For reprint orders, please contact: Review 2016/03/29 The germline/soma dichotomy: implications for aging and degenerative disease Human somatic cells are mortal due in large part to telomere shortening associated Michael D West*,1, Francois with cell division. Limited proliferative capacity may, in turn, limit response to injury Binette2, David Larocca3, and may play an important role in the etiology of age-related pathology. Pluripotent Karen B Chapman3, Charles stem cells cultured in vitro appear to maintain long telomere length through relatively Irving4 & Hal Sternberg11 high levels of telomerase activity. We propose that the induced reversal of cell aging BioTime, Inc., 1010 Atlantic Ave., Alameda, CA 94501, USA by transcriptional reprogramming, or alternatively, human embryonic stem cells 2ReCyte Therapeutics, Alameda, engineered to escape immune surveillance, are effective platforms for the industrial- scale manufacture of young cells for the treatment of age-related pathologies. Such 3OncoCyte Corporation, Alameda, cell-based regenerative therapies will require newer manufacturing and delivery technologies to insure highly pure, identified and potent pluripotency-based Cell Cure Neurosciences, Jerusalem, *Author for correspondence: Tel.: +1 510 521 3390 ext. 303 First draft submitted: 10 October 2015; Accepted for publication: 2 March 2016; Fax: +1 510 521 3389 Published online: 24 March 2016 Keywords: age-related macular degeneration • aging • bone • brown adipose tissue
• cartilage • choroid plexus • clonal embryonic progenitor cells • embryonic stem cells
• endothelial progenitor cells • pluripotent stem cells
The cellular basis of age-related
The assumption that aging is ultimately due to intrinsic changes in cells and tissues At a tissue level, human aging is often char- leads logically to the next question; namely, acterized by a progressive loss of normal what are the molecular and cellular mecha-histology. While the nature of this deterio- nisms behind the ‘clockwork' that triggers the ration obviously varies by tissue type, the downstream pathological changes? Since the term ‘morpholysis' (literally meaning ‘dis- birth of cell biology in 19th century, a logical solution of form') is generally applicable. place to begin the search for possible clock-Salient examples include osteoarthritis, age- work mechanisms was within the cell itself. related macular degeneration, skin aging In charting the modern theory of heredity, and osteoporosis. This morpholysis of aging the German naturalist August Weismann differs from that associated with trauma promulgated the simple proposition that the or infection in that age-related morpholy- molecular basis of the immortal transmission sis generally occurs in the absence of obvi- of hereditary information from generation ous external stimuli or immune infiltrates to generation is through direct transport of (i.e., osteoarthritis largely lacking the pro- molecules through germline cells [1]. In con- found immunological component typical of trast to the germline, Weismann proposed rheumatoid arthritis). Instead, age-related that the somatic cell lineages constituting the morpholysis appears to reflect, at least in other tissues of the body are cast off each gen-numerous cases, an intrinsic change in the eration as a disposable appendage, therefore particular tissue.
do not need the same replicative immortal- 10.2217/rme-2015-0033 Biotime Inc.
Regen. Med. (2016) 11(3), 331–344
Review West, Binette, Larocca, Chapman, Irving & Sternberg ity. He therefore speculated that the first appearance that could have a dominant bystander effect on tissues. of aging in evolutionary history coincided at about Perhaps only a minority of senescent cells in a tissue the time of the evolution of somatic cell types, and an could shift the balance of synthesis to degradation of associated divergence of the germline and soma repli- tissue extracellular matrix components leading to mor- cative strategies. He surmised that "death takes place pholysis (i.e. collagenolysis and elastolysis) seen in the because a worn-out [somatic] tissue cannot for ever intrinsic aging of skin [9] or other tissues.
renew itself, and because a capacity for increase by But, what is the cause of the cell senescence in the means of cell-division is not everlasting, but finite" [2]. first place? A theory for the primary mechanisms must Weismann added that age-related disease could present take into account Weismann's suggestion, now con-itself before the complete exhaustion of cell prolifera- sidered empirical fact, that not all human cell lineages tive capacity in a given tissue, "functional disturbances age. The germline lineage of cells has perpetuated life will appear as soon as the rate at which the worn-out throughout the millennia, necessarily possessing repli-cells are renewed becomes slow and insufficient" [2].
cative immortality [10,11]. This immortality is likely not Weismann's prediction that somatic cells possessed dependent on meiosis. While most metazoans repro- an inherent finite replicative capacity was finally con- duce through unlimited cycles of sexual reproduction firmed in the mid-20th century. Commonly desig- (alternating meiotic and mitotic events) that span the nated the ‘Hayflick limit' [3,4], this model system com- entire history of life on earth, some species, includ- pares the molecular properties of cells early in their ing occasional vertebrate species, can reproduce from replicative lifespan with those at or near the end of that unfertilized egg cells alone (parthenogenesis). There-lifespan to search for alterations in, for example, gene fore, it is reasonable to postulate that sexual recombina-expression, to find molecular pathways that may pro- tion or other events associated with meiosis may not be vide causal insights into age-related pathophysiology. required for the immortality of the germline [12,13]. In In support of the physiological relevance of the cell addition, a valid model of cell aging needs to account senescence model of aging, the study of segmental pre- for the potential of somatic cells to immortalize in mature aging syndromes such as Hutchinson-Gilford the course of malignant transformation such as what (progeria) and Werner Syndrome, each of which dis- occurs in the presence of viral oncogenes expressed by plays a premature onset of multiple age-related degen- papillomaviruses, adenoviruses or Epstein-Barr virus. erative diseases such as atherosclerosis, osteoporosis, If cellular aging reflected very complex mechanisms Type II diabetes, lipoatrophy and skin aging, also show or generalized entropy, how can cells immortalize at a premature onset of in vitro cell senescence of cultured relatively high frequencies in the presence in some fibroblasts compared with normal control samples [5,6].
