表現人類終端酶藉由表觀基因回復骨髓幹細胞多能分化性及抑制自然分化

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國立臺灣大學醫學院臨床牙醫學研究所牙周病學組碩士論文

Department of Periodontology, Graduate Institute of Dental Sciences College of Medicine

National Taiwan University Master Thesis

Epigenetic Alchemy for Reversion of Pluripotency and Blockage of Spontaneous Differentiation by Ectopic Expression of Telomerase

陳俊利 Chun-Li Chen

指導教授：侯連團教授洪士杰教授

Thesis Advisors: Lien-Tuan Hou, Professor Shih-Chieh Hung, Professor

中華民國 97 年 1 月

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國立臺灣大學碩士學位論文口試委員會審定書

Epigenetic Alchemy for Reversion of Pluripotency and Blockage of Spontaneous Differentiation by Ectopic

Expression of Telomerase

本論文係陳俊利君（R94422004）在國立臺灣大學醫學院

臨床牙醫學研究所牙周病學組完成之碩士學位論文，於民國

97 年 01 月 25 日承下列考試委員審查通過及口試及格，特此

證明。

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Acknowledgements

首先誠摯的感謝指導教授侯連團博士及洪士杰博士，兩位老師悉心的教導使我得以一窺幹細胞領域的深奧，不時的討論並指點我正確的方向，使我在這些年中獲益匪淺。老師對學問的嚴謹更是我輩學習的典範。

本論文的完成另外亦得感謝實驗室的孫元蕙助理大力協助。因為有妳的體諒及幫忙，使得本論文能夠更完整而嚴謹。

三年裡的日子，實驗室裡共同的生活點滴，學術上的討論、言不及義的閒扯、讓人又愛又怕的宵夜、趕作業的革命情感...，感謝眾位學長姐、

同學、學弟妹的共同砥礪，你/妳們的陪伴讓三年的研究生活變得絢麗多彩。

感謝秘心吾、李泰億、王振穎、劉錦龍、余瑞成學長們不厭其煩的指出我研究中的缺失，且總能在我迷惘時為我解惑，也感謝邱慧緣同學的幫忙，恭喜我們順利走過這三年。

父母親在背後的默默支持更是我前進的動力，沒有他們的體諒、包容，相信這三年的生活將是很不一樣的光景。

最後，謹以此文獻給我摯愛的雙親。

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Abstract

Background: Cellular senescence and spontaneous differentiation are critical phenomena that impair long-tern study of cells in vitro. The same problems are similarly encountered in the investigation of human mesenchymal stem cells (hMSCs), despite its high proliferation rate and multipotency comparing to those of differentiated cells derived from different tissues. The present study was desigmed to overcome these problems and also explored to drive hBMSCs toward stemness.

Materials and Methods: Primary hBMSCs, E6E7-transfected hBMSCs (KP cells) and Htert transfected cells (3A6) were used in the present study under regular culture condition. Flow cytometry using many stem cell markers was applied to screen the similarity of studied cell types. Major targeted cells, 3A6, were generated by insertion of phTERT-IRES2-EGFP gene fragment using Nucleofector technology. Functional analysis on differentiation and dedifferentiation of all investigationed hBMSCs, KP and 3A6 were evaluated by RT-PCR of osteogenic, neural and adipogenic gene messages cytochemical staining and in vitro mineralization. Mater embryonic transcription gene markers, such as Oct4 and Nanog etc, and germline differentiation were further examined by RT-PCR, in unstimulated and stimulated BMP4 and RA condition.

Stemness related evidence and possible mechanism were explored by measurements of demethylation level of genomic DNA with CpG island microarray and level of various

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demethylation enzymes, such as DNMT1, 3A, 3B and EZH2 by real-time PCR between 3A6 and KP cells. Further relative relashinship between 3A6 and ESC was investigated by microarray expression data sets and principal component analysis in Affymetrix^TM U133 and principal compared with public accessible array databasesat Gene Expression Omnibus (GEO) (http: //www.ncbi.nlm.nih.gov/geo/).

Results: Ectopic expression of telomerase in a previously developed hMSC line was found to enhance pluripotency and block spontaneous differentiation of the cells.

Surprisingly, the telomerase-transfected hMSCs (3A6) had a differentiation potential far beyond the normal hMSCs. They expressed trophoectoderm and germline specific markers in vitro, similar to those of embryonic stem cells, upon perturbations with BMP4 and retinoic acid, respectively. Furthermore, the telomerase-transfected hMSCs (3A6) displayed higher osteogenic and neural differentiation efficiency than their parental cells did, while there was a decrease in DNA methylation level as proved by a global CpG island methylation profile analysis. Possible underlying mechanisms were assayed by DNA methylation and its regulation enzymes. Notably, the demethylated CpG islands were found to be highly associated with development and differentiation associated genes.

Principal component analysis further pointed out the expression profile of the cells converged toward embryonic stem cells.

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Conclusions: In addition to the preservation of stem cells’ characteristics in hTERT-transduced hMSCs, our present data also demonstrated the first pilot evidence that the reversion of pluripotency and blockage of spontaneous differentiation of hMSCs could be initiated in immortalized hBMSCs cell line by ectopic expression of telomerase.

Key words: Human mesenchymal stem cells; bone marrow stromal cells; telomerase;

hTERT; pluripotency; spontaneous differentiation; CpG island; DNA methylation;

DNMT; embryonic stem cells

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Introduction

hBMSCs (human bone marrow stromal cells) based gene and cell therapy is one of the most promising and prospecting field of medicine because of their great self-renewal and versatile plasticity in vitro and in vivo (Gerson, 1999; Pittenger et al., 1999).

Discovery of adult stem cell-derived pluripotent cells may bring a tantalizing castle into reality due to their ease of collection and versatility. Two important and interesting recent reports showed that Oct4 and Nanog characteristically expressed in embryonic stem cells (ESCs) could be detected in hBMSCs such as MAPCs (Jiang et al., 2002) and MIAMIs (D'Ippolito et al., 2004). These mesodermal originated cells were proven to be able to cross the germ layer boundaries and differentiated into ectoderm and endoderm successfully.

Though the nature plasticity of hBMSCs needs to be further elucidated in vivo, these amazing results have successfully challenged the traditional concept that the differentiation capacity of hBMSCs can be only limited to their resident local environment. Beyond the differentiation limitation, the pity comes from the rare distribution of hBMSCs in bone marrow (Friedenstein et al., 1982; Wexler et al., 2003) which hampers the clinical application and makes it necessary to expand the populations of hBMSCs in vitro. In addition, there are still two major difficulties encountered during in vitro expansion: cellular senescence and spontaneous differentiation.

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Cellular senescence, also referred as cellular aging, could be defined as a cell’s diminished replicative capacity and altered functionality (Beausejour, 2007). The phenomenon of cellular senescence causes changes of cells in view of physiological, functional, and molecular parameters (decreased transcription, decreased translation and decreased proteolysis; increased abnormal proteins and increased lipofucsin; increased number of both abnormal nuclei and size of lysosomes; elevated number of chromosomal abnormalities and reduced response to hormones) (Dice, 1993) during long-term cultures.

These changes include typical Hayflick phenomenon of cellular aging, gradual decreasing proliferation potential, shortening telomere and impairment of functions (Bonab et al., 2006). Due to the nature cellular senescence, the expansion of hBMSCs would be limited up to 30~40 PDs (Banfi et al., 2000; Baxter et al., 2004; Stenderup et al., 2003) which severely impeded the application of these cells in clinical regenerative medicine. Meanwhile, literatures have demonstrated that decreased differentiation efficiency could be caused by cellular senescence in adipogenesis and osteogenesis (Bonab et al., 2006; Conget and Minguell, 1999; Digirolamo et al., 1999). Cellular senescence has been known to be generally associated with reduced expression of the human telomerase reverse transcriptase (hTERT) gene and shortened length of telomere (Bodnar et al., 1998). Most primary isolates of hBMSCs have deficient or limited detectable level of telomerase activity which contributes to limited replication life cycle

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during in vitro expansion (Bonab et al., 2006).

