Basem M. Abdallah

Adult human mesenchymal stem cell as a target for neoplastic transformation

The neoplastic process may involve a cancer stem cell. This concept has emerged largely from the careful analysis of tumour biopsy systems from haematological, breast and brain tumours. However, the experimental systems necessary to provide the cellular and molecular evidence to support this important concept have been lacking. We have used adult mesenchymal stem cells (hMSC) transduced with the telomerase hTERT gene to investigate the neoplastic potential of adult stem cells. The hTERT-transduced line, hMSC-TERT20 at population doubling level (PDL) 256 showed loss of contact inhibition, anchorage independence and formed tumours in 10/10 mice. hMSC-TERT4 showed loss of contact inhibition at PDL 95, but did not exhibit anchorage independence and did not form tumours in mice. Both lines had a normal karyotype but showed deletion of the Ink4a/ARF locus. At later passage, hMSC-TERT4 also acquired an activating mutation in KRAS. In hMSC-TERT20, expression of the cell cycle-associated gene, DBCCR1 was lost due to promoter hypermethylation. This epigenetic event correlated with acquisition of tumorigenicity. These data suggest that the adult hMSCs can be targets for neoplastic transformation and have implications for the development of novel anticancer therapeutics and for the use of hMSC in tissue engineering and transplantation protocols.

Human mesenchymal stem cells: from basic biology to clinical applications

Mesenchymal stem cells (MSC) are a group of clonogenic cells present among the bone marrow stroma and capable of multilineage differentiation into mesoderm-type cells such as osteoblasts, adipocytes and chondrocytes. Due to their ease of isolation and their differentiation potential, MSC are being introduced into clinical medicine in variety of applications and through different ways of administration. Here, we discuss approaches for isolation, characterization and directing differentiation of human mesenchymal stem cells (hMSC). An update of the current clinical use of the cells is also provided.

MicroRNA-138 regulates osteogenic differentiation of human stromal (mesenchymal) stem cells in vivo

Elucidating the molecular mechanisms that regulate human stromal (mesenchymal) stem cell (hMSC) differentiation into osteogenic lineage is important for the development of anabolic therapies for treatment of osteoporosis. MicroRNAs (miRNAs) are short, noncoding RNAs that act as key regulators of diverse biological processes by mediating translational repression or mRNA degradation of their target genes. Here, we show that miRNA-138 (miR-138) modulates osteogenic differentiation of hMSCs. miRNA array profiling and further validation by quantitative RT-PCR (qRT-PCR) revealed that miR-138 was down-regulated during osteoblast differentiation of hMSCs. Overexpression of miR-138 inhibited osteoblast differentiation of hMSCs in vitro, whereas inhibition of miR-138 function by antimiR-138 promoted expression of osteoblast-specific genes, alkaline phosphatase (ALP) activity, and matrix mineralization. Furthermore, overexpression of miR-138 reduced ectopic bone formation in vivo by 85%, and conversely, in vivo bone formation was enhanced by 60% when miR-138 was antagonized. Target prediction analysis and experimental validation by luciferase 3′ UTR reporter assay confirmed focal adhesion kinase, a kinase playing a central role in promoting osteoblast differentiation, as a bona fide target of miR-138. We show that miR-138 attenuates bone formation in vivo, at least in part by inhibiting the focal adhesion kinase signaling pathway. Our findings suggest that pharmacological inhibition of miR-138 by antimiR-138 could represent a therapeutic strategy for enhancing bone formation in vivo.

Regulation of Human Skeletal Stem Cells Differentiation by Dlk1/Pref‐1

Dlk‐1/Pref‐1 was identified as a novel regulator of human skeletal stem cell differentiation. Dlk1/Pref‐1 is expressed in bone and cultured osteoblasts, and its constitutive overexpression led to inhibition of osteoblast and adipocyte differentiation of human marrow stromal cells.

