Proteomics Approaches in the Identification of Molecular Signatures of Mesenchymal Stem Cells

Mesenchymal stem cells (MSCs) are undifferentiated, multi-potent stem cells with the ability to renew. They can differentiate into many types of terminal cells, such as osteoblasts, chondrocytes, adipocytes, myocytes, and neurons. These cells have been applied in tissue engineering as the main cell type to regenerate new tissues. However, a number of issues remain concerning the use of MSCs, such as cell surface markers, the determining factors responsible for their differentiation to terminal cells, and the mechanisms whereby growth factors stimulate MSCs. In this chapter, we will discuss how proteomic techniques have contributed to our current knowledge and how they can be used to address issues currently facing MSC research. The application of proteomics has led to the identification of a special pattern of cell surface protein expression of MSCs. The technique has also contributed to the study of a regulatory network of MSC differentiation to terminal differentiated cells, including osteocytes, chondrocytes, adipocytes, neurons, cardiomyocytes, hepatocytes, and pancreatic islet cells. It has also helped elucidate mechanisms for growth factor–stimulated differentiation of MSCs. Proteomics can, however, not reveal the accurate role of a special pathway and must therefore be combined with other approaches for this purpose. A new generation of proteomic techniques have recently been developed, which will enable a more comprehensive study of MSCs. Keywords

ATF5, a possible regulator of osteogenic differentiation in human adipose-derived stem cells

The regulatory pathways involved in maintaining the pluripotency of embryonic stem cells are partially known, whereas the regulatory pathways governing adult stem cells and their "stem-ness" are characterized to an even lesser extent. We, therefore, screened the transcriptome profiles of 20 osteogenically induced adult human adipose-derived stem cell (ADSC) populations and investigated for putative transcription factors that could regulate the osteogenic differentiation of these ADSC. We studied a subgroup of donors' samples that had a disparate osteogenic response transcriptome from that of induced human fetal osteoblasts and the rest of the induced human ADSC samples. From our statistical analysis, we found activating transcription factor 5 (ATF5) to be significantly and consistently down-regulated in a randomized time-course study of osteogenically differentiated adipose-derived stem cells from human donor samples. Knockdown of ATF5 with siRNA showed an increased sensitivity to osteogenic induction. This evidence suggests a role for ATF5 in the regulation of osteogenic differentiation in adipose-derived stem cells. To our knowledge, this is the first report that indicates a novel role of transcription factors in regulating osteogenic differentiation in adult or tissue specific stem cells. © 2012 Wiley Periodicals, Inc.

Expression of PTRF in PC3 cells modulates cholesterol dynamics and the actin cytoskeleton impacting secretion pathways

Expression of caveolin-1 is up-regulated in prostate cancer metastasis and is associated with aggressive recurrence of the disease. Intriguingly, caveolin-1 is also secreted from prostate cancer cell lines and has been identified in secreted prostasomes. Caveolin-1 is the major structural component of the plasma membrane invaginations called caveolae. Co-expression of the coat protein Polymerase I and transcript release factor (PTRF) is required for caveolae formation. We recently found that expression of caveolin-1 in the aggressive prostate cancer cell line PC-3 is not accompanied by PTRF, leading to noncaveolar caveolin-1 lipid rafts. Moreover, ectopic expression of PTRF in PC-3 cells sequesters caveolin-1 into caveolae. Here we quantitatively analyzed the effect of PTRF expression on the PC-3 proteome using stable isotope labeling by amino acids in culture and subcellular proteomics. We show that PTRF reduced the secretion of a subset of proteins including secreted proteases, cytokines, and growth regulatory proteins, partly via a reduction in prostasome secretion. To determine the cellular mechanism accounting for the observed reduction in secreted proteins we analyzed total membrane and the detergent-resistant membrane fractions. Our data show that PTRF expression selectively impaired the recruitment of actin cytoskeletal proteins to the detergent-resistant membrane, which correlated with altered cholesterol distribution in PC-3 cells expressing PTRF. Consistent with this, modulating cellular cholesterol altered the actin cytoskeleton and protein secretion in PC-3 cells. Intriguingly, several proteins that function in ER to Golgi trafficking were reduced by PTRF expression. Taken together, these results suggest that the noncaveolar caveolin-1 found in prostate cancer cells generates a lipid raft microenvironment that accentuates secretion pathways, possibly at the step of ER sorting/exit. Importantly, these effects could be modulated by PTRF expression.

