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Kassem: Skeletal (stromal) stem cells – basic biology and clinical use in tissue regeneration

Human bone marrow-derived stromal stem cells [also known as skeletal stem cells or mesenchymal stem cells (hMSCs)] are a group of clonogenic cells that are present among the bone marrow stroma as well as the stroma of other organs. hMSCs are capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes, and possibly, but still controversial, non-mesoderm-type cells (e.g. neuronal cells or hepatocytes). Moreover, hMSCs provide supportive stroma for growth and differentiation of haematopoietic stem cells and haematopoiesis. Recently, hMSCs have been employed in an increasing number of cell-based therapies for treating skeletal and non-skeletal chronic degenerative diseases.1,2

In ex vivo cultures, hMSCs exhibit characteristic markers (CD34, CD45, CD14, CD29+, CD73+, CD90+, CD105+, CD166+ and CD44+) for haematopoietic cells. However, these markers are not specific to MSCs and therefore MSCs are usually defined operationally as cells capable of ex vivo differentiation into osteoblasts, adipocytes and chondrocytes (i.e. multipotential) or forming bone and bone marrow organs, ‘an ossicle’, upon transplantation subcutaneously in immunodeficient mice. We have also recently defined the molecular signature of MSCs and identified a set of markers that can predict the in vivo phenotype of MSCs and thus be used to identify MSCs prospectively.13

Traditionally, MSCs have been isolated from bone marrow low-density mononuclear cell population based on their selective adherence to plastic surfaces. Populations with a MSC-like phenotype have been isolated from different tissues (e.g. peripheral blood, umbilical cord blood, adipose tissue and dental pulp). Tissue-specific MSCs share some basic morphological and differentiation characteristics with bone marrow-derived MSCs. However, these cells are not identical and we have reported differences in their ‘molecular signature’.4

From the laboratory to the clinic

The emerging field of regenerative medicine holds promise for treating a variety of degenerative and age-related diseases, where no specific or effective treatment is currently available, by transplanting biologically competent mature cells and tissues or by stimulating tissue-resident stem cells. Stem cells, in general, and MSCs, in particular, with their versatile growth and differentiation potential, as well as their ability to produce a large number of factors that modulate inflammation and promote tissue regeneration following injury, are ideal candidates for use in regenerative medicine protocols and are currently making their way into clinical trials. However, successful use of MSCs in therapy requires developing well-defined methods for MSC isolation, growth and differentiation.

Limited in vitro cell growth and replicative senescence of human mesenchymal stem cells

The clinical use of hMSCs requires the availability of a large number of functionally competent cells with a stable phenotype and genotype. This is usually achieved by long-term ex vivo culturing of hMSCs. However, hMSCs, in contrast to embryonic stem cells or cancer cells, exhibit a limited capacity for the ex vivo growth, a phenomenon known as ‘in vitro replicative senescence’. In addition, the proliferative capacity of hMSCs is dependent on donor age and thus compromising the ability to generate enough cells from elderly donors.5 We have also demonstrated that genetic overexpression of the human telomerase reverse transcriptase gene in hMSCs increases their telomerase activity and abolishes the replicative senescence phenotype.6 However, genetic manipulation of hMSCs is not desirable for cells used in clinical transplantation and alternative methods for ex vivo enhancement of hMSC growth (e.g. use of small chemical molecules with proliferation-enhancing abilities or enhancing cell growth using a combination of growth factors) are being tested.

Directing differentiation of mesenchymal stem cells into specific lineages

While the multipotentiality of MSCs is the basis for using the cells to generate differentiated cells for cell replacement therapy, protocols that direct the differentiation of hMSCs into a specific lineage are still inefficient and require improvement. We have identified a number of molecules and microRNAs that regulate the genetic programme of MSC differentiation into osteoblastic cells.79 Ex vivo protocols for lineage-specific differentiation of MSCs based on this knowledge are currently being developed.

Is it safe to transplant human mesenchymal stem cells?

There are concerns that transplanted, culture-expanded hMSCs may undergo spontaneous transformation and lead to cancer. However, spontaneous transformation of cultured hMSCs has not been reported and the safety record of hMSCs employed in therapy is excellent. We have recently developed an non-invasive assay for monitoring the integrity of hMSCs prior to transplantation based on Raman spectroscopy.10

Clinical perspective

The potential for hMSC use in therapy is enormous and positive results have been obtained in preclinical animal models of human diseases and from phase I/II clinical trials with very promising preliminary results in treating diverse conditions, for example graft-versus-host disease, heart failure, neural tissue and skeletal tissue regeneration (for a review, see Aldahmash et al.1). Combining basic research studies identifying the mechanisms controlling MSC proliferation and differentiation and well-designed and controlled clinical trials will bring major advances in realizing the potential of hMSC-based therapies and bringing new therapies into clinical practice.

References

1. 

Aldahmash A, Zaher W, Al-Nbaheen M, Kassem M. Human stromal (mesenchymal) stem cells: basic biology and current clinical use for tissue regeneration. Ann Saudi Med 2012; 32:68–77.

2. 

Zaher W, Harkness L, Jafari A, Kassem M. An update of human mesenchymal stem cell biology and their clinical uses. Arch Toxicol 2014; 88:1069–82. http://dx.doi.org/10.1007/s00204-014-1232-8

3. 

Larsen KH, Frederiksen CM, Burns JS, Abdallah BM, Kassem M. Identifying a molecular phenotype for bone marrow stromal cells with in vivo bone forming capacity. J Bone Miner Res 2009; 25:796–808. http://dx.doi.org/10.1359/jbmr.091018

4. 

Al-Nbaheen M, Vishnubalaji R, Ali D, et al. Human stromal (mesenchymal) stem cells from bone marrow, adipose tissue and skin exhibit differences in molecular phenotype and differentiation potential. Stem Cell Rev 2012; 9:32–43. http://dx.doi.org/10.1007/s12015-012-9365-8

5. 

Stenderup K, Justesen J, Clausen C, Kassem M. Aging is associated with decreased maximal life span and accelerated senescence of bone marrow stromal cells. Bone 2003; 33:919–26. http://dx.doi.org/10.1016/j.bone.2003.07.005

6. 

Simonsen J, Rosada C, Sernici N, et al. Telomerase expression extends lifespan and prevents senescence-associated impairment of osteoblast functions. Nat Biotechnol 2002; 20:592–6. http://dx.doi.org/10.1038/nbt0602-592

7. 

Abdallah BM, Ditzel N, Mahmood A, et al. DLK1 is a novel regulator of bone mass that mediates estrogen-deficiency induced bone loss in mice. J Bone Miner Res 2011; 26:1457–71. http://dx.doi.org/10.1002/jbmr.346

8. 

Eskildsen T, Taipaleenmäki H, Stenvang J, et al. MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo. Proc Natl Acad Sci U S A 2011; 108:6139–44. http://dx.doi.org/10.1073/pnas.1016758108

9. 

Kratchmarova I, Blagoev B, Haack-Sorensen M, Kassem M, Mann M. Mechanisms of divergent growth factor effects in mesenchymal stem cell differentiation. Science 2005; 308:1472–7. http://dx.doi.org/10.1126/science.1107627

10. 

Harkness L, Novikov SM, Beermann J, Bozhevolnyi SI, Kassem M. Identification of abnormal stem cells using Raman spectroscopy. Stem Cells Dev 2012; 12:2152–9. http://dx.doi.org/10.1089/scd.2011.0600





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