Another important topic of great interest and clinical significance may be to keep the hBMSCs in a more quiescent state because their spontaneous differentiation overtly observed by several reports. Whether in rat or human BMSCs, several literatures have perceived spontaneous emergence of some lineage-specific genes or proteins expression in culture without any induced differentiation. The phenomenon included spontaneous adipogenesis (Seshi et al., 2000), osteogenesis (Seshi et al., 2000), myogenesis (Seshi et al., 2000) and neurogenesis (Li et al., 2007; Tondreau et al., 2004; Tseng et al., 2007).

The most striking observation came from Woodbury (Woodbury et al., 2002) who found that in rat BMSCs, genes specific for all the three embryonic germ layers and germline cells were actively transcribed without any induction of differentiation. For exploring the possibility that if the gene expression of all three germ layers and germinal tissues were the consequence of mixed population of cells in primary BMSCs cultures, Woodbury (Woodbury et al., 2002) further assessed the expression of these representative genes in a clonal MSC line derived from a single cell. Interestingly, the clonally derived BMSCs recapitulated the gene expression pattern of primary BMSCs cultures, which suggested the spontaneous multidifferentiation capacity of individual BMSCs in cultures. In the present study, not only various lineage-specific genes, several developmental markers, such as Pax6, Gata4, Gata6, FoxA2 and Sox17, which designates early embryonic

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development could also be detected in primary cultured hBMSCs. Enhanced expression of these genes from P3 to P10 hBMSCs confirmed the concept of spontaneous differentiation of hBMSCs during long term culture and the concurrent downregulation of embryonic markers, such as Oct-4 and Nanog. These data implicated the loss of pluripotency at the same time. Albeit important in clinical application, the potential problem of spontaneous differentiation is less, if any, investigated.

In need of great amount of cell source for stem cell-based tissue engineering, it seems remarkably significant to crave for resolutions dealing with cellular senescence and spontaneous differentiation of hBMSC, that is, to keep the stemness of hBMSCs during long term culture. In previous study, we attempted to conquer the obstacle mentioned above by transferring of HPV16 E6/E7 genes into hMSCs isolated from bone marrow (Hung et al., 2002), and established a cell line-KP. Establishment of KP did successfully overcome the drawback of its cellular senescence in that it could be passaged over 100 PDs. Unfortunately, the phenomenon of spontaneous differentiation could not be avoided, which still caused the loss of stemness of KP during long term culture.

It has been mentioned for a long time that the role of telomerase in maintaining telomere length and immortality of embryonic stem cells (Amit et al., 2000; Thomson et al., 1998). But in hBMSCs, with exception of MAPCs (Jiang et al., 2002) and MIAMIs (D'Ippolito et al., 2004), no telomerase activity could be detected (Zimmermann et al.,

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2003), which contributed to the shortening of telomere at a rate similar to that of non- stem cells (30–120 bp/ PD) (Fehrer and Lepperdinger, 2005). hBMSCs are found to cease dividing at telomere length about 10 kb (Baxter et al., 2004). Ectopic expression of hTERT in normal human cells, however, has been documented to be a strategy to bypass cellular senescence and to extend life span of cells (Bodnar et al., 1998; Broccoli et al., 1995). In addition to cell replication, current interest and attention seems to focus on the role of hTERT in regulation stem cell biology. Overexpression of hTERT in ES cells was reported to enhance differentiation capacity toward hematopoietic lineage (Armstrong et al., 2005), and ectopic expression of hTERT in hBMSCs were described to enhance osteogenic differentiation as well (Gronthos et al., 2003a; Shi et al., 2002; Simonsen et al., 2002). Notably, a recent report revealed an interesting phenomenon that with increasing expression of lineage-determining gene, such as cbfa1, expression level of hTERT decreased upon osteogenic differentiation due to an inhibition effect of cbfa1 on hTERT transcription (Isenmann et al., 2007). The inverse relationship between the presence of hTERT and differentiation-related genes was also observed by other groups (Wang et al., 2007). An interesting question is that if this inverse relationship is still valid when the expression level of hTERT is intentionally elevated by ectopic introduction of hTERT gene to hBMSCs? In our pilot study, we did observe downregulation of a variety of lineage specific genes after hTERT transduction, and we postulated that this phenomenon

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might contribute to the reversion of stemness of hBMSCs that was elicited by ectopic expression of hTERT. The recovery of stemness by ectopic expression of hTERT, in our hypothesis, would be represented by conversion of hBMSCs from a commitment to a quiescent state with preservation of versatile differentiation capacity and enforced differentiation efficiency. In an attempt to clarify the hypothesized dedifferentiated effect on BMSCs by the insertion of hTERT, the establishment of hTERT-expressed hBMSCs is necessary. In our study, hTERT gene was not transferring into primary culture hBMSCs, but rather, to E6E7 expressed cell line-KP, due to the current evidence that hTERT alone might be insufficient to bypass the cellular senescence (Okamoto et al., 2002) and that extreme difficulty in selecting and expanding successfully transduced single cell clone in primary cultured hBMSCs. Furthermore, transducing hBMSCs with both hTERT and E6E7 had been suggested as a better strategy to circumvent cellular senescence (Okamoto et al., 2002).

With progressing of knowledge about gene regulation through epigenetic mechanism, including DNA methylation and chromatin modification, the globalized gene silencing phenomenon observed after ectopic expression of hTERT needed to be elucidated. In mammals, methylation of cytosines in cytosine guanine dinucleotide (CpG) island had been well-known to play a crucial role in mediating epigenetic gene silencing (Boyes and Bird, 1991; Watt and Molloy, 1988). Four DNA methyltransferases (DNMTs)

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have been identified so far in mammals. Among them, DNMT1 has been known to maintain the pre-existing methylation state of genome during DNA replication (Leonhardt et al., 1992). DNMT3a and DNMT3b are de novo methyltransferases which would target unmethylated CpG sites (Okano et al., 1999). DNMT2, which has been shown to exhibit weak methyltransferaseactivity in vitro (Hermann et al., 2003), would be responsible to methylate tRNA(Goll et al., 2006). In addition to DNMTs, in this study, we try to prove our hypothesis that in hBMSCs, through some mechanism, ectopic expression of hTERT may modulate stemness genes and down-regulate development- associated and lineage-determining genes, and thus increases stemness of hBMSCs by shifting from a tissue-committed state to a more quiescent state.

Meanwhile, we also try to confirm the notion that after reversion from a commitment to a more quiescent and primitive state of the hTERT-transduced hBMSCs, these cells still retain versatile differentiation potential, even shifting toward germline and trophoectoderm differentiation, which were characteristics merely to hESCs in previous studies.

Finally, we explore to verify underlying mechanism that whether alterations mentioned above after hTERT transduction are attributed to the influence of hTERT on the methylation state of the whole genome, especially the genes responsible for early development and lineage-determining. If this postulation was true, the hypermethylation

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of these genes would explain the phenomenon of gene silencing and keep hBMSCs in pluripotent status after hTERT transduction in the present study.

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I. Review of Literature

1. Cell Biology of Bone Marrow Stromal Stem Cells (BMSCs)

A. Definition of Stem Cells in Adult Bone Marrow (BM)

The basic definition of the term “stem” refers to the ability of progenitor cells to divide and produce an undifferentiated daughter cell, which is designed as “self-renewal”, and meanwhile, to produce another daughter cell that is differentiated or divides then differentiate (Sell, 2004). It is extremely hard, however, to prove that the so called “stem cells” to be able to self-renew. For this reason, the expandable population of mesenchymal cells with differentiation capacity is termed mesenchymal progenitor cells by some groups while others utilize traditional terminology based on their field of study (Sell, 2004).