Bone regeneration and stem cells

•  Introduction •  Bone fracture healing and healing problems •  Biomaterial scaffolds and tissue engineering in bone formation ‐  Bone tissue engineering ‐  Biomaterial scaffolds ‐  Synthetic scaffolds ‐  Micro‐ and nanostructural properties of scaffolds ‐  Conclusion •  Mesenchymal stem cells and osteogenesis ‐  Bone tissue ‐  Origin of osteoblasts ‐  Isolation and characterization of bone marrow derived MSC ‐  In vitro differentiation of MSC into osteoblast lineage cells ‐  In vivo differentiation of MSC into bone ‐  Factors and pathways controlling osteoblast differentiation of hMSC ‐  Defining the relationship between osteoblast and adipocyte differentiation from MSC ‐  MSC and sex hormones ‐  Effect of aging on osteoblastogenesis ‐  Conclusion •  Embryonic, foetal and adult stem cells in osteogenesis ‐  Cell‐based therapies for bone ‐  Specific features of bone cells needed to be advantageous for clinical use ‐  Development of therapeutic biological agents ‐  Clinical application concerns ‐  Conclusion •  Platelet‐rich plasma (PRP), growth factors and osteogenesis ‐  PRP effects in vitro on the cells involved in bone repair ‐  PRP effects on osteoblasts ‐  PRP effects on osteoclasts ‐  PRP effects on endothelial cells ‐  PRP effects in vivo on experimental animals ‐  The clinical use of PRP for bone repair ‐  Non‐union ‐  Distraction osteogenesis ‐  Spinal fusion ‐  Foot and ankle surgery ‐  Total knee arthroplasty ‐  Odontostomatology and maxillofacial surgery ‐  Conclusion •  Molecular control of osteogenesis ‐  TGF‐β signalling ‐  FGF signalling ‐  IGF signalling ‐  PDGF signalling ‐  MAPK signalling pathway ‐  Wnt signalling pathway ‐  Hedgehog signalling ‐  Notch signalling ‐  Ephrin signalling ‐  Transcription factors regulating osteoblast differentiation ‐  Conclusion •  Summary

Induction of Adipocyte‐Like Phenotype in Human Mesenchymal Stem Cells by Hypoxia

Human mesenchymal stem cells (hMSCs) have the capacity to differentiate along several pathways to form bone, cartilage, tendon, muscle, and adipose tissues. The adult hMSCs reside in vivo in the bone marrow in niches where oxygen concentration is far below the ambient air, which is the most commonly encountered laboratory condition. The study reported here was designed to determine whether oxygen has a role in the differentiation of hMSCs into adipocytes. Indeed, when exposed to atmosphere containing only 1% of oxygen, the formation of adipocyte‐like phenotype with cytoplasmic lipid inclusions was observed. The effect of hypoxia on the expression of adipocyte‐specific genes was determined by real‐time reverse transcription polymerase chain reaction. Interestingly, neither of the two central regulators of adipogenesis—the transcription factors peroxisome proliferator‐activated receptor γ2 (PPAR‐γ2) and ADD1/SREBP1c—was induced. Furthermore, hypoxia did not have any effect on the transcription of early (lipoprotein lipase) or late (aP2) marker genes. By the same token, neither of the mature adipocyte‐specific genes—leptin and adipophilin—was found responsive to the treatment. High level of induction, however, was observed with the PPAR‐γ–induced angiopoietin‐related gene, PGAR. The lack of an adipocyte‐specific transcription pattern thus indicates that despite accumulation of the lipid, true adipogenic differentiation did not take place. In conclusion, hypoxia appears to exert a potent lipogenic effect independent of PPAR‐γ2 maturation pathway.

Tumorigenic Heterogeneity in Cancer Stem Cells Evolved from Long-term Cultures of Telomerase-Immortalized Human Mesenchymal Stem Cells

