Cellular Lifespan and Regenerative Medicine

Abstract

Tissue engineering is a promising approach to aid in the treatment of a wide range of clinical disorders by developing replacement tissues for damaged or diseased organs. Such approaches, however, will require large and functional cell populations in order to produce a tissue that can replicate in vivo function. Most adult cells have limited replicative potential that limits their use in tissue engineering applications. Thus, cell populations with expanded lifespan or increased replicative potential are of interest. Stem-cell derived populations may allow the creation of large cell populations that have increased replicative potential over adult differentiated cells. In addition, ectopic human telomerase reverse transcriptase expression and induced bcl-2 expression can allow adult cells to proliferate more extensively than unaltered cells. However, concerns for malignant transformation exist with telomerase and bcl-2 approaches. The current states of research in these areas are reviewed as they relate to tissue engineering and the cellular lifespan.

1. Introduction

There is a great medical need for replacement tissues for damaged or diseased organs. Tissue engineering is one approach to bridge the large gap between the clinical demand and the organs and tissues that are available for transplant and repair [1-3].

In order for any engineered tissue to be effective, it must contain sufficient cells that remain functional over clinically relevant time periods. Most adult human cells have a limited lifespan. After repeated cell divisions, cells eventually enter replicative senescence, a state in which they are still viable, yet no longer divide and display reduced functionality. This presents a challenge to using differentiated cells as a cell source for tissue engineering, especially since the cells of an individual requiring an engineered tissue are likely already aged and impaired. In the case of the vasculature, cells from older, atherosclerotic tissues have demonstrated impaired function in mice [4] and in humans [5]. Researchers have therefore been exploring other avenues in order to obtain sufficiently large, functional cell populations for tissue engineering and regenerative medicine applications.

One area of interest is the use of stem cells as a cell source in order to derive a possibly ‘younger’ and more functional cell population. Although adult stem or progenitor cells are capable of substantial expansion, these cells are difficult to obtain for many tissues, such as the pancreas [6]. Pluripotent or totipotent embryonic stem cells can be expanded indefinitely, but are difficult to control in their differentiation fate, and present ethical challenges to their use. As an alternative, researchers have also explored a variety of genetic manipulations in order to expand differentiated cell populations. The use of the enzyme telomerase to extend cell lifespan is widespread, but others have used the anti-apoptotic gene bcl-2 as well as other methods [7]. Telomerase has been ectopically introduced into cells to allow population expansion beyond what would otherwise occur [8-11]. This is important as large, functional cell populations are required to create replacement tissues, and so telomerase has been used in tissue engineering applications [1]. Bcl-2 has been used to reduce cell susceptibility to apoptosis and enhance formation of functional vascular networks [7]. These approaches have been shown to effectively increase cell lifespan and allow creation of improved engineered tissues, but also introduce danger of malignant transformation in the transplanted cells.

2. Stem cells

Adult stem cells are critical for the renewal and repair of tissues in vivo. In addition, embryonic and many types of adult stem cells are self-renewing, thereby making them an attractive cell source for tissue engineering applications. Stem cells are able to proliferate more extensively to provide larger cell populations than differentiated cells. Already, adult stem cells have been used to produce functional populations of cells for a wide variety of tissue engineering applications. Lee and co-workers induced human mesenchymal stem cells into hepatocytes, and found appropriate morphology, marker expression, and in vitro function [12]. Other groups have used stem cells to explore tissue engineering of bone [13], cardiac and skeletal muscle[14], blood vessels [15], and skin [16] (for review, see Bianco and Robey [17]). Due to ethical concerns and regulatory issues, limited research has been done on human embryonic stem cells for regenerative medicine applications. One group has induced endothelial cell growth from embryonic stem cells, and found the cells to express endothelial markers, uptake acetylated low-density lipoprotein, and form microvessels in SCID mice [18]. Due to the enhanced replicative potential of stem cells over adult, differentiated populations, stem cells are an interesting and attractive cell source for tissue engineering.