Friedenstein and Owen et al. were the first to characterize cells that compose the physical stroma of bone marrow (Friedenstein et al., 1970; Friedenstein et al., 1966;

Owen, 1988). Friedenstein et al.(Friedenstein et al., 1966) observed the formation of cartilage and bone within the diffuse chamber and demonstrated that bone marrow had the potential to form bone and cartilage. Furthermore, he noticed that the osteogenic potential of bone marrow derived from a specific subgroup of cells, and when these cells were plated at low density, they rapidly adhere and could be easily separated from the

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nonadherent hematopoietic cells by repeated washing, and accordingly, these cells were termed colony-forming unit-fibroblast (CFU-f) (Friedenstein 1973). Friedenstein later illustrated that approx 30% of isolated CFU-f colonies were able to form bone alone or bone with the microenvironment necessary for the formation of hematopoietic element (Friedenstein, 1980). The first concept of “stem cells” residing in bone marrow was presented by Owen (Owen, 1978). He (Owen, 1978), based on his hypothesis, expanded and proposed a model for the stromal lineage that contained “stem cells”, “committed progenitors” and “maturing cells” compartments, and included a lineage diagram for

“stromal stem cells” that included “reticular”, “fibroblastic”, “adipocytic,” and

“osteogenic” cells as end-stage phenotypes (Sell, 2004).

The main source of MSCs is in the bone marrow, which has been conventionally regarded as an organ composed of two main systems according to distinct resided cell lineages; the hematopoietic tissue proper and the associated supporting stroma (Bianco et al., 2001). Even though, the MSCs only constitute a small percentage of whole population of bone marrow cells. Only 0.01% to 0.001% of mononuclear cells isolated on density gradient (ficoll/percoll) medium give rise to plastic adherent fibroblastic-like colonies (Pittenger et al., 1999). Even in the same donor, the numbers of bone marrow MSCs are different upon repeated puncture in view of the yield and the quality (Phinney et al., 1999).

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The BMSCs are postulated to arrive in bone marrow by three different mechanisms (Sell, 2004). First, they can enter bone marrow along with vasculature. Second, they can migrate into the marrow space after vascularization along the vessel paths (Nakahara et al., 1992; Yoo and Johnstone, 1998). In fact, the first two mechanisms differ only in the timing of the entrance of BMSCs into bone marrow, but not paths of arrival or migration.

Third, they can arrive through the blood proper, suggesting the existence of MSCs in circulation, and meanwhile the circulating MSCs endows the possibility of delivering reparative MSCs to repair adult tissue (Sell, 2004).

Besides bone marrow, MSCs are also reported to be located in other tissues of the human body (Bobis et al., 2006), such as adipose tissue (Gronthos et al., 2001), umbilical cord blood, chronionic villi of the placenta (Igura et al., 2004), amnionic fluid (Tsai et al., 2004), peripheral blood (Zvaifler et al., 2000), fetal liver (Campagnoli et al., 2001; in 't Anker et al., 2003), lung (in 't Anker et al., 2003), and even in exfoliated deciduous teeth (Miura et al., 2003). Althrough MSCs are widly distributed in the whole body, the amount of MSCs decreases with aging (Fibbe and Noort, 2003) and infirmity (Inoue et al., 1997). Their presence reaches greatest amount in neonate and reduces in number during the later lifespan, which is about one-half of the neonate stage at the age of 80 (Fibbe and Noort, 2003). The circulating fetal MSCs also reach highest number in the first trimester

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and declines during the second trimester to about 0.0001% and 0.00003% of the nucleated cells in cord blood (Campagnoli et al., 2001).

B. Heterogeneity of the BMSCs

Some evidences clearly indicated a heterogeneous nature of the BMSCs population (Kucia et al., 2005a; Kucia et al., 2005c; Ratajczak et al., 2004), which was clarified by the presence of different properties of individual colonies, such as colony sizes, growth rates and cell morphologies ranging from fibroblast-like spindle-shaped cells to large flat cells (Bianco et al., 2001). In addition, if the cultures are allowed to develop for more than 20 days, some colonies are positive for alkaline phosphatase (ALP) stain, while others are either negative or positive in the central region and negative in the periphery (Friedenstein et al., 1982). Some colonies formed nodules which can be identified by Alizarin Red S or von Kossa staining for calcium (Bianco et al., 2001). Some colonies could still accumulate fat and were identified by oil red O staining (Herbertson and Aubin, 1997) or formed cartilage which can be stained with alcian blue (Berry et al., 1992).

C. Surface Markers on BMSCs

Besides cell morphology and physiology, expression of cell surface antigens is another evidence of BMSC’s heterogeneity, and to date, no single specific marker which

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designates BMSCs has been identified (Bobis et al., 2006). BMSCs express a large number of adhesion molecules, extracellular matrix proteins, cytokines and growth factor receptors associated with their function and cell interactions within the bone marrow stroma (Devine and Hoffman, 2000). The population of BMSCs isolated from bone marrow express: CD44, CD105 (SH2; endoglin), CD106 (vascular cell adhesion molecule; VCAM-1), CD166, CD29, CD73 (SH3 and SH4), CD90 (Thy-1), CD117, STRO-1 and Sca-1 (Boiret et al., 2005; Conget and Minguell, 1999; Dennis et al., 2002;

Gronthos et al., 2003b). To another aspect, BMSCs do not possess markers typical for hematopoietic and endothelial cell lineages: CD11b, CD14, CD31, CD33, CD34, CD133 and CD45 (Pittenger et al., 1999). The absence of CD14, CD34 and CD45 enable us to distinguish BMSCs from the hematopoietic precursors (Baddoo et al., 2003). BMSCs are also known to express a set of receptors associated with matrix- and cell-to-cell adhesive interactions, like integrins αß3 and αß5, ICAM-1, ICAM-2, LFA-3 and L-selectin

(Boiret et al., 2005; Conget and Minguell, 1999; Pittenger et al., 1999).

Some surface antigens, however, may change during the culturing process due to specific culture conditions, components (especially growth factors added) in the medium and the duration prior to individual passages (Dazzi et al., 2006). The alteration of chemokine receptor expression could also occur during passage of human BMSCs (Honczarenko et al., 2006). CCR1, CCR7, CCR9, CXCR4, CXCR5 and CXCR6 were

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expressed at the second passage of BMSCs but all of these molecules disappeared at the 12-16^th passages accompanied with the disability of the cells to migrate towards specific chemokine attractants (Honczarenko et al., 2006). Meanwhile, loss of the expression of chemokine receptors was accompanied with a decrease in the expression of adhesion molecules, such as ICAM-1, ICAM-2, VCAM-1 and CD157 (Honczarenko et al., 2006).

Moreover, the changes of BMSCs’ phenotype were relevant to increasing cell cycle arrest and induction of apoptotic pathway (Honczarenko et al., 2006). With the progression of cellular differentiation of BMSCs, the alteration of one surface antigen has also been observed that CD166 (activated leukocyte cell adhesion molecule) presented on undifferentiated BMSCs is absent from the cells that differentiate toward osteogenic lineage (Bruder et al., 1998).

The antibody STRO-1 (Simmons and Torok-Storb, 1991) has, so far, been the most useful antibody for identifying and selecting for positively MSCs in bone marrow (Sell, 2004). The STRO-1 marker was successfully used to isolate the CFU-f cells from marrow (Simmons et al., 1994), and STRO-1-selected cells had been shown to be osteogenic (Gronthos et al., 1994), chondrogenic, adipogenic, and hematopoiesis supportive (Dennis et al., 2002). The STRO-1 staining could be used to characterize BMSCs, and sort cells by magnetic activated cell sorting (Dennis et al., 2002; Gronthos et al., 1994; Tamayo et al., 1994).