Long-term cultures of telomerase-transduced adult human mesenchymal stem cells (hMSC) may evolve spontaneous genetic changes leading to tumorigenicity in immunodeficient mice (e.g., hMSC-TERT20). We wished to clarify whether this unusual phenotype reflected a rare but dominant subpopulation or if the stem cell origin allowed most cells to behave as cancer stem cells. Cultures of the hMSC-TERT20 strain at population doubling 440 were highly clonogenic (94%). From 110 single-cell clones expanded by 20 population doublings, 6 underwent detailed comparison. Like the parental population, each clone had approximately 1.2 days doubling time with loss of contact inhibition. All retained 1,25-(OH)(2) vitamin D(3)-induced expression of osteoblastic markers: collagen type I, alkaline phosphatase, and osteocalcin. All shared INK4a/ARF gene locus deletion and epigenetic silencing of the DBCCR1 tumor suppressor gene. Despite in vitro commonality, only four of six clones shared the growth kinetics and 100% tumorigenicity of the parental population. In contrast, one clone consistently formed latent tumors and the other established tumors with only 30% penetrance. Changing the in vitro microenvironment to mimic in vivo growth aspects revealed concordant clonal heterogeneity. Latent tumor growth correlated with extracellular matrix entrapment of multicellular spheroids and high procollagen type III expression. Poor tumorigenicity correlated with in vitro serum dependence and high p27(Kip1) expression. Aggressive tumorigenicity correlated with good viability plus capillary morphogenesis on serum starvation and high cyclin D1 expression. Thus, hMSC-TERT20 clones represent cancer stem cells with hierarchical tumorigenicity, providing new models to explore the stem cell hypothesis for cancer.

Patients With High Bone Mass Phenotype Exhibit Enhanced Osteoblast Differentiation and Inhibition of Adipogenesis of Human Mesenchymal Stem Cells

Genetic mutations in the LRP5 gene affect Wnt signaling and lead to changes in bone mass in humans. Our in vivo and in vitro results show that activated mutation T253I of LRP5 enhances osteogenesis and inhibits adipogenesis. Inactivating mutation T244M of LRP5 exerts opposite effects.

Demonstration of the presence of independent pre-osteoblastic and pre-adipocytic cell populations in bone marrow-derived mesenchymal stem cells.

Mesenchymal stem cells (MSC) are defined as plastic-adherent, clonal cells that are common progenitors for osteoblasts and adipocytes. An inverse relationship between bone and fat has been observed in several clinical conditions and has been suggested to be caused by re-directing MSC differentiation into one particular lineage. However, this inverse relationship between bone and fat is not consistent and under certain in vivo conditions, bone and fat can change independently suggesting separate precursor cell populations. In order to test for this hypothesis, we extensively characterized two plastic-adherent clonal MSC lines (mMSC1 and mMSC2) derived from murine bone marrow. The two cell lines grew readily in culture and have undergone more than 100 population doublings with no apparent differences in their growth rates. Both cell lines were positive for the murine MSC marker Sca-1 and mMSC1 was also positive for CD13. Both cell lines were exposed to in vitro culture induction of osteogenesis and adipogenesis. mMSC1 and not mMSC2 were only able to differentiate to adipocytes evidenced by the expression of adipocyte markers (aP2, adiponectin, adipsin, PPARgamma2 and C/EBPa) and the presence of mature adipocytes visualized by Oil Red O staining. On the other hand, mMSC2 and not mMSC1 differentiated to osteoblast lineage as demonstrated by up-regulation of osteoblastic makers (CBFA1/RUNX2, Osterix, alkaline phosphatase, bone sialoprotein and osteopontin) and formation of alizarin red stained mineralized matrix in vitro. Consistent with the in vitro results, mMSC2 and not mMSC1, were able to form bone in vivo after subcutaneous implantation in immune-deficient (NOD/SCID) mice. Our data suggest that contrary to the current belief, bone marrow contains clonal subpopulations of cells that are committed to either osteoblast or adipocyte lineage. These cell populations may undergo independent changes during aging and in bone diseases and thus represent important targets for therapy.

Human bone-marrow-derived mesenchymal stem cells: biological characteristics and potential role in therapy of degenerative diseases

Mesenchymal stem cells (MSC) are a group of cells present in bone-marrow stroma and the stroma of various organs with the capacity for mesoderm-like cell differentiation into, for example, osteoblasts, adipocytes, and chondrocytes. MSC are being introduced in the clinic for the treatment of a variety of clinical conditions. The aim of this review is to provide an update regarding the biology of MSC, their identification and culture, and mechanisms controlling their proliferation and differentiation. We also review the current status of their clinical use. Areas in which research is needed to enhance the clinical use of MSC are emphasized.