3. Telomere hypothesis & Hayflick's limit

Adult, differentiated cells have a finite replicative potential. This limit is termed the Hayflick limit, and is due to eukaryotic cells' inability to completely replicate their chromosomes with cell division. The ends of chromosomes are capped by non-coding telomeres, which consist of thousands of linear repeats of the 6-base pair sequence 5′-TTAGGG-3′ [19]. Due to the nature of DNA polymerases, the final ∼30-120 base pairs of the telomeres cannot be replicated with cell division, and these losses accumulate with repeated cell divisions. When the telomeres are shortened below a critical length, replicative senescence (phase M1) results – the Hayflick limit [20]. Cells are senescent, yet remain viable, and can be stimulated to replicate by infection with viral oncogenes [21]. After an additional ∼20 population doublings (PDs), cells encounter a second checkpoint, termed M2 or crisis (figure 1). At this point, massive cell death due to chromosomal fusions occurs. A series of changes must take place, including acquisition of telomerase activity, to allow the cell to proceed through M2 and achieve immortality, thus becoming malignant.

Figure 1.

Relationship of telomere length to cellular replicative age. Telomere length gradually shortens in somatic cells until the checkpoints M1 and M2 are reached. Ectopic telomerase expression or malignant transformation can permit cell growth to continue past these checkpoints. Germline cells, progenitor cells, and cells from dyskeratosis congenita (DKC) patients show different rates of telomere shortening.

4. Telomerase

Telomeres provide a ‘mitotic clock’ that allows sufficient cell replication to enable organism survival during a single generation, while reducing the likelihood that cells will undergo malignant transformation. Eukaryotic organisms evolved a mechanism of restoring telomeres at the ends of chromosomes, in order to maintain telomere length between generations. Telomerase consists of an RNA subunit (hTR), which is expressed in most cells, and an enzymatic subunit (hTERT), which is only expressed in gametes and pluripotent stem cells, and to lesser extents in adult progenitor stem cells. hTR contains an RNA template for the 6bp telomeric repeat, while hTERT provides enzymatic activity to synthesize telomeres. Progenitor stem cells express a low level of telomerase, which is presumably enough to allow them to perform their role as stem cells over the expected lifespan of the organism.

Without telomerase activity, cells can replicate in vitro for ∼30-70 population doublings (PDs) before senescence, depending on cell type and growth conditions. This is insufficient for some tissue engineering applications, especially since cells harvested from adults may have a much shorter effective lifespan. The keratinocytes in skin grafts were shown to have shorter telomeres than non-expanded cells and than non-expanded cells in old patients [22]. In order to avert senescence, ectopic expression of hTERT has been induced in a wide variety of cell types, including smooth muscle, fibroblasts, osteoblasts, endothelial and epithelial cells. Interestingly, some cells are able to replicate indefinitely with hTERT expression, while others require expression of other proteins as well. Bodnar and colleagues extended the lifespan of fibroblasts and retinal epithelial cells by 20-40 population doublings [8] with hTERT, Yang induced ectopic hTERT in a wide variety of endothelial cells and found an increase in population doubling level (PDL) of at least 2.5-fold [9], while Simonsen and co-workers infected human bone marrow stromal cells with hTERT and found an increase in PDL of ∼230 [10]. These studies demonstrated normal growth of the hTERT-transduced cell populations, with normal karyotype and cell function.

4.1. Risk of Carcinogenesis

Although telomerase presents an appealing method of extending cell proliferation, the potential for malignant transformation is of concern. Telomerase is active in all major types of cancer, occurring in 87% of human malignancies [11] as well as some pre-malignant lesions such as in the breast or colon [23]. Acquisition of telomerase activity must occur for a cell to bypass M2 and become malignant. Thus, inducing expression of hTERT must be done with caution in order to avoid any chance of introducing malignant or pre-malignant cells into a patient.