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D. Morphology, Growth and Expansion of BMSCs

BMSCs that initially adhere to plastic culture dish have fibroblastic appearance and shift to symmetrical colony after plating for 5-7 days (Sell, 2004). Although various culture protocols of BMSCs have been published, these cells displayed most rapid proliferation and retained their optimal multipotency when cultured at low density (Sekiya et al., 2001). Culture density not only affects growth but also cell morphology of BMSCs. Tropel et al. observed that BMSCs displayed a spindle-like shape at low density, but they started to grow in several layers and become flat in cell shape with torn ends at confluence (Tropel et al., 2004). Growth of BMSCs could be characterized by the occurrence of three phases (Bruder et al., 1997; Colter et al., 2001): (1) an opening lag phase of 3-4 days; (2) a rapid expansion (log phase); (3) a stationary phase (growth plateau).

BMSCs have been known to maintain in culture for 20-30 population doublings with their capacity of differentiation under proper conditions (Friedenstein et al., 1970).

Recent studies even showed that BMSCs could be maintained in culture for more than 50 population doublings (Ramalho-Santos et al., 2002) which indicates the great proliferative potential of these cells. According to a study examining the profile of BMSCs cell cycle, major part of these cells remained in the G0/G1 phase and only 10%

in phase S (Conget and Minguell, 1999). Notwithstanding a report had observed that

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BMSCs could maintain normal karyotype and telomerase activity even at passage 12 (Pittenger et al., 1999), extensive subcultiavation of BMSCs impaired their functionality and confronted them with the crisis of senescence and apoptosis (Conget and Minguell, 1999).

A number of cytokines, growth factors, hormones and other molecules have been known to impact on proliferation of BMSCs (Bobis et al., 2006). PDGF, FGF-2 and EGF have been proved to be potent mitogens for BMSCs (Bruder et al., 1997). In contrast, the addition of interferon-alpha and interleukin 4 to the culture could raise opposite results (Bruder et al., 1997; Jeong et al., 2005). The proliferative activity of BMSCs has been shown to be in proportion to their increased potential of late differentiation potential (Prockop et al., 2003).

E. Cross-over Plasticity of BMSCs

According to the previous study results, it was believed that BMSCs could differentiate only to mesodermal tissues (Bobis et al., 2006). Many recent data, however, has revealed that these cells could differentiate into various kind of lineages rather than only mesodermal origin, such as hepatocytes and even neurons (Jiang et al., 2002; Pittenger et al., 1999). The former hypothesis claimed that the plasticity of BMSCs was attributed to the stochastic repression/induction model that various sets of BMSCs occurred upon a

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series of gene silencing events during development (Dennis and Charbord, 2002).

Whatsoever, the stochastic repression/induction model was not generally accepted due to different data from other researchers, and there were alternative explanations of the phenomenon of stem cell plasticity. Based on the previous data of transdifferentiation, three important explanations have been proposed (Kucia et al., 2005a). First, some authors assumed that the alteration of BMSCs phenotype was caused by cell fusion with other lineages of cells (Alvarez-Dolado et al., 2003; Terada et al., 2002; Ying et al., 2002), and the fused cells retained signatures of both parental cells. The drawbacks of this hypothesis, however, come from the facts that cell fusion is an extremely rare phenomenon and preferentially to cells with polyploidity, such as hepatocytes, Purkinje cells and skeletal muscle cells (Kucia et al., 2005a). Additionally, several recent publications demonstrating plasticity of BMSCs also excluded the possibility that cell fusion was a cause of cell transdifferentiation (Almeida-Porada et al., 2004; Harris et al., 2004; Jang and Sharkis, 2004; Shefer et al., 2004; Wurmser et al., 2004).

Second, epigenetic alterations might occur in BMSCs in response to external stimuli under some circumstances, and thus modulate the expression of some early development and lineage-determining genes. Such a mechanism could be happened during reproductive/therapeutic cloning because some studies observed that nuclei isolated from differentiated somatic cells were plastic and might be reprogrammed and trans-

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differentiated when injected into cytoplasm of enucleated oocyte (Hochedlinger and Jaenisch, 2003; Kucia et al., 2005a). Accordingly, when stem cells isolated from original physiological environment are exposed to stress factors associated to culture condition in vivo, epigenetic alteration may possibly take placed (Kucia et al., 2005a).

The most convincing explanation of BMSCs, plasticity may originate from the concept that in addition to hematopoietic stem cells, other scarce subpopulations of versatile tissue-committed stem cells (TCSCs) and even more primitive pluripotent stem cells (PSCs) perhaps accumulate in bone marrow during ontogenesis (Kucia et al., 2006a;

Kucia et al., 2005a; Kucia et al., 2005b; Kucia et al., 2006b; Kucia et al., 2005c; Kucia et al., 2007; Kucia et al., 2006d; Ratajczak et al., 2007). The basis of this model emphasizes the presence of heterogeneous population of stem cells at different differentiation level from PSCs to TCSCs, and these cells are reservoir of primitive cells for tissue repair and organ regeneration, which can migrate from BM to peripheral blood after tissue injury or organ damage (Kucia et al., 2005a). The establishment of this model challenges the former concept that pluripotent stem cell is characteristic only to embryonic stem cell during embryogenesis. Recently observations of several studies support the presence of positive embryonic markers, such as Oct-4 (Niwa et al., 2000), Nanog (Mitsui et al., 2003) and SSEA (Muramatsu and Muramatsu, 2004) in the PSCs of BM. Among these embryonic markers, Oct-4 is the most pivot in that it is an essential embryonic

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transcription factor that plays a critital role in specification of ES cells (Boiani and Scholer, 2005; Hay et al., 2004) and is downregulated during development (Kucia et al., 2007). To date, various groups have identified Oct-4 positive stem cells in BM, such as very small embryonic-like (VSEL) cells, multipotent adult progenitor (MAPC) cells, multipotent mesenchymal stromal (MSC) cells and marrow-isolated adult multilineage inducible (MIAMI) cells. These later cell types derived from BM not only display potential in vitro and in vivo to differentiate into cells from all three germ layers (D'Ippolito et al., 2004; Jiang et al., 2002); mesoderm, ectoderm and endoderm.

Unexpectedly and strikingly, BM has also been recently identified as a potential sources of precursors of germ cells (Johnson et al., 2005; Nayernia et al., 2006). In addition to bone marrow, Oct-4 positive stem cells have also been discovered in many other tissues (Kucia et al., 2007; Ratajczak et al., 2007) such as epidermis (Dyce et al., 2004; Yu et al., 2006), bronchial epithelium (Ling et al., 2006), myocardium (Mendez-Ferrer et al. 2006), pancreas (Danner et al., 2007; Kruse et al., 2006), testes (Guan et al., 2006; Kanatsu- Shinohara et al., 2004), retina (Koso et al., 2006) and amniotic fluid (De Coppi et al., 2007). These findings not only consolidate the theory of developmental deposition of Oct-4 positive PSCs in developing organs (Ratajczak et al., 2007), but also suggest that these cells might circulate in the peripheral blood and shuttle between BM and other organs during tissue injury or organ damage (Eghbali-Fatourechi et al., 2005; Gomperts

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et al., 2006; Kucia et al., 2004; Kucia et al., 2006c; Palermo et al., 2005; Togel et al., 2005).

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2. Aging and Senescence of MSCs

A. Definition of aging mesenchymal stem cells (MSCs)

Due to the aforementioned rare distribution of adult mesenchymal stem cells (MSCs), in vitro cultivation and expansion of MSCs cannot be avoided for the purposes of experimental study. Although much promise of clinical applications of MSCs has been proposed, the senescence, or cellular aging of MSCs during long-term culture significantly impeded the propagation of their therapeutic use. Consequently, the resulting loss of proliferation and functionality of MSCs caused by cellular aging contrasts with those of the immortal embryonic stem cells, which results in the major hindrance of clinical utilization of adult MSCs. The aging discussed here is restricted to

“in vitro aging”, which happens during prolonged cultivation in vitro. Cellular aging, also referred as cellular senescence, is generally defined as a cell’s diminished replicative capacity and altered functionality (Beausejour, 2007). The phenomenon of cellular senescence was firstly described as replicative senescence by Hayflick (Hayflick and Moorhead, 1961) and defined as “an essentially irreversible arrest of cell division”.

