differences in the ability of BMSCs to withstand the stresses of long-term expansion culture in vitro. This is especially important for the screening of BMSC populations for potential use as cellular therapies in the future, especially considering the desire to use autologous cells for these therapies when possible. Below, we review the effects of in vitro aging on the function of BMSCs. Characterization of in vitro-aged BMSCs There is significant controversy regarding whether the effects observed as a result of long-term cell culture really represent changes associated with cell aging in vivo. Nevertheless, the functional quality of harvested BMSCs may not depend so much on donor age as it does on the conditions of ex vivo culture, and understanding the effects of cellular aging in vitro is necessary for the screening and development of BMSC populations for their potential use as cellular therapies. Furthermore, it is important to consider that differences in BMSC donor age may also be translated to differences in the ability of BMSCs to withstand the stresses of long-term expansion culture in vitro. BMSCs undergoing long-term expansion in culture display enlarged morphology, decreased expression of BMSC-specific surface antigens, and a decline in differentiation potential [73]. Proposed mechanisms underlying these expansion-related changes include changing proportions of subpopulations within a heterogeneous BMSC population, acquisition of mutations and other cellular defects, impairment of selfrenewal resulting in differentiation, telomere loss and resulting proliferative decline, and replicative senescence [73]. Stem cell senescence is a pre-death state in which the cell stops cycling and self-renewal capacity is extinguished. Senescence occurs in many cell types and appears to have evolved as a protective pathway to prevent malignant transformation and cancer formation in cells that have accumulated high levels of exposure to environmental stress. There is no doubt that once in culture, BMSCs undergo replication senescence, by some estimates between 50 and 90 days post-harvest [57,74]. The mechanisms leading to senescence in BMSCs have been explored in cells from systemic lupus erythematosus (SLE) patients that exhibit accelerated functional decline as part of the SLE phenotype. In these cells, expression of the senescence-related gene p16 is increased [75]. Other studies looking at senescence-related genes upregulated in long-term culture have observed increases in not only p16, but also p21 and p53 [76]. The tumor suppressor retinoblastoma (RB) genes have also been shown to play a role in BMSC function and senescence [77], and many cytokines and growth factors impact BMSC proliferation in culture, among them IL-1, IL-3, IL-4, IL-6, IL-8, IL-17, EGF, FGF-2, FGF4, and FGF-8, HB-EGF, HGF, IGF-1, PGDF, RANTES, TGF-β1, TGF-β2, TGFβ3, and VEGF [78]. Other factors that affect BMSC growth in vitro include, but are not limited to, plating density, media and additives such as serum, culture substrates, and ambient oxygen tension. Indeed, resistance to oxidative stress is also critical in BMSC aging and is likely a huge factor in turning on senescence during extended culture. For example, BMSCs exhibit morphological changes in their mitochondria, accompanied by decreased antioxidant capacity and increased intracellular ROS, as a result of serial passaging [49,52]. With serial passaging in culture, BMSCs also exhibit decreased differentiation potential. In particular, the balance between differentiation to the osteogenic versus adipogenic lineages is disrupted, although the direction of this shift is controversial. This change is thought to be driven by accumulated oxidative stress and dysregulation of key differentiation regulatory factors such as Runx2, C/EBPα, and PPARγ [76,79]. Disagreement about whether senescent BMSCs are more or less osteogenic is likely a result of differing culture conditions from lab to lab and the inadequacy of in vitro assays to fully characterize osteogenesis. For example, an increased rate of cell death can result in a higher degree of

alizarin red staining, lending the false appearance of enhanced osteogenic differentiation. In one well-controlled study employing a rat model, extremely long-term passaging resulted in diminished in vitro differentiation potential overall, with complete loss of osteogenic potential and decreased adipogenic potential [52]. However, whether this translates to similar effects in vivo remains to be determined. Defects in BMSC in vitro migratory ability have also been noted by multiple groups as a result of in vitro aging [52], which is not surprising given the declining maintenance of the cytoskeleton and focal adhesion machinery that is observed with serial passaging [49]. Conversely, surface markers expressed by BMSCs in culture include Stro-1, CD13, CD29, CD44, CD73, CD90, CD105, CD106, and CD146 [80], and expression of BMSC surface markers persists even after the loss of differentiation potential in vitro [52,81]. Specific changes in gene expression have been observed during long-term BMSC culture regardless of chronologic age of the cell donor. Generally, these changes include down-regulation of genes related to differentiation, focal adhesion organization, cytoskeletal maintenance, and mitochondrial function [52]. Also, as discussed above, upregulation of the tumor suppressor p16 has been shown to accompany BMSC senescence [82]. Senescence-associated gene expression markers regulated with age in BMSCs also include