Osteocyte-induced angiogenesis via VEGF–MAPK-dependent pathways in endothelial cells

Recently, it has been suggested osteocytes control the activities of bone formation (osteoblasts) and resorption (osteoclast), indicating their important regulatory role in bone remodelling. However, to date, the role of osteocytes in controlling bone vascularisation remains unknown. Our aim was to investigate the interaction between endothelial cells and osteocytes and to explore the possible molecular mechanisms during angiogenesis. To model osteocyte/endothelial cell interactions, we co-cultured osteocyte cell line (MLOY4) with endothelial cell line (HUVECs). Co-cultures were performed in 1:1 mixture of osteocytes and endothelial cells or by using the conditioned media (CM) transfer method. Real-time cell migration of HUVECs was measured with the transwell migration assay and xCELLigence system. Expression levels of angiogenesis- related genes were measured by quantitative real-time polymerase chain reaction (qRT-PCR). The effect of vascular endothelial growth factor (VEGF) and mitogen-activated phosphorylated kinase (MAPK) signaling were monitored by western blotting using relevant antibodies and inhibitors. During the bone formation, it was noted that osteocyte dendritic processes were closely connected to the blood vessels. The CM generated from MLOY4 cells-activated proliferation, migration, tube-like structure formation, and upregulation of angiogenic genes in endothelial cells suggesting that secretory factor(s) from osteocytes could be responsible for angiogenesis. Furthermore, we identified that VEGF secreted from MLOY4-activated VEGFR2–MAPK–ERK-signaling pathways in HUVECs. Inhibiting VEGF and/or MAPK–ERK pathways abrogated osteocyte-mediated angiogenesis in HUVEC cells. Our data suggest an important role of osteocytes in regulating angiogenesis.

Breast cancer cells induce osteolytic bone lesions in vivo through a reduction in osteoblast activity in mice

Bone metastases are severely debilitating and have a significant impact on the quality of life of women with metastatic breast cancer. Treatment options are limited and in order to develop more targeted therapies, improved understanding of the complex mechanisms that lead to bone lesion development are warranted. Interestingly, whilst prostate-derived bone metastases are characterised by mixed or osteoblastic lesions, breast-derived bone metastases are characterised by osteolytic lesions, suggesting unique regulatory patterns. This study aimed to measure the changes in bone formation and bone resorption activity at two time-points (18 and 36 days) during development of the bone lesion following intratibial injection of MDA-MB-231 human breast cancer cells into the left tibiae of Severely Combined Immuno-Deficient (SCID) mice. The contralateral tibia was used as a control. Tibiae were extracted and processed for undecalcified histomorphometric analysis. We provide evidence that the early bone loss observed following exposure to MDA-MB-231 cells was due to a significant reduction in mineral apposition rate, rather than increased levels of bone resorption. This suggests that osteoblast activity was impaired in the presence of breast cancer cells, contrary to previous reports of osteoclast-dependent bone loss. Furthermore mRNA expression of Dickkopf Homolog 1 (DKK-1) and Noggin were confirmed in the MDA-MB-231 cell line, both of which antagonise osteoblast regulatory pathways. The observed bone loss following injection of cancer cells was due to an overall thinning of the trabecular bone struts rather than perforation of the bone tissue matrix (as measured by trabecular width and trabecular separation, respectively), suggesting an opportunity to reverse the cancer-induced bone changes. These novel insights into the mechanisms through which osteolytic bone lesions develop may be important in the development of new treatment strategies for metastatic breast cancer patients.

