Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells

Okamoto, Takeshi; Aoyama, Tomoki; Nakayama, Tomitaka; Nakamata, Takeharu; Hosaka, Taisuke; Nishijo, Koichi; Nakamura, Takashi; Kiyono, Tohru; Toguchida, Junya

doi:10.1016/s0006-291x(02)00661-7

Cited by 248 publications

(232 citation statements)

References 33 publications

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“…The loss of differentiation activity in this case is likely to be because of the influence of the viral oncoprotein E6E7, (35,36) although E6E7 has been shown to immortalize some cell types without loss of differentiation potential. (18,37) We have previously shown that cementoblast progenitors are present in BDFCs and that they are phenotypically different from osteoblasts. (10) In this study, we successfully isolated a cementoblast cell line, BCPb8, which seems to behave similarly to the cementum progenitors present in BDFCs, both in vitro and in vivo.…”

Section: Discussionmentioning

confidence: 99%

Immortalization of Cementoblast Progenitor Cells With Bmi-1 and TERT

Saito

Handa

Kiyono³

et al. 2005

Journal of Bone and Mineral Research

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A cementoblast progenitor cell line designated BCPb8 was successfully isolated from dental follicle cells immortalized with Bmi-1 and hTERT. BCPb8 showed the potential to differentiate into cementoblasts on implantation into immunodeficient mice. BCPb8 was confirmed to be the first established cementoblast progenitor cell line and will provide a useful model for investigating cementogenesis. Introduction:The dental follicle is the mesenchymal tissue surrounding the developing tooth germ. During tooth root development, progenitor cells present in the dental follicle are believed to play a central role in the formation of periodontal components (cementum, periodontal ligament, and alveolar bone). However, little more is known about the biology of these progenitors. Previously, we observed that cultured bovine dental follicle cells (BDFCs) contained putative cementoblast progenitors. To further analyze the biology of these cells, we attempted to isolate cementoblast progenitors from immortalized BDFC through expression of the polycomb group protein, Bmi-1, and human telomerase reverse transcriptase (hTERT). Materials and Methods: BDFCs were transduced with replication-deficient retroviruses carrying human Bmi-1(LXSN-Bmi-1), and hTERT (LXSH-hTERT) for immortalization. Single cell clones were established from immortalized BDFC, and differentiation into cementoblasts was assessed by implantation into immunodeficient mice. Results and Conclusion: BDFCs expressing Bmi-1 and hTERT showed an extended life span-90 population doublings more than normal BDFCs-and still contained cells with the potential to differentiate into cementoblasts on implantation into immunodeficient mice. From these cells, we established a clonal cell line, designated BCPb8, which formed cementum-like tissue that was reactive to the anti-cementum-specific monoclonal antibody 3G9 and expressed mRNA for bone sialoprotein, osteocalcin, osteopontin, and type I collagen on implantation. Thus, by using Bmi-1 and hTERT, we succeeded in immortalizing cementoblast progenitor cells from BDFC without affecting differentiation potential. The BCPb8 cell line is the first immortalized clonal cell line of cementoblast progenitors and could be a useful tool not only to study cementogenesis but also to develop regeneration therapy for patients with periodontitis.

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Section: Discussionmentioning

confidence: 99%

Immortalization of Cementoblast Progenitor Cells With Bmi-1 and TERT

Saito

Handa

Kiyono³

et al. 2005

Journal of Bone and Mineral Research

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“…Activation of telomerase alone is sufficient for immortalization of human foreskin fibroblasts. HPV16 E6 and E7 have been used to inhibit p53 and RB, respectively, to prolong the life span of marrowderived MSCs (Okamoto et al, 2002;Takeda et al, 2004), endometrial gland cells (Kyo et al, 2003), mammary epithelial cells, and keratinocytes (Kiyono et al, 1998). Bmi-1 also has been used to inhibit p16 INK4a transcription to prolong the life span of marrow-derived MSCs.…”

Section: Is Successful Prolongation Of Ucbmscs Life Span Without Inhimentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

“…E6 also accelerates degradation of p53, which induces the cdk inhibitor p21 (Sekiguchi and Hunter, 1998). This system in which p16 INK4a /RB is inhibited and telomerase is activated is highly efficient in extending the life span of hMSCs (Okamoto et al, 2002).In the present study, we investigated the growth regulatory mechanism of UCBMSCs and attempted to establish UCBMSCs with hTERT (UCBTERTs) to overcome their limited life span. Introduction of hTERT alone was sufficient to extend the life span of UCBMSCs in vitro, and this technique for prolonging the life span of UCBMSCs will be a useful tool.…”

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confidence: 98%

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Immortalization of Human Fetal Cells: The Life Span of Umbilical Cord Blood-derived Cells Can Be Prolonged without Manipulating p16^INK4a/RB Braking Pathway