tumor suppressors like PARG1 and CDKN2B (up-regulated), the growth factor PTN (down-regulated), and MCM3, required for initiation of DNA replication and cell cycle progression (down-regulated) [73]. Studies of gene expression changes upon replicative senescence and of differences in the gene expression of BMSCs from young versus old donors have shown overlap [57]. One study that revealed differential expression of a number of homeobox genes regulating mesodermal proliferation and stature in young versus old BMSC donors (including MEOX2, which is dysregulated in fibroblasts of HGPS patients), found significant overlap between genes up- or down-regulated with donor age and during serial passaging [57]. This suggests that in fact the process of replicative senescence may mirror changes that occur during in vivo aging. The biggest concern in terms of culture-related genomic changes is that BMSCs might undergo malignant transformation during in vitro manipulation or after transplantation/implantation; thus far, this has not been observed in animal studies [52] or clinical trials [73]. Karyotyping and complete genomic hybridization could be used to screen for some changes, but are not sufficiently sensitive. Given the critical role of epigenetic regulation in the developmental program of cells, it is important to consider the effects of cellular aging on the epigenome. Epigenetic dysregulation as a result of BMSC aging has been shown to silence genes involved in self-renewal and result in dysregulated differentiation [83]. Decreased expression of histone deacetylases (HDACs) has been observed in BMSCs undergoing cellular senescence in vitro, a process that can be induced with application of HDAC inhibitors [84]. Analyses of DNA methylation changes upon long-term culture of BMSCs show overall maintenance of methylation patterns but highly significant changes at specific promoter regions, particularly in homeobox genes and regulators of cell differentiation [74]. These changes were found to significantly overlap with changes observed during in vivo aging. In summary, BMSC aging ex vivo, as evidenced by senescence and decreased differentiation capacity, is underpinned by several factors, including telomere shortening, oxidative stress, and less dynamic cytoskeletal remodeling. Specific BMSC functions to monitor in vitro for regenerative applications include proliferation rate, genomic stability, differentiation potential and senescence, as well as paracrine effector secretion and immunomodulatory activity [85,86]. Tools to monitor and prevent in vitro BMSC aging Immunological characterization of BMSCs has been recommended by the ISCT to allow more expanded use of these cells for clinical

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applications, in particular taking advantage of their innate immunomodulatory properties [87]. More definitive characterization, including immunophenotyping, functional assays, and gene expression studies, will be especially important in the context of BMSC aging. Compared to gene expression assays, methylation profiling may actually be a more sensitive approach for monitoring age-related changes, considering that epigenomic studies are always of two copies of DNA and gene expression studies are of many copies of mRNA and thus more difficult to correctly interpret [73]. Also, senescence-associated gene expression signatures have been detected upon serial passaging of BMSCs; such genetic markers may prove useful as biomarkers for monitoring BMSC function in vitro [88]. Genetic engineering of cells is one possible approach for preventing BMSC aging in vitro. Some groups have attempted to combat replicative senescence or improve BMSC potency by induced ectopic expression of telomerase [89,90]. However, this is inadvisable for clinical applications given the small but possible risk of malignant transformation, and/or induced tendency toward osteogenesis [91–93]. Similarly, expression of pluripotency-inducing genes has been used to prolong the selfrenewal and differentiation capacity of BMSCs in long-term culture [94]. In accordance with senescence-associated overexpression of p16, knockdown of p16 in BMSCs from SLE patients rescues their prematurely senescent phenotype [75]. Silencing of RB2, meanwhile, increases BMSC proliferation rate and enhances clonogenicity [77]. However, silencing of RB genes disrupts differentiation to osteogenic, chondrogenic, and adipogenic lineages. Addition of 3% hydrogen gas to expansion cultures has also been shown to extend the replicative life span of BMSCs with maintenance of differentiation potential and paracrine effector secretion; interestingly, this effect was not due to hydroxyl radical scavenging [95]. Antioxidants have been shown, however, to improve BMSC survival and function. In a rat model, BMSCs cultured in serum from aged animals displayed decreased survival and differentiation potential [96]. It was determined that this effect was due to increased intracellular production of ROS and subsequent damage of cell proteins. This effect could be reversed by lowering oxidative stress in in vitro cultures or administering antioxidants in vivo, a result that has been observed by several groups [94]. Another in vitro culture technique that has been shown to prevent age-related changes during expansion is suspension culture; however, only certain subpopulations of BMSCs can be successfully cultured in this manner [97]. Pharmacological approaches and specific metabolites may also be employed as tools to prevent BMSC