Toward biomimetic materials in bone regeneration : functional behavior of mesenchymal stem cells on a broad spectrum of extracellular matrix components

Bone defect treatments can be augmented by mesenchymal stem cell (MSC) based therapies. MSC interaction with the extracellular matrix (ECM) of the surrounding tissue regulates their functional behavior. Understanding of these specific regulatory mechanisms is essential for the therapeutic stimulation of MSC in vivo. However, these interactions are presently only partially understood. This study examined in parallel, for the first time, the effects on the functional behavior of MSCs of 13 ECM components from bone, cartilage and hematoma compared to a control protein, and hence draws conclusions for rational biomaterial design. ECM components specifically modulated MSC adhesion, migration, proliferation, and osteogenic differentiation, for example, fibronectin facilitated migration, adhesion, and proliferation, but not osteogenic differentiation, whereas fibrinogen enhanced adhesion and proliferation, but not migration. Subsequently, the integrin expression pattern of MSCs was determined and related to the cell behavior on specific ECM components. Finally, on this basis, peptide sequences are reported for the potential stimulation of MSC functions. Based on the results of this study, ECM component coatings could be designed to specifically guide cell functions.

Rho GTPases mediate the mechanosensitive lineage commitment of neural stem cells

Adult neural stem cells (NSCs) play important roles in learning and memory and are negatively impacted by neurological disease. It is known that biochemical and genetic factors regulate self-renewal and differentiation, and it has recently been suggested that mechanical and solid-state cues, such as extracellular matrix (ECM) stiffness, can also regulate the functions of NSCs and other stem cell types. However, relatively little is known of the molecular mechanisms through which stem cells transduce mechanical inputs into fate decisions, the extent to which mechanical inputs instruct fate decisions versus select for or against lineage-committed blast populations, or the in vivo relevance of mechanotransductive signaling molecules in native stem cell niches. Here we demonstrate that ECM-derived mechanical signals act through Rho GTPases to activate the cellular contractility machinery in a key early window during differentiation to regulate NSC lineage commitment. Furthermore, culturing NSCs on increasingly stiff ECMs enhances RhoA and Cdc42 activation, increases NSC stiffness, and suppresses neurogenesis. Likewise, inhibiting RhoA and Cdc42 or downstream regulators of cellular contractility rescues NSCs from stiff matrix- and Rho GTPase-induced neurosuppression. Importantly, Rho GTPase expression and ECM stiffness do not alter proliferation or apoptosis rates indicating that an instructive rather than selective mechanism modulates lineage distributions. Finally, in the adult brain, RhoA activation in hippocampal progenitors suppresses neurogenesis, analogous to its effect in vitro. These results establish Rho GTPase-based mechanotransduction and cellular stiffness as biophysical regulators of NSC fate in vitro and RhoA as an important regulatory protein in the hippocampal stem cell niche.

Autologous human kidney proximal tubule epithelial cells (PTEC) modulate dendritic cell (DC), T cell and B cell responses

This is a comprehensive study of human kidney proximal tubular epithelial cells (PTEC) which are known to respond to and mediate the pathological process of a range of kidney diseases. It identifies various molecules expressed by PTEC and how these molecules participate in down-regulating the inflammatory process, thereby highlighting the clinical potential of these molecules to treat various kidney diseases. In the disease state, PTEC gain the ability to regulate the immune cell responses present within the interstitium. This down-regulation is a complex interaction of contact dependent/independent mechanisms involving various immuno-regulatory molecules including PD-L1, sHLA-G and IDO. The overall outcome of this down-regulation is suppressed DC maturation, decreased number of antibody producing B cells and low T cell responses. These manifestations within a clinical setting are expected to dampen the ongoing inflammation, preventing the damage caused to the kidney tissue.

EphB4 localises to the nucleus of prostate cancer cells

The EphB4 receptor tyrosine kinase is over-expressed in a variety of different epithelial cancers including prostate where it has been shown to be involved in survival, migration and angiogenesis. We report here that EphB4 also resides in the nucleus of prostate cancer cell lines. We used in silico methods to identify a bipartite nuclear localisation signal (NLS) in the extracellular domain and a monopartite NLS sequence in the intracellular kinase domain of EphB4. To determine whether both putative NLS sequences were functional, fragments of the EphB4 sequence containing each NLS were cloned to create EphB4NLS-GFP fusion proteins. Localisation of both NLS-GFP proteins to the nuclei of transfected cells was observed, demonstrating that EphB4 contains two functional NLS sequences. Mutation of the key amino residues in both NLS sequences resulted in diminished nuclear accumulation. As nuclear translocation is often dependent on importins we confirmed that EphB4 and importin-α can interact. To assess if nuclear EphB4 could be implicated in gene regulatory functions potential EphB4-binding genomic loci were identified using chromatin immunoprecipitation and Lef1 was confirmed as a potential target of EphB4-mediated gene regulation. These novel findings add further complexity to the biology of this important cancer-associated receptor.