examples of a single viral oncogene? Initial studies comparing young and senescent cells A decade after Hayflick's report of human cell aging, for alterations in functionality showed that while young Olovnikov proposed the hypothesis that the dichotomy cells were capable of dynamic alterations in the expres- of immortality/mortality in the germ-line/soma was sion of secreted proteins such as proteases and protease due to an enzyme capable of maintaining the struc-inhibitors, senescent cells appeared to be arrested in ture of chromosome ends (telomeres) in germline and a nonproliferative state, but paradoxically, one char- cancer cells, while somatic cells lacked the activity and acterized by the secretome of activated young cells as a result experienced a progressive loss of telomeric rather than quiescent ones. Since the normal main- DNA with each cell doubling eventually leading to tenance mode of most stromal cells in vivo is quies- senescence [14,15]. This telomere hypothesis of cellular cence, abnormally ‘activated' senescent cells would be aging and immortalization, is summarized in Figure 1. unable to proliferate in response to tissue damage, and Telomere length is represented as terminal restriction potentially express secreted proteins that could lead to fragment (TRF) length which is the average combined alterations in tissue maintenance. This inflammatory- length of all chromosome telomeric repeats along with like state of gene expression combined with cell cycle subtelomeric sequences between the restriction sites and arrest is commonly designated senescence-associated the telomere end. Therefore, a TRF length of 5000 bp secretory phenotype (SASP). Generally, activation may have 3000 bp of subtelomeric DNA and 2000 bp markers are associated with growth factor or cytokine- of telomeric repeats on average, but one or more telo- induced proliferation and are downregulated with qui- meres that have become uncapped through a critical escence. The resulting profound fibroclastic nature of loss of repeats thereby triggering a p53-dependent senescent cells displaying the SASP phenotype in what DNA damage checkpoint cell cycle arrest.
would otherwise be quiescence-inducing conditions TRF length is reported to be approximately 15 kbp may be due in part to constitutively high expression of in human sperm cells, and to shorten in somatic cell interstitial collagenase and plasminogen activators [7,8] lineages approximately 50–200 bp with each round of Regen. Med. (2016) 11(3)
future science group The germline/soma dichotomy: implications for aging & degenerative disease Review Stem cellsconditionally T´ase (+) Somatic cells(T´ase [-]) TRF length (kbp)
Immortalization (T´ase [+]) Figure 1. Telomere dynamics from embryogenesis to M1, M2 and immortalization. Somatic cells generally
repress telomerase activity early in embryogenesis and as a result progressively lose telomere length following
differentiation from the germline. By contrast, while perhaps oscillating during germ cell differentiation, the
germline cell lineage maintains an immortal continuity from generation to generation. Finally, the abnormal
reactivation of telomere maintenance during oncogenesis, typically after the inactivation of tumor suppression
and extension of telomere loss to the point of multiple critically short telomeres, stabilizes telomere length in
most malignant tumor types. This maintenance is most frequently through the reactivation of the telomerase
catalytic component.
TRF: Terminal restriction fragment.
cell division until senescence occurs at about 5–7 kbp ignated ‘ALT') and an ability to maintain telomere (Hayflick limit). Telomeric attrition in somatic cells length over extended doublings [21,22]. Since these early lacking telomerase activity is therefore proposed to observations, telomere dynamics have been the subject function as a ‘replicometer', recording cell doublings of extensive investigation. Based on studies of com-as opposed to a ‘chronometer', measuring metabolic parative zoology, the emerging evolutionary theory is time. The telomere theory received experimental sup- that, at least for some taxa, the repression of telom- port with studies demonstrating that the Hayflick phe- erase activity combined with relatively short telomere nomenon did indeed appear to reflect a mitotic rather lengths in the soma was selected for as it reduced the than chronologic clock [16], and later that telomere risk of malignancy in long-lived species. This model shortening could be observed in somatic cell types is therefore proposed to be a type of antagonistic compared with germline cells during human aging pleiotropy [23].