Researchers have evaluated hTERT-transformed cells for a variety of malignant characteristics, such as the ability to grow in soft agar, form tumors in nude mice, or exhibit altered expression of proto-oncogenes or cell cycle regulatory proteins. The endothelial populations studied by Yang and co-workers [9] did not grow in soft agar or have altered phosphorylation of the retinoblastoma protein (RB), a critical cell cycle checkpoint control that blocks cell growth when hypophosphorylated . Jiang and colleagues used fibroblasts and retinal pigment epithelial cells expressing ectopic hTERT, and found that despite cell lifespan reaching population doubling levels twice that of controls, hTERT cells did not grow in soft agar or form tumors in mice [24]. Furthermore, hTERT cells maintained normal growth control and cell density in culture. Our group studied smooth muscle cells from elderly donors that were infected with hTERT, and found stable growth kinetics, lack of growth in soft agar, and lack of tumor formation in nude mice [25]. Karyotype abnormalities were detected in hTERT cells, but at similar or lower rates as compared to control cells, suggesting that these chromosomal abnormalities were unrelated to telomerase induction. In bovine endothelial cells, hTERT expression was able to produce an immortalized population, but pRB phosphorylation status was not dysregulated. In addition, although levels of p16 and p21 were repressed, hTERT expression was unable to maintain inactivation of p16^INK4A, a cyclin-dependent kinase inhibitor that must be inactivated for malignant transformation to occur [26]. The interplay between these and other regulatory proteins, telomerase, and cell growth behavior is diagrammed in Figure 2. Bone marrow stromal cells expressing hTERT maintained normal osteoblastic marker expression and normal karyotype, and did not form tumors in nude mice. In addition, no growth abnormalities were observed up to 230 PDs [10].

Figure 2.

Interplay between key cell cycle regulatory proteins, telomerase and cell growth behavior. Key interactions are explained in the text.

In contrast, some cell populations have been found to be more susceptible to malignant transformation, and telomerase can increase this susceptibility. In a study of hTERT-infected cells from Barrett's esophagus epithelium, cells demonstrated a modest increase in growth in soft agar by 3-6 fold [27]. In T-lymphocytes transduced with hTERT, cellular lifespan was extended by up to 25 PDLs, but a population of binucleated cells appeared in hTERT cultures [28]. Wang infected mammary epithelial cells with hTERT, but discovered induced expression of the oncogene c-myc at levels comparable to those found in malignant cell lines[29]. Furthermore, since c-myc is a regulator of hTERT expression, hTERT expression continued even after ectopic hTERT was removed by Cre-lox recombination [29]. This is of further concern since Kiyono et al showed that this same cell population requires p16 inactivation before immortalization [30]. Thus, ectopic hTERT expression in these cells may have contributed to c-myc activation. Although most reports do not indicate overt malignant transformation of hTERT cells, it is evident that telomerase is associated with growth dysregulation in some populations. Hence, use of lifespan extension by telomerase must be examined on a case-by-case basis, since not all cell types appear to be equally susceptible to transformational changes following lifespan extension.

It is important to note that while telomerase is tied to malignancy, it is not an oncogene. As reviewed by Harley [31], oncogenes promote growth deregulation in cells, which telomerase does not. Telomerase only functions to maintain telomere length, thus permitting cell growth. Indeed, the vast majority of studies show that cell cycle checkpoint controls, such as contact growth inhibition, are maintained in the setting of hTERT over-expression. Nonetheless, telomerase warrants special attention due to its ties to cancer.

4.2. Transient Delivery of Telomerase

In order to overcome the risks of hTERT-transformed cell lines, researchers have pursued methods of inducing transient ectopic hTERT expression. This approach would allow the expansion of a cell population to the required level with the help of telomerase, and then remove the ectopic hTERT gene before implantation. Several groups have utilized Cre-lox recombination, and in a variant of this method, Ungrin and colleagues [32] demonstrated the ability to invert hTERT transcripts in human fibroblasts between active and inactive states by using anti-parallel loxP sites. In this report, telomerase activity could thus be turned on to expand the cell population as needed, and then inactivated or removed before implantation.

