Therapeutic Properties of Superoxide Dismutase 3 and Mesenchymal Stromal Cells in Peripheral Ischemia

The aim of this study was to characterize the cellular mechanisms leading to the beneficial effect of anti-oxidative gene therapy and pro-angiogenic stem cell therapy in acute peripheral ischemia. Post-ischemic events aim to re-establish tissue blood perfusion, to clear cellular debris, and to regenerate lost tissue by differentiation of satellite cells into myoblasts. Although leukocytes have an essential role in clearing cellular debris and promoting angiogenesis, they also contribute to tissue injury through excessive ROS production. First, we investigated the therapeutic properties of extracellular superoxide dismutase (SOD3) gene transfer. SOD3 was shown to reduce oxidative stress, to normalize glucose metabolism, and to enhance cell proliferation in the ischemic muscle. Analysis of the mitogenic Ras-Erk1/2 pathway showed SOD3 mediated induction offering a plausible explanation for enhanced cell proliferation. In addition, SOD3 reduced NF-κB activity by enhancing IκBα expression thus leading to reduced expression of inflammatory cytokines and adhesion molecules with consequent reduction in macrophage infiltration. Secondly, we sought to determine the fate and the effect of locally transplanted mesenchymal stem/stromal cells (MSCs) in acute ischemia. We showed that a vast majority of the transplanted cells are cleared from the injury site within 24 hours after local transplantation. Despite rapid clearance, transplantation was able to temporarily promote angiogenesis and cell proliferation in the muscle. Lack of graft-derived growth factor expression suggests other than secretory function to mediate this observed effect. In conclusion, both SOD3 and MSCs could be utilized to alleviate peripheral ischemia induced tissue injury. We have described a previously unidentified growth regulatory role for SOD3, and suggest a novel mechanism whereby transplanted MSCs enhance the reparative potential of the recipient tissue through physical contacts.

Factors Regulating Chondrogenic Differentiation

Chondrogenesis is a co-ordinated differentiation process in which mesenchymal cells condensate, differentiate into chondrocytes and begin to secrete molecules that form the extracellular matrix. It is regulated in a spatio-temporal manner by cellular interactions and growth and differentiation factors that modulate cellular signalling pathways and transcription of specific genes. Moreover, post-transcriptional regulation by microRNAs (miRNAs) has appeared to play a central role in diverse biological processes, but their role in skeletal development is not fully understood. Mesenchymal stromal cells (MSCs) are multipotent cells present in a variety of adult tissues, including bone marrow and adipose tissue. They can be isolated, expanded and, under defined conditions, induced to differentiate into multiple cell lineages including chondrocytes, osteoblasts and adipocytes in vitro and in vivo. Owing to their intrinsic capability to self-renew and differentiate into functional cell types, MSCs provide a promising source for cell-based therapeutic strategies for various degenerative diseases, such as osteoarthritis (OA). Due to the potential therapeutic applications, it is of importance to better understand the MSC biology and the regulatory mechanisms of their differentiation. In this study, an in vitro assay for chondrogenic differentiation of mouse MSCs (mMSCs) was developed for the screening of various factors for their chondrogenic potential. Conditions were optimized for pellet cultures by inducing mMSC with different bone morphogenetic proteins (BMPs) that were selected based on their known chondrogenic relevance. Characterization of the surface epitope profile, differentiation capacity and molecular signature of mMSCs illustrated the importance of cell population composition and the interaction between different populations in the cell fate determination and differentiation of MSCs. Regulation of Wnt signalling activity by Wnt antagonist sFRP-1 was elucidated as a potential modulator of lineage commitment. Delta-like 1 (dlk1), a factor regulating adipogenesis and osteogenesis, was shown to exhibit stage-specific expression during embryonic chondrogenesis and identified as a novel regulator of chondrogenesis, possibly through mediating the effect of TGF-beta1. Moreover, miRNA profiling demonstrated that MSCs differentiating into a certain lineage exhibit a specific miRNA expression profile. The complex regulatory network between miRNAs and transcription factors is suggested to play a crucial role in fine-tuning the differentiation of MSCs. These results demonstrate that commitment of mesenchymal stromal cells and further differentiation into specific lineages is regulated by interactions between MSCs, various growth and transcription factors, and miRNA-mediated translational repression of lineage-specific genes.

Identification and characterization of new genes and pathways regulating spermatogonial cell fate

Spermatogenesis, i.e sperm production in the seminiferous tubules of the testis, is a complex process that takes over one month to complete. Life-long ability of sperm production ultimately lies in a small population of undifferentiated cells, called spermatogonial stem cells (SSCs). These cells give rise to differentiating spermatogonia, which are committed to mature into spermatozoa. SSCs represent a heterogeneous population of cells and many aspects of their basic biology are still unknown. Understanding the mechanisms behind the cell fate decision of these cells is important to gain more insights into the causes of infertility and testis cancer. In addition, an interesting new aspect is the use of testis-derived stem cells in regenerative medicine. Our data demonstrated that adult mouse testis houses a population of Nanog-expressing spermatogonia. Based on mRNA and protein analysis these cells are enriched in stage XII of the mouse seminiferous epithelial cycle. The cells derived from this stage have the highest capacity to give rise to ES cell-like cells which express Oct4 and Nanog. These cells are under tight non- GDNF regulation but their fate can be dictated by activating p21 signalling. Comparative studies suggested that these cells are regulated like ES cells. Taken together these data imply that pluripotent cells are present in the adult mammalian testis. CIP2A (cancerous inhibitor of PP2A) has been associated with tumour aggressiveness and poor prognosis. In the testis it is expressed by the descendants of stem cells, i.e. the spermatogonial progenitor cells. Our data suggest that CIP2A acts upstream of PLZF and is needed for quantitatively normal spermatogenesis. Classification of CIP2A as a cancer/testis gene makes it an attractive target for cancer therapy. Study on the CIP2A deficient mouse model demonstrates that systemic inhibition of CIP2A does not severely interfere with growth and development or tissue or organ function, except for the spermatogenic output. These data demonstrate that CIP2A is required for quantitatively normal spermatogenesis. Hedgehog (Hh) signalling is involved in the development and maintenance of many different tissues and organs. According to our data, Hh signalling is active at many different levels during rat spermatogenesis: in spermatogonia, spermatocytes and late elongating spermatids. Localization of Suppressor of Fused (SuFu), the negative regulator of the pathway, specifically in early elongating spermatids suggests that Hh signalling needs to be shut down in these cells. Introduction of Hh signalling inhibitor resulted in an increase in germ cell apoptosis. Follicle-stimulating hormone (FSH) and inhibition of receptor tyrosine kinases resulted in down-regulation of Hh signalling. These data show that Hh signalling is under endocrine and paracrine control and it promotes germ cell survival.