in vivo [17], even in stem cell compartments such as The foregoing observations documented the cor- candidate hematopoietic stem cells [18]. Similar pro- relation of telomere dynamics with that predicted gressive shortening of telomeres was then observed in by the Olovnikov hypothesis. The causal connec-cultured human fibroblasts during passaging in vitro tion with senescence in vitro was demonstrated most to senescence [19], while stabilization of telomeres was convincingly by the exogenous expression of TERT observed during the immortalization of cells by onco- in mortal differentiated cell types leading to their genic viruses [20]. Furthermore, the replicative immor- direct immortalization, apparently without transfor- tality of cancer cell lines correlated with the abnormal mation [24]. Additional data linking the biology to expression of telomerase activity (or more rarely, an in vivo age-related degenerative disease resulted from alternative mechanism of telomere maintenance des- the observation of increased incidence of damaged or future science group Review West, Binette, Larocca, Chapman, Irving & Sternberg shortened telomeres in cultured cells from patients In summary, human somatic cell types typically with Werner syndrome [25] and progeria [26]. A com- display a mortal phenotype characterized by progres- mon link between the two aging phenotypes having sive telomere shortening when cultured in vitro and distinct mutated genes (WRN and LMNA) may be presumably during aging in vivo. Telomeres appear the mutual interaction of the encoded proteins of each to be not merely a marker of cell aging, but may play gene with TRF2. The rescue from senescence in these a critical causative role, at least to some cell and tis-progeroid cell types through the exogenous expression sue types. Nevertheless, many other cellular processes of TERT [27] provides further experimental support have been implicated in aging and cell aging in par-of the relevance of telomere shortening or damage to ticular. These include alterations in such processes as aging in vivo. Also providing support are experiments autophagy, mitochondrial DNA mutations and other aimed at modifying telomere dynamics in mice. The mitochondrial changes, free radical damage, deregu-breeding of Terc-knockout mice with Wrn mice leads lated nutrient sensing, epigenetic alterations, non-cell to animals displaying human age-related pathologies autonomous and endocrine/paracrine mediators and not normally expressed in aged mice [28]. Also, trans- others [41]. Further research is required to clarify the genic mice overexpressing Tert together with tumor order of cause and effect for each of these changes, suppressor genes has been reported to extend normal some of which could be linked to telomere dynamics lifespan [29]. In conclusion, growing experimental data and some potentially to other developmental changes support the hypothesis that the several degenerative such as the embryonic-fetal transition where intrinsic diseases observed in common in normal aging and tissue regeneration may be repressed. However, on the accelerated aging syndromes may also have common assumption that the germline lineage of cells escapes etiology in telomere dysfunction and may therefore many of these age-related alterations with time, the logically be rescued by restoring youthful (long telo- culture of this entirely unique class of cells may pro- mere) cell function in the target tissues afflicted with vide a means of manufacturing young cells for the such pathology.
treatment of age-related degenerative disease.
The delineation of some of the molecular pathways triggering cell senescence resulting from telomeric The pluripotent stem cell manufacturing
attrition or other genotoxic cascades such as those platform
caused by oxidative stress on telomeres in telomerase-
In contrast to the mortal phenotype of cultured human negative cells [30] (collectively referred to as senescence somatic cells, human pluripotent stem (hPS) cells such in this review), allowed for improved markers to deter- as human embryonic stem (hES) cells or human- mine if senescence occurs in vivo and if it correlates induced pluripotent stem (hiPS) cells uniquely dis-with age-related degenerative disease. Early observa- play replicative immortality when propagated in vitro. tions showed that aged, as opposed to young tissue, This appears to result from a relatively high level of shows a greater percentage of cells in the primary cul- telomerase activity [42]. Upon differentiation in vitro, ture with a morphology like that of senescent cells [31]. TERT appears to be repressed relatively early with all This is difficult to reproduce however in comparing continuously propagating differentiated or partially the replicative lifespan of cultures from individuals of differentiated cell cultures lacking telomerase activ-differing ages, likely because of the overgrowth of the ity and showing progressive telomere shortening and cultures by the youngest cells in the culture [32]. The a finite replicative lifespan when cultured in vitro [43]. use of markers of senescence such as lipofuscin [33] and This natural replicative immortality of most pluripo-secondary lysosomal enzymes [34], or more modern tent stem cells appears to reflect less the ability of these counterparts of lysosomal markers such as senescence- cells to normally self-renew (a defining characteristic associated β-galactosidase assay at pH6 (SAβ-gal) [35], of a stem cell) than their nearness to the branch point markers of cell cycle arrest such as those in the telo- of germline and somatic cells on the ontological tree. mere damage-induced p53–p21–pRB pathway, or Indeed, the concept of the self-renewal of stem cells stress-induced p16–pRB pathway [36] or direct imaging being equated with replicative immortality, at least in of damaged telomeres such as senescence-associated the case of the human species, is probably misplaced. DNA damage response foci (SDFs) or alternatively While mouse stem cells and numerous other mouse telomere dysfunction-induced foci (TIF) detected by cell types spontaneously immortalize at a relatively the presence of γ-H2AX (a histone H2A variant) [37] high frequency, human cell types typically do not, have proven useful in this regard. TIFs, for instance, and there has yet to be a functional human stem cell have been reported to be useful markers for normal type observed to maintain telomere length and display somatic cell senescence in vitro [38] as well as premature replicative immortality in vitro. While hES and hiPS senescence in Werner syndrome [39] and progeria [40].
cells are clearly an exception, it is important to note Regen. Med. (2016) 11(3)
future science group The germline/soma dichotomy: implications for aging & degenerative disease Review that they are not normally stem cells in vivo. That is, formed to determine whether the use of epithelial cells there is no niche of pluripotent stem cells in the nor- as opposed to connective tissue fibroblasts as somatic mal human body that have the potential to self-renew cell sources truly influences the reprogramming of or differentiate, unless one considers the ephemeral telomere length.