Notably, arrest of growth in senescent cells to over time would negatively influence the ability of renewable tissues to replace damaged or dysfunctional cells and thus reduced their capacity for tissue repair (Beausejour, 2007; Smith and Pereira-Smith, 1996;

Stanulis-Praeger, 1987). In addition, recent reports also revealed that these metabolically

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active senescent cells might accumulate over time (Herbig et al., 2006) and secret biologically active molecules which would affect environment of normal tissue and behavior of neighboring cells (Krtolica et al., 2001).

B. Influence of aging on MSCs

Various changes of physiological, functional, and molecular parameters have been described in senescent stem cells, and these changes included typical Hayflick phenomenon of cellular aging, gradual decreasing proliferation potential, telomere shortening and impairment of functions (Bonab et al., 2006). It has been reported that several characteristics of senescent stem cells could be regarded as markers of aged MSCs. Firstly, some authors noticed that aged MSCs exhibited larger size (Baxter et al., 2004; Mauney et al., 2004; Stenderup et al., 2003) with more pseudopodia (Mauney et al., 2004), further spreading and marked intracellular actin stress fibers than non-senescent parts (Stenderup et al., 2003). In fact, it has been long discovered that increase in cell size is often associated with senescence (Hayflick and Moorhead, 1961). The MSCs from older patients or young patients do display different morphology in culture that MSCs from young donors exhibit spindle-type morphology and this characteristic fades over long-term cultivation (Baxter et al., 2004). In contrast, when MSCs are immortalized by either SV40 (Negishi et al., 2000) or telomerase (Kobune et al., 2003), these cells shift considerably to a smaller size than original cells they derived from.

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Secondly, another significant phenomenon occurs in senescent MSCs is a gradual decreasing proliferation potential. When actual age of a culture is recorded by population doublings (PDs), it has been reported that a single-cell-derived colonies of MSCs can be expanded up to as many as 30~50 PDs in about 10~18 weeks (Bonab et al., 2006; Colter et al., 2000; Stenderup et al., 2003). Moreover, it has also been revealed that growth curve relationship between cumulative PDs and duration of culture demonstrates a relatively linear decreasing rate of PDs with the progression of time. An appreciable decrease in the number of PDs was seen in the latter period of culture (more than 130 days in culture), implicating that proliferative potential of MSCs decreased remarkedly after 120 days in vitro expansion (Bonab et al., 2006).

Thirdly, when it comes to aging MSCs, one of the most puzzling issues might be the differentiation potential of aged MSCs. Differentiation into various lineages has been used as a marker for the multipotential nature of these cells (Pittenger et al., 1999), and such multipotency of MSCs seems to change with age. Alteration of osteogenic and adipogenic differentiation capacity of MSCs with age, especially the osteogenic potential of aged MSCs have been extensively investigated. Although there is conflicting evidence with some groups reporting no change, majority of studied disclose an age-related decreased potential of osteogenic differentiation potential of MSCs in their late passages (Bonab et al., 2006; Conget and Minguell, 1999; Digirolamo et al., 1999). Regarding to

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adipogenetic differentiation, it has been revealed early (Meunier et al., 1971) that aged MSCs lose its osteogenic potential and gain adipogenetic potential which termed

“ adipogenetic switch” (Ross et al., 2000). Some recent reports, however, do not observe such changes, and even others observe a decreased adipogenesis of aged MSCs (Bonab et al., 2006). Although no definite statement regarding age-related effects on differentiation potential could be made in spite of some considerable researches (Sethe et al., 2006), a recent study indicated that the efficiency of differentiation into local tissue (homing) of transplanted MSCs was found to be severely decreased following culture (Rombouts and Ploemacher, 2003). These data elicited a fundamental question that if in vitro differentiation data could be unequivocally applied in vivo.

Fourthly, besides aforementioned alteration of aged MSCs, some senescent markers in vitro were also utilized to detect the aging process of MSCs. Among these markers, bata-galactosidase (beta-GAL) activity at pH 6 was reported to be associated with cellular senescence in vitro (Sethe et al., 2006), and one theory suggests that beta-GAL activity is associated with the RAS pathway (Minamino et al., 2003) and with lysosomal dysfunction (Kurz et al., 2000). One further study (Stenderup et al., 2003) demonstrated that beta-GAL activity increased in late-passage MSCs, but there was no difference between MSCs from young and aged donors. Another group (Park et al., 2005) also found that not only beta-GAL, but also p53 and p16/RB increased in prolonged

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cultivation of human MSCs. While beta-GAL was regarded as a reliable marker for senescence in low-density culture and seemed to be correlated with aging in vivo (Dimri et al., 1995; Sethe et al., 2006), its wide application in vivo was limited (Severino et al., 2000).

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3. Role of Telomere Length and Telomerase in MSCs

A. The telomere structure and telomerase

Telomeres are specialized chromatin structures at the ends of eukaryotic chromosomes that prevent the ends of chromosomes from being recognized as a DNA break, which cap and protect every eukaryotic chromosome end against chromosomal fusion, recombination, and terminal degradation (Blasco, 2007; Chan and Blackburn, 2002;

Hiyama and Hiyama, 2007). Telomeric DNA consists of short guanine-rich repeat sequences in all eukaryotes with linear chromosomes. In vertebrate, telomeres are composed of TTAGGG repeats bound by a protein complex called shelterin (Blasco, 2007; de Lange, 2005; Liu et al., 2004a) which have roles in chromosome protection and in the regulation of telomere length. Telomere length in human somatic cells is remarkably heterogeneous among individuals, ranging from 5 to 20 kb, according to age, organ, and the proliferative history of each cell (Hiyama and Hiyama, 2007; Wright and Shay, 2005). During the process of DNA synthesis and cell division, telomere becomes shortened as results of incomplete replication of linear chromosomes, that is, the “end- replication problem” (Hiyama and Hiyama, 2007). The progressive shortening of telomere is one of the molecular mechanisms of the aging because when telomeres reach a certain length, cells generally stop dividing and enter chromosome senescence and loss of cell viability (Blasco, 2005; Collins and Mitchell, 2002; Harley et al., 1990; Wright

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and Shay, 2005). A theory suggest the possibility that telomere dysfunction could be viewed as a specialized form of DNA damage and thus contributes to the aging of human stem cells (Sharpless and Depinho, 2007). Telomere shortening caused by the absence of adequate telomerase activity occurs with cell proliferation and finally results in an alteration of telomere structure, which is sensed by the cells as a DNA double-strand break (Sharpless and Depinho, 2007). To prevent degradation by exonucleases or processing as damaged DNA, the G-strand overhang can fold back and invade the double-stranded region of the telomere, thereby generating a looped structure known as the telomere loop or T-loop (Griffith et al., 1999), which is reinforced with TRF2 and other telomeric DNA-binding proteins named shelterin (de Lange, 2005; Hiyama and Hiyama, 2007). Telomere repeats are generated by a cellular reverse transcriptase known as telomerase (Blasco, 2007; Chan and Blackburn, 2002). A recent study with highly purified telomerase extracts has demonstrated that the telomerase enzyme contains two molecules. These compose of the telomerase reverse transcriptase subunit (Tert) and the telomere-associated RNA molecule (Terc), as well as one molecule of dyskeratin (Blasco, 2007; Cohen et al., 2007), a protein known to stabilize the telomerase complex (Collins and Mitchell, 2002). Telomerase can add telomeric repeats onto the chromosome ends, and prevents the replication-dependent loss of telomere and cellular senescence in highly proloferative cells of the germline and in the majority of cancers (Blasco, 2005).