Manufacturing and use of human placenta-derived mesenchymal stromal cells for phase I clinical trials: Establishment and evaluation of a protocol

Background/Aim. Mesenchymal stromal cells (MSCs) have been utilised in many clinical trials as an experimental treatment in numerous clinical settings. Bone marrow remains the traditional source tissue for MSCs but is relatively hard to access in large volumes. Alternatively, MSCs may be derived from other tissues including the placenta and adipose tissue. In an initial study no obvious differences in parameters such as cell surface phenotype, chemokine receptor display, mesodermal differentiation capacity or immunosuppressive ability, were detected when we compared human marrow derived- MSCs to human placenta-derived MSCs. The aim of this study was to establish and evaluate a protocol and related processes for preparation placenta-derived MSCs for early phase clinical trials. Methods. A full-term placenta was taken after delivery of the baby as a source of MSCs. Isolation, seeding, incubation, cryopreservation of human placentaderived MSCs and used production release criteria were in accordance with the complex regulatory requirements applicable to Code of Good Manufacturing Practice manufacturing of ex vivo expanded cells. Results. We established and evaluated instructions for MSCs preparation protocol and gave an overview of the three clinical areas application. In the first trial, MSCs were co-transplanted iv to patient receiving an allogeneic cord blood transplant as therapy for treatmentrefractory acute myeloid leukemia. In the second trial, MSCs were administered iv in the treatment of idiopathic pulmonary fibrosis and without serious adverse effects. In the third trial, MSCs were injected directly into the site of tendon damage using ultrasound guidance in the treatment of chronic refractory tendinopathy. Conclusion. Clinical trials using both allogeneic and autologous cells demonstrated MSCs to be safe. A described protocol for human placenta-derived MSCs is appropriate for use in a clinical setting, relatively inexpensive and can be relatively easily adjusted to a different set of regulatory requirements, as applicable to early phase clinical trials.

Mapping the regulatory sequences controlling 93 breast cancer-associated miRNA genes leads to the identification of two functional promoters of the Hsa-mir-200b cluster, methylation of which is associated with metastasis or hormone receptor status in advanced breast cancer

MicroRNAs (miRNAs) are small non-coding RNAs of 20 nt in length that are capable of modulating gene expression post-transcriptionally. Although miRNAs have been implicated in cancer, including breast cancer, the regulation of miRNA transcription and the role of defects in this process in cancer is not well understood. In this study we have mapped the promoters of 93 breast cancer-associated miRNAs, and then looked for associations between DNA methylation of 15 of these promoters and miRNA expression in breast cancer cells. The miRNA promoters with clearest association between DNA methylation and expression included a previously described and a novel promoter of the Hsa-mir-200b cluster. The novel promoter of the Hsa-mir-200b cluster, denoted P2, is located 2 kb upstream of the 5′ stemloop and maps within a CpG island. P2 has comparable promoter activity to the previously reported promoter (P1), and is able to drive the expression of miR-200b in its endogenous genomic context. DNA methylation of both P1 and P2 was inversely associated with miR-200b expression in eight out of nine breast cancer cell lines, and in vitro methylation of both promoters repressed their activity in reporter assays. In clinical samples, P1 and P2 were differentially methylated with methylation inversely associated with miR-200b expression. P1 was hypermethylated in metastatic lymph nodes compared with matched primary breast tumours whereas P2 hypermethylation was associated with loss of either oestrogen receptor or progesterone receptor. Hypomethylation of P2 was associated with gain of HER2 and androgen receptor expression. These data suggest an association between miR-200b regulation and breast cancer subtype and a potential use of DNA methylation of miRNA promoters as a component of a suite of breast cancer biomarkers.