appearance of the inner cell mass and epiblast of the SCNT is not only technically challenging, its wide- human blastocyst a ‘niche'. Therefore, human pluripo- spread use would require a large supply of human tent stem cells appear to differ from all reported human oocytes. Therefore, the simplification of the reprogram-fetal and adult-derived stem cells in regard to their ming protocol to only the exogenous administration of simultaneous potential for replicative immortality and defined transcriptional regulators to produce iPS cells pluripotency. There have been numerous reports of the has streamlined the process in a manner that makes it initial characterization of immortal and pluripotent practical to implement in most laboratories [52]. While cells from human fetal or adult sources [44,45], but to telomere length extension back to embryonic length is date, there is scant or contradictory confirmation in not a universal property of aged somatic cells repro-the literature [46].
grammed using all iPS cell protocols, it nevertheless can be achieved [43]. As in the case of the exogenous Rescue from developmental aging
expression of TERT, transcriptional reprogramming Cells clinically useful for transplantation would appears to also rescue Werner syndrome and progeria require a cost-effective method of solving the prob- fibroblasts from senescence [53,54], further supporting lem of histoincompatibility. The demonstration that the potential utility of the technology in reversing somatic cell nuclear transfer (SCNT) is capable of age-related pathology in the diseases held in common reprogramming mammalian somatic cells to pluripo- between normal aging and these segmental progeroid tency, indeed, pregnancies and live births [47], led to disorders.
the logical question of its use to ‘reverse the develop- Since the immortality of pluripotent stem cells mental aging' of aged human somatic cells and the enables the indefinite expansion of master cell banks generation of patient-specific young cells of all types and subsequently differentiating working cell banks for use in regenerative medicine [48]. The ‘age-reversal' into scalable young and diverse somatic cell types, it of somatic cells reprogrammed by SCNT or iPS cell may also allow for complex targeted homogeneous technologies can be observed through the resetting genetic modifications similar to that used in the pro-telomere length, reversal of progerin accumulation in duction of transgenic mice. A commonly overlooked reprogrammed cells from progeria patients, restoring advantage of immortal master cell banks is that they cells to an embryonic state and even live births in non- allow cells that have been successfully targeted with human species as well as by other markers of aging as a genomic sequence modification to be clonally described in this review. However, while it is the the- expanded into new master cell banks that can subse- sis of this review that this ‘age-reversal' as applied to quently be modified again, indeed endlessly, yielding reprogrammed human somatic cells is valid, the rigor- highly sophisticated modifications with homogeneity. ous demonstration of the fact would require human Any genetic drift or unintended alterations can then experimentation difficult or unethical to undertake. easily be identified using current genomic sequencing Therefore, the authors use the term ‘the reversal of technologies [55]. These modifications can include ones the developmental aging' of cells to refer to only that intended to induce allogeneic immunotolerance toler-which has been documented to date in the literature, ance of a graft such as the overexpression of PD-L1 in other words, the restoration to pluripotency and ES and CTLA4-Ig [56], or the elimination of HLA class I cell telomere length to aged somatic cells.
genes [57] that provides the additional benefit of poten- Initially, in regard to Dolly, the first mammal cloned tially allowing an off-the-shelf product simplifying from a somatic (mammary epithelial) cell, reports sug- regulatory approval. Therefore, the unique property gested that the animal was born with abnormally short of hPS cells to propagate robustly and indefinitely
(presumably not reset) telomere length [49]. However, facilitates a strategy of producing cGMP-grade thera-
numerous subsequent studies in bovine and other peutically useful master cell banks that allow the con-
species have shown telomere extension and extended tinuous and unlimited manufacture of product of a
somatic cell replicative lifespans to even longer lengths uniform genotype.
than that observed normally [50]. Similar results have
been observed in other laboratories though differences The differentiation of hPS cells into
may be linked to the choice of the somatic cell source therapeutic products
or other unidentified parameters in the protocols In addition to the benefit of the immortal prolifera-
used [51]. Further experimentation will need to be per-
tion of undifferentiated hPS cells, the cells also have future science group Review West, Binette, Larocca, Chapman, Irving & Sternberg the advantage of their potential to cascade into all cells and then for covalent cross-linking of the matrix the 1000-plus somatic cell types in the human body components to occur to a defined degree of rigidity.
including embryonic progenitors (i.e. cells displaying a Thiol-modified hyaluronan and gelatin hydro- phenotype characteristic of that prior to the embryonic- gels can be produced using polyethylene diacrylate fetal transition). Cells and tissues from the developing crosslinkers. When the components are mixed in mammal in the period of embryonic development, but the presence of cells, a viscoelastic hydrogel forms in before fetal development, show a remarkable potential approximately 20 min at room temperature where the for tissue regeneration including scarless wound repair compliance (stiffness) of the hydrogel can be varied such as well studied in the skin [58]. Some multicel- from 25 to 3500 Pa by altering the amount of cross- lular organisms, including some vertebrates, show a linker. Such combination cell–matrix formulations profound ability to regenerate complex tissues such as appear to increase cell survival for cells transplanted limbs after amputation. Vertebrates that display such a into such diverse tissues as the liver, brain, heart as well profound regenerative capacity generally do not show as other tissues [67]. An alternative strategy is the assem-age-dependent loss of the regenerative potential [59]. bly of cells onto decellularized tissues. The latter strat-Therefore, it seems likely that gene expression patterns egy has the advantage of a more robust organ-shaped of the developing embryo are maintained through construct, but generally has the challenge of integrating some undefined process of heterochrony in these ani- cells into a tight 3D matrix construct, and therefore of mals compared with humans. The ability to manufac- sufficient cellularity to function adequately in vivo.