Another approach has been adenoviral-mediated telomerase delivery, which provides the advantage of transient viral delivery with fewer risks of insertional mutagenesis as compared to retroviral therapy. Rudolph [33] used adenoviral delivery of the RNA component of telomerase (mTR) to mTR-null mice to restore telomere function and improve liver function in animals with induced liver failure. A protein termed protection of telomeres-1 (POT1) has recently been identified, and found to induce the lengthening of telomeres in a telomerase-dependent manner [34]. hPOT1 may be useful for extending cellular lifespan if telomerase expression alone is insufficient. Other methods to control hTERT expression may be the introduction of suicide genes along with telomerase. Chemical or other signals can be used to induce cell death if malignant activity is detected. However, with all of these methods, the risk of any remaining ectopic telomerase expression is concerning. In addition, it must be certain that malignant transformation does not begin during induced hTERT expression. These concerns make any telomerase approach for extension of cellular lifespan risky unless adequate safeguards are in place and regulatory challenges met.

5. Bcl-2

The bcl-2 family of genes is involved in the regulation of apoptosis. B-cell lymphoma 2 (bcl-2) is a proto-oncogene originally identified in B-cell lymphomas at a chromosomal break point. The bcl-2 family now contains over 20 members, which are both pro- and anti-apoptotic. Some of the family members promote, while others prevent, cell death via caspase-mediated apoptosis. Bcl-2 itself is anti-apoptotic, and has attracted interest as a means of prolonging cell survival. Bcl-2 has been found to protect neuronal precursor cells from death due to toxic exposure to retinoic acid [35] and protect beta cells from damage from reactive oxygen and reactive nitrogen species [36]. In hybridoma and CHO cells, bcl-2 overexpression was found to reduce cell death, in one case increasing cell viability from 20% to 90% [37].

The protective effects of bcl-2 have been explored for regenerative medicine applications. In an in vivo rat model, bcl-2-transduced cardiomyoblasts were injected in a collagen matrix into a rat heart after myocardial infarction [38]. Hearts with bcl-2 cells demonstrated improved signals with bioluminescence imaging, yet equivalent fractional shortening and ejection fraction compared to non-bcl-2 infected cells. Human umbilical vein endothelial cells (HUVEC) infected with a retrovirus containing bcl-2 showed delayed apoptosis and improved ability to induce microvessel formation in an immunodeficient mouse model [7]. These bcl-2 transduced cells were found to form functional arterial, venular and capillary endothelial cells that expressed endothelial markers, formed tight junctions and retained fluid within vessels [39]. However, untransduced endothelial cells derived from either adult peripheral blood or umbilical cord blood progenitor cells were found to engraft a skin substitute equivalently to HUVEC that were transduced with bcl-2 [16].

However, bcl-2 is not used without concern – in B-cell lymphomas a transposition of this gene results in constitutive expression and resultant resistance to apoptosis. This extended survival allows for activation of the c-myc oncogene and eventually leads to malignancy. Therefore, tissue engineering approaches utilizing induced bcl-2 expression must be undertaken with care and fully evaluated from a safety standpoint.

6. Growth conditions and storage

Proper growth conditions can greatly impact cell proliferation and survival. One key point is that many cell types in in vitro culture do not continually divide until limited by telomere length. Contact inhibition will inhibit cell growth of many cell types, including smooth muscle and endothelium. Cells can also be sensitive to culture media, serum concentration and lot, growth factor concentrations, cell density, and other factors. This is especially true with stem cell populations, and mesenchymal stem cells (MSC) are quite sensitive. Lin [40] reported that a new culture medium for MSC accelerated growth and extended lifespan compared to growth rates quoted in the literature. In addition, in vitro culture can expose cells to various stresses that may limit lifespan, including oxidative stress from high oxygen tensions or free radical generation, glycosylation of protein moieties from high ambient levels of glucose in medium, or radiation damage.