senescence in culture. For example, histone acetyltransferase (HAT) inhibitors may prevent BMSC senescence associated with decreased HDAC activity [84]. Also, lysophosphatidic acid (LPA), critical for membrane phospholipid synthesis, has been shown to play a role in BMSC senescence, and pharmacologic antagonism of the LPA receptor pathway achieves an anti-aging effect in cultured BMSCs, with enhanced proliferation, clonogenicity, and plasticity [98]. Isothiocyanates, such as those naturally occurring in cruciferous vegetables, have been employed in low doses to delay BMSC aging in vitro, largely through reducing oxidative stress and protecting BMSCs from chemically induced oxidative damage [99]. Select growth factors and medium supplements have also been used to maintain BMSC self-renewal and differentiation potential with limited success. In particular, FGF-2, PDGF-BB, ascorbate, and EGF have been shown to enhance proliferation and delay senescence of BMSCs in vitro, although these treatments did not maintain osteogenic or adipogenic differentiation potential [81,100]. Similarly, BMSC neurotropic activity, which makes BMSCs attractive candidates for stroke therapy and other neurologic therapies but is blunted by extended in vitro culture [101], can be restored with administration of various growth factors [102]. It is important to note, however, that this was effective only after long term culture of young BMSCs and not old BMSCs [102]. Meanwhile, exogenous expression of genes for growth factors such as VEGF has also been used to extend the life span of BMSCs in vitro [90].

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In terms of signaling, it is also noteworthy that another mechanism underlying decreased BMSC differentiation with age that could be addressed in vitro is sirtuin activity. BMSC-specific knockout of SIRT1 results in decreased nuclear accumulation of β-catenin and β-catenindependent gene activation, which is critical for BMSC differentiation [103]. Mechanical stimulation could also be another means of regulating differentiation potential changes in culture. One study showed that mechanical strain represses adipogenesis of a mesenchymal stem cell line in vitro, even in the presence of excess adipogenic supplements [104]. Another reported similar results on mechanical regulation of adipogenesis-related signaling in BMSCs in vitro. The findings showed that mechanical strain repressed C/EBPβ expression, contributing to decreased adipogenesis and increasing the capacity of the endoplasmic reticulum to handle stress, which improved overall cellular health [105]. Considering the suppression of BMSC adipogenesis by mechanical strain in vitro, the question has also arisen as to whether direct mechanical stimulation via forces applied to bone during exercise could play a role in promoting osteogenesis [106]. Finally, enhanced in vitro functions of BMSCs (e.g., proliferation, migration) have been achieved by culturing them on BMSC-derived ECM [107]. In addition, culture on ECM from young animals has been shown to restore self-renewal capacity and osteogenic differentiation of BMSCs from old animals, with a significant reduction of intracellular levels of reactive oxygen species [53]. Much further research is required to determine if such approaches could be effective on MSCs in vivo. However, initial results seem promising. For example, researchers have found that pre-conditioning of older human BMSCs with certain growth factors such as bFGF and VEGF can restore their efficacy to that of younger BMSCs in models of myocardial infarction [66,67]. Conclusion In conclusion, BMSCs are a population of progenitor cells in the bone marrow that can self-renew, differentiate into a limited number of cell types, support hematopoietic cells, and regulate immune activity. Their immunophenotypic features are not universally agreed upon, and cell populations currently classified as BMSCs may represent heterogeneous cell populations and/or subpopulations of the BMSC lineage. In vivo evidence is accumulating to suggest that BMSCs can be affected by aging and stem cell age can impact mammalian life span/health span. It is possible that aging of BMSCs and/or the reaction of BMSCs to age-related changes to environmental stimuli, such as the ECM and circulating metabolites, could perpetuate aging or age-related diseases, but further work is required to determine this. Meanwhile, outcomes of natural chronological aging of BMSCs are still not clear, but in general it appears that with chronological age, BMSCs decrease in frequency and undergo a decline in progenitor cell functions such as proliferation and differentiation. However, these activities need to be investigated further within in vivo environments. The deficiency in the characterization of BMSCs and studies that monitor the in vivo activity of aged BMSCs represent areas where further research is needed. Also, expansion in vitro disrupts the biology of BMSCs, and this must also be addressed. BMSCs senesce with prolonged culture in vitro, and some features of their in vitro aging mirror those of chronological aging in vivo. Some researchers have reported in vitro treatments that improve aged BMSC performance. Improving the conditions of ex vivo culture and discovering better markers of BMSC aging may help to suppress and monitor in vitro aging. Acknowledgement The authors acknowledge research support from the Commonwealth of Pennsylvania Department of Health (SAP 4100050913).

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