ture primitive human embryonic progenitor cell types
could potentially allow easy access to candidate cell Potential therapeutic applications in
populations with this phenotype.
age-related degenerative disease
However, the vast pluripotency of hPS cells is as Assuming that previously-mentioned vertebrates with much a challenge as it is opportunity. Strict quality a profound capacity for tissue regeneration do so control standards enforced by the US FDA for cells because they are either transiently in a developmen-used in human therapeutics demand a high degree of tal stage prior to the embryonic–fetal transition or are reproducibility in manufacturing identified and puri- displaying a heterochronous retention of embryonic fied cell types [60]. Since the fate space of hPS cells patterns of gene expression in adulthood, the ques-exceeds 1000 distinguishable cell types, demonstrating tion arises whether hPS cell-derived clonal embryonic purity and identity will likely be challenging [61].
progenitor cell lines with long initial telomere length A simple solution to this challenge is to scale prod- also displaying a pre-fetal pattern of gene expression uct from single cells in transit from pluripotency to could prove useful in regenerating tissues afflicted
fully differentiated cells. These cultures also known with age-related degenerative disease. We will briefly
as clonal embryonic progenitor cell lines, have been discuss some salient examples of where such therapies
reported to be expandable in greater than 200-fold may prove interventional.
diversity [62], with numerous lines being multipo-
tent, capable of making, for instance, site-specific Skin aging
osteochondral cell types [63], meningeal and choroid Dermal fibroblasts are one of the most common cell
plexus cells [64] as well as other therapeutically useful types utilized in cell aging research [9]. Evidence of up
cell types. The relatively long initial telomere length to 15% of dermal fibroblasts displaying senescence in
of the parental hPS cell master cell banks gives clonal aging primates has been reported [68]. The morpholy-
lines, while mortal, a correspondingly long replicative sis in aging skin can be profound, with a marked loss
lifespan facilitating industrial scale-up [65].
and fragmentation of collagen fibers and elastolysis. The study of the secretome of aging dermal fibroblasts Injectable biocompatible matrices
showed that while young cells are capable of becom- When most anchorage-dependent somatic cell types ing activated in the presence of serum growth factors are formulated in saline and injected into solid tissue, and entering the cell cycle while up-regulating metallo-they typically undergo massive anoikis (apoptosis) [66]. proteinases such as interstitial collagenase (MMP1) [7] Therefore, a prerequisite for the aforementioned cell- or upon removal of such stimulation returning to qui- based therapies directed at solid tissues are formula- escence, exiting the cell cycle and down-regulating tions of cells combined with critical extracellular MMP1, senescent cells differ in that they are blocked matrix components such as hyaluronic acid and type I in a terminally activated, yet nonproliferating state [8]. collagen. Since cell matrices also require a degree of Besides contributing to the aged appearance to the rigidity to prevent anoikis, it is necessary for matrix skin, dermal fibroblast senescence may contribute to components to be injected safely in the presence of impaired wound healing and chronic nonhealing ulcers Regen. Med. (2016) 11(3)
future science group The germline/soma dichotomy: implications for aging & degenerative disease Review in the aged. The production of young fibroblasts may the world, afflicting over 30 million people [76]. The facilitate wound healing in the elderly. Consistent with wet form of the disease can be effectively prevented this is the observation that adenoviral-based exogenous with regular administration of angiogenesis inhibitors expression of TERT in a rabbit model of skin aging led such as ranibizumab or aflibercept, however, this form to markedly improved wound repair [69]. In addition of the disease accounts for only about 10% of disease to proliferation-competent cells, wound healing could prevalence in the USA. Some 90% of patients have the be facilitated by the induction of scarless wound repair dry form represented by slow progression of geographic such as that observed in the embryonic phases of devel- atrophy which can also cause blindness and for which opment [70]. The isolation of such cells from pluripotent there is currently not approved therapy in the USA. stem cells and their use in stimulating rapid and scarless The etiology of this age-related degenerative disease wound repair in the aged could greatly reduce the costs is thought to be the age-related loss or dysfunction of of managing chronic wounds in these patients [58].
retinal pigment epithelial (RPE) cells [77,78] that nor-mally provide diverse types of support for the neural retina and produce a natural angiogenesis inhibitor Osteoarthritis is an age-related degenerative disease. (PEDF) [79], potentially reflecting the senescence of the The pain associated with the disorder is a leading com- cells [80]. Consistent with this is the observation of a plaint in the elderly and the most common disease of loss of PEDF (also known as EPC-1) expression in cells the joint [71]. While the etiology appears to be multi- grown to senescence in vitro [81]. The derepression of factorial, aging is a leading risk factor [72]. Articular angiogenesis inhibition in the aging retina may there-chondrocytes are generally considered to be essentially fore lead to increased neovascularization and the wet postmitotic throughout adulthood, nevertheless, at form of the disease. The loss of RPE cells, or reduced sites of cartilage injury, clonal proliferation of chon- phagocytosis or autophagy may account in part for the drocytes has been observed, potentially leading to dry form of the disease as well [82].