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Therefore, the immortality of cancer cells, germ-line cells and embryonic stem cells are postulated to be possibly associated with telomerase activity and telomere maintenance (Hiyama and Hiyama, 2007). Except for stem cells and lymphocyte, the telomerase activity of most human somatic cells is gradually diminished after birth and thus, telomere length shortens with each cell division (Hiyama and Hiyama, 2007). To ensure proper telomere function and avoid the activation of DNA damage pathways which may result in replicative senescence or cell death, a critical length of telomere repeats is required (Hiyama and Hiyama, 2007). Low levels of telomerase activity had been detected in human adult stem cells including haematopoietic and non-haematopoietic stem cells such as neuronal, skin, intestinal crypt, mammary epithelial, pancreas, adrenal cortex, kidney and mesenchymal stem cells, and the telomerase activity explained the prolonged poliferative capacity and the mechanism that maintained telomere length through many cell divisions (Hiyama and Hiyama, 2007). Different from the majority of human stem cells, cells that undergo rapid expansion, such as committed haematopoietic progenitor cells, activated lymphocytes, or keratinocytes, and in tissues with a low cell turnover such as the brain, displayed an upregulated telomerase activity (Haik et al., 2000).

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B. Telomere elongation mechanisms

The majority of tumors and immortal cell lines has high levels of telomerase to sustain their immortal growth by preventing telomere shortening and bypassing senescence and apoptosis (Blasco, 2005). In contrast, the adult organism has only limited amount of telomerase activity, which results in the attrition of telomeric DNA during aging and also disability to compensate for the progressive telomere shortening that occurs as cells divide during tissue regeneration (Blasco, 2005; Blasco, 2007; Collins and Mitchell, 2002;

Harley et al., 1990). Even though, in the lack of telomerase activity, some immortal cell lines and tumors are still able to maintain or elongate their telomeres through activation of another mechanism known as alternative lengthening of telomeres (ALT) (Dunham et al., 2000; Muntoni and Reddel, 2005). In yeast and mammals, ALT has been shown to involve homologous recombination events between telomeric sequences (Blasco, 2007;

Dunham et al., 2000; Lundblad, 2002; Muntoni and Reddel, 2005). Heterogeneous telomere lengths are characteristics of ALT-positive cells that both very short and very long telomeres are present at the same time (Blasco, 2007; Dunham et al., 2000;

Lundblad, 2002; Muntoni and Reddel, 2005). Mechanisms of ALT, however, cannot rescue the viability of Terc-deficient mice, which suggests that this later mechanism do not function to overcome the crisis of telomere shortening of most multicellular organisms (Blasco, 2007). In addition, it is noticed that ALT is mostly restricted to Terc-

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deficient mice, as well as immortal cell lines and tumors, which suggests the existence of a mechanism that actively represses ALT in normal cells (Blasco, 2007). Several recent reports revealed that Pot1 and TRF2 or TRF2-interacting proteins such as WRN, which are components of shelterin complex, could influence telomere recombination and thus are potential regulator of ALT (Blanco et al., 2007; Blasco, 2007; Laud et al., 2005; Wu et al., 2006). Other reports also implicated that both subtelomeric DNA methylation (Benetti et al., 2007; Gonzalo et al., 2006) and histone methylation at telomeres (Benetti et al., 2007) were potent repressors of telomere recombination and ALT activation.

C. Telomere and Telomerase in MSCs

The phenotype of replicative senescence in cultured MSCs is dependent on the studied species (Hiyama and Hiyama, 2007). In regard to human MSCs, they are reported to cease dividing early at around 30~50 population doublings (Bonab et al., 2006; Colter et al., 2000; Stenderup et al., 2003). On the contrary, murine MSCs that have high telomerase activity can be passaged for more than 100 population doublings (Meirelles Lda and Nardi, 2003). The rate of telomere shortening of hMSCs has been reported to be about 30-120 bp/ PD (Fehrer and Lepperdinger, 2005; Stenderup et al., 2003).

Interestingly, one study, at least, reported that bone marrow-derived hMSCs maintained long telomeres without the upregulation of telomerase activity for more than 100

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population doublings undr culture with basic FGF (Yanada et al., 2006). Moreover, the relationship between ectopic expression of telomerase and differentiation efficiency of hMSCs had been discussed. It was implicated that forced telomerase expression in hMSCs led to an extend life span and enhanced differentiation potential that the efficiency of telomerase-overexpressing cells to form bone in vivo was greatly enhanced (Shi et al., 2002; Simonsen et al., 2002). On the other hand, mMSCs knocked-down of their telomerase activity completely failed to differentiate into adipocyte or chondrocyte, even in early passages (Hiyama and Hiyama, 2007; Liu et al., 2004b). Recently, a report unraveled that subtelomeric DNA hypomethylation would facilitate telomere elongation in mammalian cells, and this result also suggested that such epigenetic modification of chromatin might occur in hMSCs (Gonzalo et al., 2006). It is highly possible that telomerase is required for both cell replication and differentiation, and thus the differentiation potential and regenerative capacity of hMSCs may be attributed to the minimum level of telomerase activity expressed in hMSCs (Hiyama and Hiyama, 2007).

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4. Epigenetic Regulation of Development and Stem Cell Differentiation

A. Epigenetic signature of pluripotency

As the progression of biotechnology, it is capable of elucidating the profile of gene expression and their relative abundance in a particular cell type, but this information provides us little about the genes that are not actively transcribed in cells (Spivakov and Fisher, 2007). What is more importantly, it is hard for using expression profiling directly to discriminate between genes that are subject to active repression and those that are not transcribed simply due to the absence or limitation of activating proteins (Spivakov and Fisher, 2007). For this reason, we would not figure out how tissue-specific genes, that will be required for executing later stages in development, are prevented from expression by ES cells, although the potential for their expression is retained (Spivakov and Fisher, 2007). To solve this problem, some underlying mechanisms of gene control in stem cells have been proposed, and they are collectively referred to as epigenetic regulation. These control mechanisms encompass a range of different properties that have been shown to affect gene expression without changes in DNA sequence (Spivakov and Fisher, 2007).

Major epigenetic mechanisms include DNA cytosine methylation, histone modifications such as acetylation and methylation of histone tails, and small non-coding RNA controlled pre- and post-transcriptional regulation of gene expression (Wu and Sun, 2006). Moreover, epigenetic information is known to be able to transmitted through

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sequential rounds of cell division (Nakatani et al., 2006; Richards, 2006) because epigenetic marks, including methylated DNA (Jaenisch and Bird, 2003) and modified histones (Henikoff et al., 2004) are propagated at S phase. It is the feature of epigenetic inheritance that lead to the postulation that chromatin has a central role in maintaining transcriptional patterns during development (Spivakov and Fisher, 2007).

