Telomerase deficiency impairs differentiation of mesenchymal stem cells

https://doi.org/10.1016/j.yexcr.2003.10.031Get rights and content

Abstract

Expression of telomerase activity presumably is involved in maintaining self-replication and the undifferentiated state of stem cells. Adult mouse bone marrow mesenchymal stem cells (mMSCs) are multipotential cells capable of differentiating into a variety of lineage cell types, including adipocytes and chondrocytes. Here we show that the lacking telomerase of mMSC lose multipotency and the capacity to differentiate. Primary cultures of mMSCs were obtained from both telomerase knockout (mTR−/−) and wild-type (WT) mice. The MSCs isolated from mTR−/− mice failed to differentiate into adipocytes and chondrocytes, even at early passages, whereas WT MSCs were capable of differentiation. Consistent with other cell types, late passages mTR−/−MSCs underwent senescence and were accompanied by telomere loss and chromosomal end-to-end fusions. These results suggest that in addition to its known role in cell replication, telomerase is required for differentiation of mMSCs in vitro. This work may be significant for further potentiating adult stem cells for use in tissue engineering and gene therapy and for understanding the significance of telomerase expression in the process of cell differentiation.

Introduction

A key function of telomerase is to add telomere repeats during cell division. Most somatic cells from humans and other mammals lack telomerase activity and undergo senescence after only a limited number of replications. Decreased telomerase activity results in telomere shortening, ultimately leading to telomere loss and chromosome end-to-end fusions, as well as cellular senescence [1]. Forced expression of telomerase prevents cellular senescence and extends the life span of cultured cells [2], [3], demonstrating a crucial role of telomerase in cell replication. Telomerase activity remains high in stem cells and precursor cells [4], [5], which are capable of infinite self-replication and differentiation into a variety of cell types [6], [7]. The presence of telomerase in stem cells suggests that it likely plays an important role in maintaining stem cells in an undifferentiated state. Indeed, the growth rate of telomerase-deficient ES cells was severely reduced [8], underlining the importance of telomerase in self-replication and proliferation of stem cells.

Bone marrow contains both hemopoietic stem cells and stemlike cells that are precursors of nonhemopoietic tissues, which were initially called plastic-adherent cells or colony-forming-units fibroblasts (CFUs) because of their unique characteristics [9], [10]. These cells also have been called mesenchymal stem cells or marrow stromal cells (MSCs) [10], [11], [12]. Recently, MSCs isolated by their adherence to tissue culture glass or plastic have been demonstrated to exhibit multipotency in both in vitro differentiation and in vivo transplantation [13], [14], [15], [16], [17], [18], [19], [20] and have great significance in both cell and gene therapy [10], [11], [21]. MSCs retain the ability to differentiate into a variety of cell types, including adipocytes, chondrocytes, osteoblasts, and cardiac and skeletal muscle cells, as well as neurons and astrocytes. Telomerase activity was detected in both early and late passage human MSCs (hMSCs), indicating that passaged hMSCs retain their telomerase activity [15]. Telomerase activity typically is repressed after differentiation of immortal cells and stem cells [22], [23], [24], [25]. However, telomerase activation and telomere lengthening have been observed in other types of cell differentiation [26], [27]. We investigated whether telomerase is required for cell differentiation by comparing the in vitro differentiation capacities of mouse bone marrow mesenchymal stem cells (mMSCs) obtained from telomerase RNA gene knockout (TR−/−) mice [28] with that of wild-type mice. The mTR−/− tissues and cells exhibited no detectable telomerase activity [28].

Section snippets

Isolation and culture of mMSCs

The mMSCs were isolated by their adherence to plastic and then passed in culture, as described [14], [15], [29], [30], with a slight modification. Tibias and femurs were dissected from muscle and connective tissue of adult wild-type and telomerase-null (TR−/−) mice [28]. The ends of the bones were cut, and marrow was flushed with 10 ml of ice-cold Dulbecco's modified Eagle's medium (DMEM) (low glucose, Gibco/BRL) containing 20% fetal bovine serum (FBS) (Sigma) using a 26-g needle and syringe.

Results

We first compared cell proliferative potential of different passages of mMSCs from the first generation (G1) and late generation (G3 and G4) mTR−/− mice to those of wild-type (WT) mice. Fig. 1A shows genotyping by 4-primer PCR of G1, G3 mTR−/−, and WT mice from which mMSCs were obtained. Crude bone marrow cells flushed from WT and mTR−/− mice contained mMSCs and were isolated by their adherence to plastic culture dishes. By taking advantage of this unique feature of mMSCs, we removed

Discussion

It has been shown that plating MSCs at the low densities possibly produces the greatest replicative potential [30]. Copurified with mesenchymal stem cells, MAPCs can differentiate into a variety of cell lineages in vitro and proliferate extensively without loss of differentiation potential [36]. The relatively enriched mMSCs from WT mouse in the present study maintain the ability to proliferate extensively and to differentiate into other types of cells in vitro, confirming their stem cell

Acknowledgements

This work was supported in part by Women and Infants Hospital/Brown Faculty Research Fund to D.L.K. and from the Ministry of Science and Technology, Spain, and the European Union to M.A.B.

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