senescence. Alternatively, stresses on articular chon- Linking the loss of healthy RPE cells to this devas- drocytes from sarcopenia or damage to the meniscus tating disease led to the proposal that RPE cell grafts may lead to fragmentation of telomeres. The resulting may protect against progression of one or both forms of senescent cells, like dermal fibroblasts, may overexpres- the disease. Initial attempts sourced cells from donated sion metalloproteinases compared with metallopro- eyes, autologous peripheral retina or from cells scaled teinase inhibitors, and the result is a cascade of disease, in vitro. While some efficacy was reported, the lim-proliferation and more disease.
ited scalability of RPE due to de-differentiation and During the aging process, degeneration of the inter- senescence led to a search for an alternative source [83].
vertebral disc can lead to considerable pain and disabil- Pluripotent stem cells have been reported to be a ity as well, including low back pain. Like other connec- robust source of young RPE cells [84,85]. Animal pre- tive tissues, cell senescence markers such as SAβ-gal, clinical studies demonstrate potential safety and effi-DNA damage checkpoint markers, metalloproteinases cacy in the Royal College of Surgeons (RCS; London, and shortened telomere length are observed [73] that UK) rat model retinal degeneration [86]. At least three mimick cell senescence changes in nucleus pulposis clinical studies using hES-derived RPE cells are under-cells aged in vitro [74]. In the case of the loss of articular way, one utilizing iPS cell-derived grafts currently on cartilage such as commonly occurs in osteoarthritis, or hold for quality control issues related to drift in geno-the loss of structure to the intervertebral discs, patients type [87]. Since hPS cell-derived RPE cells appear to could potentially benefit from the transplantation of have little immunogenicity and perhaps even to be hPS cell-derived progenitors to those respective tissues. immunosuppressive [88], unmodified cells may be tol-The use of hPS cell-derived clonal embryonic progeni- erated long-term with only transient immunosuppres- tor cell lines has been shown to lead to scalable pro- sion. Human clinical trials utilizing doses expected to genitors of diverse site-specific skeletal tissues that have be potentially therapeutic should soon yield useful data potential to regenerate structure in animal models [75]. to evaluate this application of hPS cell-derived grafts Further studies will be required to determine the util- for an age-related degenerative disease.
ity of these novel and diverse embryonic cell types in
contrast to the more commonly studied adult-derived Age-related neurodegenerative disorders
mesenchymal stem cells.
The aging of the CNS is correlated with several degen-erative diseases such as Parkinson's disease (PD), Age-related macular degeneration
Alzheimer's disease (AD), and stroke [89]. The focal Age-related macular degeneration (AMD) remains a and profound loss of dopaminergic neurons in the leading cause of blindness in many countries around midbrain in PD, and evidence of potential efficacy of future science group Review West, Binette, Larocca, Chapman, Irving & Sternberg transplanted fetal-derived neurons, has led to a search sure to pathogens, or endocrine factors. In addition, for hPS-derived cells potentially useful in treating many early studies have used relatively crude mea-PD [90]. Animal nonclinical data support the poten- sures of mean TRF length on a Southern blot that is tial safety and utility of such cells [91]. Human clinical complicated by individual variations in the subtelo-trials of parthenogenetic pluripotent stem cell-derived meric X region of the fragments and/or incomplete dopaminergic neurons for the treatment of PD are cur- enzymatic digestion. Newer assay methods such as rently underway with initial results expected within qPCR-based assays, quantitative FISH (Q-FISH) in the decade. By contrast to PD, the pathology of AD interphase cells [100] or the measurement of individual is diffusely disseminated. However, localized degenera- telomeres [101] when implemented on a large scale may tion of the choroid plexus has been implicated in the provide more precise and reproducible results. Never-pathogenesis of the disorder. The transplantation of theless, it seems clear that telomere length decreases progenitors to the choroid plexus to increase the turn- with age in PBLs, reaching a length in centenarians over rate of cerebral spinal fluid may therefore provide a comparable to that observed in fibroblasts grown to the novel therapeutic strategy for AD. In the case of stroke, Hayflick limit in vitro [102].
nonclinical studies of the utility of embryonic neuroep- While the pathological impact of senescence in blood ithelium in regenerating damaged neocortex, as well as cells is still largely unknown, the correlation of short the use of hPS cell-derived cells to express neurotrophic telomeres in CD8+ lymphocytes in AIDS [103], suggests factors such as BDNF are currently underway.
that senescence may explain the similarities of AIDS and age-related immunodeficiencies [104]. In addition, Age-related metabolic disorders
it is common knowledge that aged individuals make Aging is often associated with profound alterations in relatively poor bone marrow stem cell donors [105]. metabolism such as generalized loss of subcutaneous Therefore, whether to provide young proliferation-fat, central visceral obesity, Type II diabetes, hyper- competent blood cells for adoptive immunotherapy or tension and atherosclerosis. It has been proposed that for reconstituting the entire hematopoietic system, hPS the age-dependent loss of white subcutaneous fat leads cell-derived grafts may benefit aged patients.
to decreased clearance of triglycerides and increased In addition, it has been suggested that circulating deposition in ectopic sites including coronary arter- endothelial progenitor cells (EPCs) originate from ies [92]. Progeria shows an almost a complete loss of bone marrow stem cells. These cells are believed to cir-peripheral subcutaneous adipose tissue also associated culate and repair injured endothelium throughout the with Type II diabetes and coronary disease [93] and body [106]. Since circulating EPC telomere length has similar alterations are seen in Werner syndrome [94].