B. The roles of chromatin modifications on self-renewal and differentiation of stem cells

Post-translational modification of core histones and methylation of genomic DNA have been revealed to be associated with both chromatin and transcriptional status of genes (Fischle et al., 2003; Hsieh, 2000; Meshorer and Misteli, 2006). During ES-cell differentiation, besides changes in the global genome activity, alterations of histone- modification patterns also occur (Lee et al., 2004). For example, during differentiation, an increase in the silenced chromatin mark tri-methylated residue K9 of histone H3 (H3- triMeK9) and a decrease in the global levels of acetylated histone H3 and H4 (Keohane et al., 1996; Lee et al., 2004; Meshorer et al., 2006), which is usually associated with active chromatin regions, are noted (Meshorer and Misteli, 2006). These findings all suggest that the chromatin of ES-cell is overall either in a more active state or marked with activity-associated histone modifications, and that when differentiation of ES-cell occurs,

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the chromatin is transitioned to a transcriptionally less-permissive status (Meshorer and Misteli, 2006). Several repressed heterochromatin marks, such as H3-triMeK9, H3- MeK27, H3-diMeK27, H4-diMeK20 and H4-triMeK20, are found to elevate during RA- induced mouse ES-cell differentiation (Martens et al., 2005). Moreover, the inhibition of mouse ES-cell differentiation after treatment with histone deacetylase (HDAC) inhibitor, trichostatin A (TSA), also indicates the functional relevance to global histone deacetylation during ES-cell differentiation (Lee et al., 2004). Local histone modifications, along with global changes, are believed to be important for the proper control of differentiation-specific genes (Meshorer and Misteli, 2006). Representative example is the promoter of the ES-cell marker, Oct4, which is observed to be enriched for the active mark, H3-triMeK4, in undifferentiated rather than in differentiating ES cells (Lee et al., 2004). Similar findings are also shown for λ5-VpreB1 (Szutorisz et al.,

2005), B-cell differentiation determining gene, and NFM (Kimura et al., 2004), neuronal differentiation determining gene, which all contain active chromatin marks in undifferentiated ES cells despite their inactivity. These observations do indicate that the maintenance of transcriptionally competent chromatin is an active process mediated by histone modifications. The histone modifications help to preserve the pluripotent state of ES cells and mark the transcriptionally competent loci expressed later in the differentiating process (Szutorisz and Dillon, 2005). In addition, there is current evidence

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of epigenetic regulation which depicts the existence of the temporary inactivation of differentiation-specific genes in pluripotent cell types (Reik, 2007). These observations unravel that genes required during development and differentiation are those in the homeobox (Hox), distal-less homeobox (Dlx), paired box (Pax) and sine-oculis-related homeobox (Six) gene families. These genes are held repressed in pluripotent ES cells by the Polycomb group (PcG) protein repressive system in mice and humans (Reik, 2007).

PcG protein repressive system marks the histones associated with these genes by inducing methylation of the lysine residue at position 27 of the histone H3 (H3K27) (Azuara et al., 2006; Boyer et al., 2006; Lee et al., 2006). It is also found that when ES cells lose the expression of EED (embryonic ectoderm development), a component of the PcG-protein repressive complex (PRC), the developmental genes are partly derepressed and ES cells are prone to spontaneous differentiation (Azuara et al., 2006; Boyer et al., 2006).

PcG-protein repressive complexes (PRCs) are a subclass of histone modification enzymes that are highly conserved throughout the evolution (Valk-Lingbeek et al., 2004).

Polycomb repressive complex 2 (PRC2) is found to contain both histone deacetylase (HDAC) and histone methyltransferase (HMT) activity, which link hypoacetylation and H3-K9/K27 methylation (Valk-Lingbeek et al., 2004). In another aspect, PRC1 recognizes the H3-K27 methylation mark established by PRC2 through its conserved

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chromodomain and takes part in stable maintenance of PRCs mediated gene silencing effect (Wu and Sun, 2006). Chromatin modifying activities opposite to epigenetic control mediated through PRCs also drawed much attention. Testis specific TAF (TBP- associated factor) associated trithorax (trx) action (tri-methylation of H3-K4) had been demonstrated to counteract PcG-mediated repression to allow terminal differentiation of Drosophila male germ cell precursors (Chen et al., 2005; Wu and Sun, 2006).

Some developmental genes, however, were present within bivalent chromatin regions which contain both inactivating marks (methylated H3K27) and activating marks (H3K4) (Bernstein et al., 2006; Szutorisz et al., 2005). The bivalent chromatin marks were demonstrated that when the PRCs expressions were downregulated during differentiation and the repressive marks had been removed, these genes were automaticaly poised for transcriptional activation through the H3K4 methylation mark (Reik, 2007). Although epigenetic silencing by PRCs could be mitotically heritable (Ringrose and Paro, 2004), these marks could also be rapidly removed by enzymatic demethylation of H3K27 (Klose et al., 2006). Therefore, in contrast to the terminal silencing achieved by the DNA methylation, developmental genes that were silenced by PRCs in pluripotent tissues required repressive marks to be rapidly and flexibly moved when differentiation begain (Reik, 2007). Another group of genes which encoded pluripotency-sustaining transcription factors, such as Oct4 and Nanog, were required for

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early development or for germ-cell development only. These pluripotency-associated genes expressed by ES cells but silent during the differentiation of these cells were also known to be mediated by epigenetic regulation with a defined kinetics of acquiring repressive histone modifications and DNA methylation (Feldman et al., 2006; Reik, 2007).

Albeit silence of genetic element could be achieved through histone modifications, these easily reversible modifications were not good gatekeeper for long-term silence (Shi et al., 2004; Takeuchi et al., 2006). Thus, prolonged silence of genetic element must be mediated by an additional epigenetic mechanism. An important component of this process is DNA methylation (Miranda and Jones, 2007). It was noticed that within gene promoters, even when the repressive marks were removed, DNA methylation still prevented the reactivation of silent genes (McGarvey et al., 2007). It is also found that DNA methylation was important for many cellular processes including the silence of repetitive elements, X-inactivation, imprinting and development. The roles of DNA methylation in these processes ensured the daughter cells to retain the same expression pattern as the precursor cells (Miranda and Jones, 2007). DNA methylation is a covalent modification in which the 5’ position of cytosine is methylated in a reaction catalyzed by DNA methyltransferases (DNMTs) with S-adenosyl-methionine as the methyl donor (Miranda and Jones, 2007). In mammals, methylation of cytosines in cytosine guanine

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dinucleotide (CpG) island has been known to play a crucial role in mediating epigenetic gene silencing through two possible mechanisms. First, it has been proved that cytosine methylation can directly silence gene expression by inhibiting DNA binding factors to its recognition gene area (Watt and Molloy, 1988). Second, some authors (Boyes and Bird, 1991) and other investigators also prove that methylated-CpG can recruit Methyl-CpG- binding proteins (MBPs), which in turn function to silence transcription and modify the surrounding chromatin structure. In mammals, four DNA methyltransferases (DNMTs) have been identified. Among them, DNMT1 has been known to maintain the pre-existing methylation state of genome during DNA replication (Leonhardt et al., 1992). DNMT3a and DNMT3b are de novo methyltransferases which will target unmethylated CpG sites (Okano et al., 1999). DNMT2, which has been proved to have weak methyltransferase activity in vitro (Hermann et al., 2003), is responsible to methylate tRNA (Goll et al., 2006).

In differentiating ES cells, increase of CpG island DNA methylation with enhanced expression of DNMTs was also noticed (Kremenskoy et al., 2003; Shen et al., 2006), and the deletion of three major DNMTs would cause hypomethylation and thorough blockage of differentiation of ES cells (Carlone et al., 2005; Jackson et al., 2004). These findings plus the fact that global methylation marks are erased during early embryogenesis and then increase during in vitro expansion (Maitra et al., 2005) suggest that differential

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changes in CpG island DNA methylation profile may serve as an indicator of

“primitiveness” or un-commiment status of stem cells. What’s noteworthy, several studies (Hattori et al., 2004; Taylor and Jones, 1979; Tsuji-Takayama et al., 2004) even observed the phenomenon of dedifferentiation caused by treating cells with demethylation agent 5-azacytidine (5-AzaC), which implied that DNA demethylation might cause a reversion of cells to a more pluripotent state (Meshorer and Misteli, 2006).

In 1979, Taylor and Jones (Taylor and Jones, 1979) firstly described the phenomenon that by treating C3H/10T1/2 cells with the demethylating agent 5-azacytidine (5-AzaC), differentiate into striated muscle cells, adipocytes and chondrocytes was found to be induced. The authors explained that the conversion of these cells to new phenotypes was caused by a reversion of cells toward a primitive pluripotent state and subsequently gave rise to other lineages. In consistent with this viewpoint, both partially differentiated ES cells (Tsuji-Takayama et al., 2004) and trophoblast stem cells (Hattori et al., 2004) treated with 5-AzaC were found to be induced toward trend of dedifferentiation as well.