Terai

Uyama

Sugiki

et al. 2005

MBoC

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Human umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) are expected to serve as an excellent alternative to bone marrow-derived human mesenchymal stem cells. However, it is difficult to study them because of their limited life span. To overcome this problem, we attempted to produce a strain of UCBMSCs with a long life span and to investigate whether the strain could maintain phenotypes in vitro. UCBMSCs were infected with retrovirus carrying the human telomerase reverse transcriptase (hTERT) to prolong their life span. The UCBMSCs underwent 30 population doublings (PDs) and stopped dividing at PD 37. The UCBMSCs newly established with hTERT (UCBTERTs) proliferated for >120 PDs. The p16 INK4a /RB braking pathway leading to senescence can be inhibited by introduction of Bmi-1, a polycomb-group gene, and human papillomavirus type 16 E7, but the extension of the life span of the UCBMSCs with hTERT did not require inhibition of the p16 INK4a /RB pathway. The characteristics of the UCBTERTs remained unchanged during the prolongation of life span. UCBTERTs provide a powerful model for further study of cellular senescence and for future application to cell-based therapy by using umbilical cord blood cells. INTRODUCTIONHuman mesenchymal stem cells (hMSCs) can be a useful source of cells for transplantation for several reasons: they have the ability to proliferate and differentiate into mesodermal tissues, and they entail no ethical or immunological problems (Caplan, 1991;Prockop, 1997;Caplan and Bruder, 2001). hMSCs have been studied extensively over the past 3 decades, and numerous independent research groups have successfully isolated hMSCs from a variety of sources, most commonly, from the bone marrow (Owen, 1988;Umezawa et al., 1992;Jaiswal et al., 1997;Makino et al., 1999;Pittenger et al., 1999;Sekiya et al., 2004). Umbilical cord blood (UCB) contains circulating stem/progenitor cells, and the cells contained in UCB are known to be distinct from those contained in bone marrow and adult peripheral blood (Mayani and Lansdorp, 1998). Isolation, characterization, and differentiation of clonally expanded hMSCs derived from UCB (UCBMSCs) have been reported (Goodwin et al., 2001;Lee et al., 2004), and UCBMSCs have been found to have multipotency, and the immunophenotype of the clonally expanded cells is consistent with that reported for bone marrow mesenchymal stem cells. Even now, most UCB is regarded as medical waste in the delivery rooms. Aspirating bone marrow from patients is, however, an invasive procedure, and the proliferation and differentiation capacity of hMSCs decreases with the donor age (D'Ippolito et al., 1999). Therefore, the applications of UCB should be further expanded.UCBMSCs will be useful sources for cell transplantation, however, it is difficult to study and apply them because of their limited life span. One of the reasons for this is that normal human cells undergo a limited number of cell division in culture and then enter a nondividing state called "senescence" (Hayflick, 1976;Campisi...

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“…A hiMSC 10 was maintained in Dulbecco's modified Eagle's medium (Sigma-Aldrich Inc., St Louis, MO, USA) supplemented with 10% fetal bovine serum (JRH Biosciences, Lenexa, KS, USA) .…”

Section: Cell Culturementioning

confidence: 99%

Integration-free and stable expression of FVIII using a human artificial chromosome

et al. 2011

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Human artificial chromosome (HAC) has several advantages as a gene therapy vector, including stable episomal maintenance that avoids insertional mutations and the ability to carry large gene inserts. To examine the copy number effect on the gene expression levels and its stability for a long-term culture for a future application in gene therapy, we constructed a HAC vector carrying the human factor VIII (FVIII) complementary DNA, FVIII-HAC in Chinese hamster ovary (CHO) cells. One and more copies of FVIII gene on the HAC were expressed in the copy-number-dependent manner in the CHO cells. The HAC with 16 copies of FVIII, FVIII (16)-HAC, was transferred from CHO hybrids into a human immortalized mesenchymal stem cell using microcell-mediated chromosome transfer. The expression levels of HAC-derived FVIII transgene products were compared with transfected FVIII plasmids. The former showed expression levels consistent with those of the original clones, even after 50 population doublings, whereas the latter showed a remarkable decrease in expression despite unvarying DNA content, indicating that the gene on the HAC is resistant to gene silencing. These results suggest that the HAC-mediated therapeutic gene-expression system may be a powerful tool for stable expression of transgenes, and possibly for industrial production of gene products.

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Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells

Cited by 248 publications

References 33 publications

Immortalization of Cementoblast Progenitor Cells With Bmi-1 and TERT

Immortalization of Cementoblast Progenitor Cells With Bmi-1 and TERT

Immortalization of Human Fetal Cells: The Life Span of Umbilical Cord Blood-derived Cells Can Be Prolonged without Manipulating p16^INK4a/RB Braking Pathway

Integration-free and stable expression of FVIII using a human artificial chromosome

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Clonal heterogeneity in differentiation potential of immortalized human mesenchymal stem cells

Cited by 248 publications

References 33 publications

Immortalization of Cementoblast Progenitor Cells With Bmi-1 and TERT

Immortalization of Cementoblast Progenitor Cells With Bmi-1 and TERT

Immortalization of Human Fetal Cells: The Life Span of Umbilical Cord Blood-derived Cells Can Be Prolonged without Manipulating p16INK4a/RB Braking Pathway

Integration-free and stable expression of FVIII using a human artificial chromosome

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Immortalization of Human Fetal Cells: The Life Span of Umbilical Cord Blood-derived Cells Can Be Prolonged without Manipulating p16^INK4a/RB Braking Pathway