been observed to decrease with age [107] and endothe- In addition to the age-dependent loss of subcuta- lial dysfunction has been implicated in a number of neous fat, there is a profound loss of brown adipose age-related changes in the vascular system including tissue with age [95]. It appears likely that these age- those associated with the highest risk of mortality in dependent redistributions of adipose tissue play an the USA [108], hPS cell-derived EPCs may also be a use-important role in aspects of the metabolic syndrome ful regenerative strategy. Old cows transplanted with in aging. The transplantation of subcutaneous fat or nuclear transfer-derived fetal liver CD34+ fetal hema-brown adipocytes may provide a novel modality to topoietic stem cells showed engraftment even without inducing weight loss and increasing triglyceride and ablation, and apparent incorporation of derivative cells glucose clearance [96]. Pluripotent stem cells may pro- into vascular endothelium [109].
vide a useful source of these cells if they can be reliably
manufactured in a highly purified and identified state. Conclusion
The transplantation of the cells in HyStem hydrogels The rapid increase in the number of elderly people
may promote survival of these grafts and promote around the world places an ever-increasing strain on
differentiation [97].
the healthcare infrastructure of many industrialized countries. The greatest expense is related to current The hematopoietic system
treatment modalities for chronic disease. In the USA The ease of access of blood cells for TRF length analy- alone, chronic disease accounts for some 80% of the sis has led to a plethora of studies of telomere length US$3 trillion expense annually [112]. Since most cur-as a function of age, age-related diseases [98] and even rent therapeutic regimens for chronic age-related lifestyle practices [99]. Many of these studies do not, diseases are largely palliative, they represent a major however, control for complex changes in composition source of the economic burden [110]. This demographic of circulating peripheral blood lymphocytes (PBLs) in trend underscores the growing need to understand response to such environmental factors such as expo- aging on a cellular and molecular level and to intro- Regen. Med. (2016) 11(3)
future science group The germline/soma dichotomy: implications for aging & degenerative disease Review duce novel cost-effective therapies. Understanding the lar tissue compare to adult or fetal-derived cell grafts? unifying cellular and molecular pathways underly- Another important safety question will be how safe are ing these pathologies could lead to innovative strate- cell grafts containing undefined cellular contaminants gies that could both alleviate human suffering and that while not pluripotent, may nevertheless be mul-reduce healthcare expenditures by collapsing the tipotent embryonic progenitors. What are those pro-time of morbidity, reducing frailty and increasing the genitors, do they survive in ectopic sites, and do they independence of the aged.
continue to differentiate to what would have been their It is possible that such unifying pathways can be iden- normal differentiated state? tified by examining the molecular mechanisms aging
holds in common with segmental progeroid syndromes. Future perspective
In particular, one intriguing avenue of study is where The rising number of elderly in the industrialized coun-
such age-related diseases as osteoporosis, skin aging, tries such as the USA and the associated escalation in
coronary disease, cataracts, Type II diabetes and lipoat-
the incidence of age-related degenerative diseases chal- rophy (diseases held in common between progeroid lenges the resources of our current healthcare system. syndromes and normal aging) are linked to genes that Therefore, there exists a significant and growing need for encode proteins with an apparent function in maintain- understanding the molecular and cellular underpinnings ing telomeres and hence related to cell replicative lifes- of aging and providing novel modalities for the cost- pan. However, the gerontological community has yet to effective treatment of its sequelae. An emerging therapeu-come to a consensus on these mechanisms [111].
tic strategy harnesses the natural replicative immortality Pluripotent stem cells appear to be naturally immor- of germline cells as it occurs in pluripotent stem cells, tal progenitors to all somatic cell lineages. To meet the as a renewable source of transplantable young cells that needs of many millions of individuals with chronic can be used to restore healthy function to aging tissues. degenerative conditions, we propose that master cell If modified to generate allogeneic off-the-shelf products, banks of pluripotent cells genetically modified to such technology has the potential to greatly reduce the escape immune surveillance and make off-the-shelf human and economic costs of chronic degenerative dis-universally transplantable allogeneic cells is likely the ease. Such therapies will require methods for the repro-most feasible strategy to manufacture cost-effective ducible manufacture of highly defined and purified product. These master cell banks can then be utilized cell types as well as biocompatible matrices to promote to produce clonal embryonic progenitors to simplify reliable regeneration of normal 3D tissue in vivo.
control of the quality (identity and purity) and the use of biocompatible matrices to increase survival of the Financial & competing interests disclosurecells and reduce undesired migration from the graft site This work was funded by BioTime, Inc., OrthoCyte Corpora-may increase the efficacy and safety of the therapies.