MicroRNAs as novel regulators of skeletal homeostasis

The human skeleton is composed of bone and cartilage. The differentiation of bone and cartilage cells from their bone marrow progenitors is regulated by an intrinsic network of intracellular and extracellular signaling molecules. In addition, cells coordinate their differentiation and function through reciprocal cell‐to‐cell interactions. MicroRNAs (miRNAs) are small, single‐stranded RNA molecules that inhibit protein translation by binding to messenger RNAs (mRNAs). Recent evidence demonstrates the involvement of miRNAs in multiple biological processes. However, their role in skeletal development and bone remodeling is still poorly understood. The aim of this thesis was to elucidate miRNA‐mediated gene regulation in bone and cartilage cells, namely in osteoblasts, osteoclasts, chondrocytes and bone marrow adipocytes. Comparison of miRNA expression during osteogenic and chondrogenic differentiation of bone marrow‐derived mesenchymal stem cells (MSCs) revealed several miRNAs with substantial difference between bone and cartilage cells. These miRNAs were predicted to target genes essentially involved in MSC differentiation. Three miRNAs, miR‐96, miR‐124 and miR‐199a, showed marked upregulation upon osteogenic, chondrogenic or adipogenic differentiation. Based on functional studies, these miRNAs regulate gene expression in MSCs and may thereby play a role in the commitment and/or differentiation of MSCs. Characterization of miRNA expression during osteoclastogenesis of mouse bone marrow cells revealed a unique expression pattern for several miRNAs. Potential targets of the differentially expressed miRNAs included many molecules essentially involved in osteoclast differentiation. These results provide novel insights into the expression and function of miRNAs during the differentiation of bone and cartilage cells. This information may be useful for the development of novel stem cell‐based treatments for skeletal defects and diseases.

To divide or differentiate? : nestin-mediated regulation of Cdk5 in cell fate decisions

The molecular functions of the non-cell cycle-related Cyclin-dependent kinase 5 (Cdk5) have been of primary interest within the neuroscience field, but novel undertakings are constantly emerging for the kinase in tissue homeostasis, as well as in diseases such as diabetes and cancer. Although Cdk5 activation is predominantly regulated by specific non-cyclin activator protein binding, additional mechanisms have proved to orchestrate Cdk5 signaling in cells. For example, the interaction between the intermediate filament protein nestin and Cdk5 has been proposed to determine cellular fate during neuronal apoptosis through nestin-dependent adjustment of the sensitive balance and turnover of Cdk5 activators. While nestin constitutes a crucial regulatory scaffold for appropriate Cdk5 activation in apoptosis, Cdk5 itself phosphorylates nestin with the consequence of filament reorganization in both neuronal progenitors and differentiating muscle cells. Interestingly, the two proteins are often found coexpressed in various tissues and cell types, proposing that nestin-mediated scaffolding of Cdk5 and its activators may be applicable to other tissue systems as well. In the literature, the molecular functions of nestin have remained in the shade, as it is mostly exploited as a marker protein for progenitor cells. In light of these studies, the aim of this thesis was to assess the importance of the nestin scaffold in regulation of Cdk5 actions in cell fate decisions. This thesis can be subdivided into two major projects: one that studied the nature of the Cdk5-nestin interplay in muscle, and one that assessed their role in prostate cancer. During differentiation of a myoblast cell line, the filament formation properties of nestin was found to be crucial in directing Cdk5 activity, with direct consequences on the process of differentiation. Also the genetic knockout of nestin was found to influence Cdk5 activity, although differentiation per se was not affected. Instead, the genetic ablation of nestin had broad consequences on muscle homeostasis and regeneration. While the nestin-mediated regulation of Cdk5 in muscle was found to act in multiple ways, the connection remained more elusive in cancer models. Cdk5 was, however, established as a significant determinant of prostate cancer proliferation; a behavior uncharacteristic for this differentiation-associated kinase. Through complex and simultaneous regulation of two major prostate cancer pathways, Cdk5 was placed upstream of both Akt kinase and the androgen receptor. Its action on proliferation was nonetheless mainly exerted through the Akt signaling pathway in various cancer models. In summary, this thesis contributed to the knowledge of Cdk5 regulation and functions in two atypical settings; proliferation (in a cancer framework) and muscle differentiation, which is a poorly understood model system in the Cdk5 field. This balance between proliferation and differentiation implemented by Cdk5 is ultimately regulated (where present) by the dynamics of the cytoskeletal nestin scaffold.