C. hTERT and DNMTs

In literatures, only two interesting reports significantly observed the relationship between the expression of hTERT and DNMTs. Young (Young et al., 2003) discovered a previously unknown function of hTERT that its ectopic expression in normal human fibroblasts would activate the DNMT1 promoter activity and maintain the DNMT1

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activity even during serial subcultivation. Although the underlying mechanism is still unknown that whether through direct effect of telomerase protein or through its telomerase activity. Results of this report revealed that hTERT could inhibit the expression of genes related to cellular aging. Another study observed similar findings but with some different phenomenon (Casillas et al., 2003). The authors (Casillas et al., 2003) found that upregulation of mRNA and protein expression level of DNMT1, DNMT3a and DNMT3b did occur after cellular tansformation by SV40 T/t-antigen, hTERT and H-ras.

They also observed a significant increase of three major DNMTs enzyme activity in transformed human fetal lung fibroblast following extended population doubling. They suggested that up-regulation of the three major DNMTs in neoplastic cell lines could in part explain the hypermethylation-mediated gene silencing occurs in human cancers.

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5. Summaries and Conclusions

Although an infinite life span and absolute pluripotency are merely characteristics to embryonic stem cells (ESCs), the ethical considerations almost obstruct their possible clinical application of ESCs. Hence it seems that human mesenchymal stem cells (hMSCs) derived from bone marrow are significant sources of patient and disease- specific stem cells. Furthermore, cellular senescence and spontaneous differentiation of hMSCs, however, are often encountered, which severely impede expansion and wide application of hMSCs clinically. For circumventing these drawbacks found in ESCs or hBMSCs, a new approach should be explored. Based on deficiency in the issue discussed above, we developed a human telomerase reverse transcriptase (hTERT) overexpressed model in a previously immortalized hMSC line. In our prelimary study, we found some interesting observations that ectopic expression of hTERT elicited dedifferentiation phenomenon of hMSCs. Therefore in the current study, we try to unravel relationships between dedifferentiation effect of hTERT, and reverse pluripotency and block spontaneous differentiation in hMSCs. Underlying epigenetic mechanisms involved in their gene regulation and dedifferentiation are also investigated. The results of current study hopefully would take a great step forward in establishing the feasibility and applicability of adult stem cells in future clinical applications.

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II. Hypothsis and Specific aims of this study

Hypothesis of this study

After successful transfection of hTERT gene segment, hMSCs would bypass cellular senescence, block spontaneous differentiation, and shift back to ESCs’ characteristics.

The latter changes were associated with alterations in DNA methylation patterns.

Aims of this study

1. To characterize the nature of hBMSCs after gene transfection with hTERT regarding to osteogenic, neurogenic gene expression.

2. To explore transcriptional gene markers associated with ESCs and test whether hTERT-transfected hMSCs regarding to osteogenic, neurogenic gene expression display similar gene markers by RT-PCR and real time PCR.

3. To investigate differentiation potential and potency of hTERT-transfected hMSCs by functional gene expressions, histochemical staining and in vitro mineralization.

4. To evaluate whether hTERT-transfected hMSCs possess the capacity similar to germline and trophoectoderm differentiation.

5. The roles of DNA methylation-modification factors, such as DNA methyltransferases (DNMTs) responsible in the reversion of hMSCs to a pluipotency state, would also be explored.

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III. Research experiments

A. Introduction

Bone marrow mesenchymal stem cells (MSCs) are considered one of the most promising and prospective resources for cell and gene therapy because of their great self-renewal and versatile plasticity in vitro and in vivo (Pittenger et al., 1999). However, there are still two major hindrances, cellular senescence and spontaneous differentiation, encountered during in vitro expansion of MSCs (Woodbury et al., 2002). Cellular senescence could be defined as diminished replication, altered functionality (Beausejour, 2007), and deteriorated potential for differentiation (Bonab et al., 2006). Spontaneous differentiation, known as the emergence of lineage-specific markers without any directed differentiation, would diminish the proportion of undifferentiated stem cells, and therefore compromised the benefit of human MSCs (hMSCs) for clinical application.

Thus, identifying methods for inhibiting senescence and spontaneous differentiation, and reversing hMSCs to a more primitive state has attracted great research interest.

In a previous attempt to immortalize hMSCs with increased life span, we have established a cell line-KP by transferring HPV16 E6E7 genes into hMSCs (Hung et al., 2002). Though KP successfully overcomes the drawback of cellular senescence and could be passaged over 100 population doublings (PDs), the phenomenon of spontaneous differentiation could not be avoided (Hung et al., 2004). Telomerase, known to maintain

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the telomere length, has been indicated to play a role in self-renewal and pluripotency of embryonic stem cells (ESCs) (Amit et al., 2000). However, hMSCs express no

telomerase activity with telomere shortening in a rate similar to non-stem cells (30–120 bp/ PD), and cease to divide when the telomere length is less than 10 kb (Baxter et al., 2004). Besides, ectopic expression of human telomerase reverse transcriptase (hTERT), the catalytic component of telomerase, has been proved not only to bypass cellular

senescence and extend life span (Bodnar et al., 1998), but also to influence differentiation potential (Shi et al., 2002).

In mammals, DNA methylation of cytosines in cytosine guanine dinucleotide (CpG) islands, known to mediate epigenetic gene silencing (Boyes and Bird, 1991; Watt and Molloy, 1988), plays pivotal roles in embryonic development (Hashimshony et al., 2003;

Siegmund et al., 2007; Weber et al., 2007) and ESC differentiation (Meshorer and Misteli, 2006). For example, treating ESCs or somatic cells with demethylation agent such as 5- azacytidine (5-AzaC) resulted in dedifferentiation, thereby pointing out the association of DNA methylation with the differentiation state (Hattori et al., 2004; Taylor and Jones, 1979; Tsuji-Takayama et al., 2004). These results also imply methods that reverse the differentiation state of stem or progenitor cells will induce changes in DNA methylation patterns (Meshorer and Misteli, 2006).

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In this study, we hypothesized, after ectopic expression of hTERT, hMSCs would bypass senescence and block spontaneous differentiation with changes in DNA methylation patterns. Meanwhile, we also tried to prove the heightened differentiation potential of hTERT-transfected hMSCs by directing germline and trophoectoderm differentiation. Finally, the roles of DNA methylation-modification factors, such as DNA methyltransferases (DNMTs) in the reversion of hMSCs to a pluipotency state would be explored.

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B. Materials and Methods 1. Cell Cultures

Primary hMSCs were obtained from the Tulane Center for Preparation and Distribution of Adult Stem Cells (http://www.som.tulane.edu/gene_therapy/distribut.shtml). The cells were grown in alpha minimal essential medium ( MEM;GIBCO/BRL, Carlsbad, CA; http://www.invitrogen.com) supplemented with 16.6% fetalbovine serum (FBS), 100 U/ml penicillin, 100 µg/ml streptomycin, and 2mM L-glutamine (GIBCO/BRL) at 37°C under 5% CO2 atmosphere. The medium was changed twice per week and a subculture was performed after they reached about 80 % confluency.

The hMSC strain (KP) was developed by transfection with the type 16 human papilloma virus proteins E6E7 as described previously (Hung et al., 2004). This strain is grown in DMEM-LG (Gibco, Grand Island, NY; http://www.invitrogen.com) supplemented with 10% FBS, 100 U/ml penicillin, 100 µg/ml streptomycin, and 2mM L-glutamine. The medium was changed twice per week and a subculture was performed at 1:3 to 1:5 split every week. Using flow cytometry, cells express CD29, CD44, CD73, CD90, CD105, SH2, and SH3, but otherwise, they lack expression of CD34 and CD166.