Cellular lifespan following cryopreservation will be important to tissue engineering applications. Cells die during cryopreservation due either to freezing or to apoptosis, which occurs over the hours and days after thawing. For endothelial cells, recovery after cryopreservation is ∼60-80% with viability of 65-80% [41]. The lag phase in cell growth is initially longer after thawing but growth returns to normal within one passage [41]. In an attempt to improve cell recovery, Fujita and co-workers treated hepatocytes with an antioxidant and a caspase-3 inhibitor, and found that both led to increased viability and cellular function [42]. Improving cell survival after cryopreservation and maximizing cell proliferation with optimized growth conditions are all methods to effectively extend the functional lifespan of a cell population.

7. ALT – Alternative lengthening of telomeres

Besides telomerase, another mechanism of increasing telomere length exists for eukaryotic cells. Termed Alternative Lengthening of Telomeres, or ALT, this mechanism(s) is poorly understood and the genes involved are unknown [43]. The existence of ALT is inferred based on an absence of telomerase detection with analytical methods (i.e. telomeric repeat amplification protocol) together with telomere lengths ranging from short to very long in replicating cells [44]. Unlike telomerase, which lengthens the shortest telomeres and keeps telomere length relatively constant, ALT leads to heterogeneous telomere length. The mechanism(s) behind ALT are unknown, although Wang and co-workers presented data suggesting that homologous recombination may be involved [45]. Another study demonstrated that a unique DNA sequence inserted into the telomere of one chromosome was rapidly copied to other chromosomes, thereby hinting at a mechanism involving homologous recombination [46]. Immortalized cell lines that have been examined to date all utilize either telomerase or ALT to lengthen their telomeres. It is interesting to note that ALT occurs more frequently in tumors arising from mesenchymal tissues, while tumors arising from epithelial tissues utilize telomerase more frequently (see Henson et al [47] for a review).

8. Clinical

In addition to tissue engineering, cellular lifespan is of relevance for other types of regenerative medicine therapies, and several genetic disorders. Dyskeratosis congenita (DKC) is a premature aging disease caused by a rare genetic defect in dyskerin, which causes – among other things – abnormal production of the RNA component of telomerase [48]. Patients die at a young age from bone marrow failure or pulmonary complications [49]. Treatment options for DKC at present are limited, although a gene therapy approach utilizing telomerase may be promising and a mouse knockout model has been developed that may facilitate such approaches [50]. Duchenne's muscular dystrophy (DMD) is a disease suitable to telomerase genetic engineering. DMD is an X-linked recessive disorder that is due to a defect in dystrophin. The disease leads to progressive wasting of striated muscle, including the heart and diaphragm, and patients die of cardiorespiratory failure by their early 20s. In DMD patients, myoblasts from even from very young patients (5-6yo) have abnormally shortened telomeres, due to extensive cellular turnover in efforts to replace damaged myofibers. One treatment approach has been ex vivo gene therapy, in which myoblasts are removed, modified to express hTERT, and then transplanted back into the patient [51]. For this application, either a stem cell population with sufficient potential must be isolated or a way to bypass the normal replicative limit must be identified. Seigneurin-Venin et al [52] used cells from muscle biopsy of DMD patients, which were then infected with hTERT and SV40 large T antigen in order to immortalize the cells. Cultures remained contact inhibited and the additional introduction of H-ras was required for malignant transformation. Although malignant potential remains for these cells, this is nonetheless an interesting and promising approach.

9. Conclusion

Effective tissue engineering requires large, functional cell populations. Adult human cells have limited replicative potential that presents challenges to using such cells in an engineered tissue, and necessitates cell populations with expanded lifespan. Researchers have explored stem cell-derived populations, ectopic telomerase expression, and induced bcl-2 expression to obtain cell populations with increased replicative potential. Despite concerns for malignant transformation with telomerase and bcl-2, these are promising approaches for tissue engineering that have been shown to allow for the creation of improved engineered tissues.