tion, ReCyte Therapeutics and OncoCyte Corporation (Al- In this review, we propose that hPS cells derived ameda, CA, USA), and Cell Cure Neurosciences, Jerusalem, from either preimplantation embryos or produced Israel. F Binette, H Sternberg and MD West are employees of from somatic cells induced into pluripotency (assum- BioTime, Inc. (Alameda, CA, USA). D Larocca is an employee ing the latter have reset telomere length back to of ReCyte Therapeutics (Alameda, CA, USA). KB Chapman is germline length) are comparable to those providing an employee of OncoCyte Corporation (Alameda, CA, USA). a lifetime of functionality to the tissues in the devel- C Irving is an employee of Cell Cure Neurosciences (Jerusalem, oped human. If products manufactured from these Israel). F Binette is also an employee of OrthoCyte Corporation cells retain the regenerative potential observed in the (Alameda, CA, USA). ReCyte Therapeutics, OncoCyte Corpo-embryonic phases of development, they may have even ration, Cell Cure Neurosciences and OrthoCyte Corporation more benefit to the patient than fetal or adult-derived are subsidiaries of BioTime Inc. The authors have no other rele-cells. Ultimately, the safety and efficacy of any one vant affiliations or financial involvement with any organization clinical applications of the foregoing will depend on or entity with a financial interest in or financial conflict with the complexities of individual pathology, genetic back- the subject matter or materials discussed in the manuscript ground and the unknown variable of heterochronic apart from those disclosed.
transplantation. Ongoing clinical trials of hES and No writing assistance was utilized in the production of this hiPS cell-derived therapeutics will likely yield data use- ful in evaluating the safety and efficacy in defined dis-ease states. However, many questions will remain to be Open accessanswered. Important questions relate to the safety of This work is licensed under the Attribution-NonCommercial-cellular grafts in a heterochronic setting. That is, how NoDerivatives 4.0 Unported License. To view a copy of this li-do young or even embryonic progenitors to a particu- future science group Review West, Binette, Larocca, Chapman, Irving & Sternberg Executive summary The cellular basis of age-related pathology
• The advent of cell biology in the 19th century led to the detailed description of the cellular composition of
tissues in the course of aging and the question of whether aging is caused by changes intrinsic or extrinsic to cells.
• August Weismann proposed that somatic cells, as opposed to germline cells have an intrinsic clocking mechanism that limits their replicative capacity while the germline maintains the immortal propagation of individuals.
• The finite replicative capacity of human somatic cells was first demonstrated by Hayflick in the 1960s and cells from premature aging disorders such as progeria and Werner syndrome were shown to have a shortened cell lifespan.
• The senescence of cells may lead to pathology either directly through the loss of cells or from associated alterations in gene expression such as the senescence-associated secretory phenotype.
• The telomere hypothesis first proposed by Olovinokov now has significant support as a fundamental cause of the dichotomy of mortality and immortality in somatic versus germline cells, respectively.
The pluripotent stem cell manufacturing platform
• Pluripotent stem cells express relatively high levels of TERT and telomerase activity and generally maintain
telomeres at a length comparable to that of sperm, but repress TERT expression upon differentiation into somatic cell types.
Rescue from developmental aging
• Somatic cell nuclear transfer techniques, like normal reproduction, are capable of resetting telomere length
and cell lifespan to aged somatic cells.
• The reprogramming of aged somatic cells by the administration of exogenous transcriptional regulators can reverse the developmental aging of the cells (i.e., reset telomere length as well as differentiation to pluripotency).
• The use of immortal and pluripotent master cell banks allows endless rounds of precise genetic modification followed by clonal expansion, with subsequent differentiation into diverse therapeutically useful cell types.
The differentiation of human pluripotent stem cells into therapeutic products
• The use of pluripotent stem cells to derive therapeutic cell formulations is complicated by the vast diversity
and relative lack of precise markers of the thousands of derivative cell types. The long telomere length of pluripotent stem cells allows the clonal expansion of primitive embryonic progenitors simplifying manufacturing protocols.
Injectable biocompatible matrices
• Chemically modified hyaluronic acid and collagen-based matrices allow in vivo covalent cross-linking of cells in
3D constructs, thereby increasing postengraftment survival while decreasing migration to undesired ectopic sites.
Potential therapeutic applications in age-related degenerative disease
• Skin aged in vivo shows markers consistent with a role of cell senescence in the pathological changes
observed. The identification of clonal embryonic progenitors that display an embryonic, as opposed to fetal/adult phenotype may promote a scarless regenerative potential.
• Senescent markers in the weight-bearing joints of patients with osteoarthritis or intervertebral joint degeneration may benefit from embryonic progenitors to those tissues capable of engrafting and regenerating healthy histology.
• Age-related macular degeneration is characterized by a loss and/or dysfunction of the retinal pigment epithelium. The replacement of such cells which play an important role in inhibiting angiogenesis as well as supporting the function of the neural retina may therefore provide an important therapeutic strategy to slow the progression of the dry form of the disease.
• Age-related neurodegenerative disorders such as Parkinson's disease may benefit from the transplantation of midbrain dopaminergic neurons. Clinical trials using human pluripotent stem cell-derived formulations are expected to begin trials within the decade.
• Metabolic disorders such as Type II diabetes and central obesity may benefit from the transplantation of brown adipocytes. The formulation of such cells in an injectable matrix such as that comprised of cross-linkable hyaluronic acid and collagen may provide the means of establishing a therapeutic dose of these cells to lower circulating glucose levels and cause weight loss.
• Nucleated blood cells are well documented for age-related telomeric attrition. In addition, protocols are well established for the transplantation of hematopoietic stem cells. The transplantation of histocompatible young hematopoietic stem cells may benefit many patients currently needing such therapy.
Regen. Med. (2016) 11(3)
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