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The contribution of microRNA-mediated posttranscriptional regulation on the final proteome in differentiating cells remains elusive. Here, we evaluated the impact of microRNAs (miRNAs) on the proteome of human umbilical cord blood-derived unrestricted somatic stem cells (USSC) during retinoic acid (RA) differentiation by a systemic approach using next generation sequencing analysing mRNA and miRNA expression and quantitative mass spectrometry-based proteome analyses. Interestingly, regulation of mRNAs and their dedicated proteins highly correlated during RA-incubation. Additionally, RA-induced USSC demonstrated a clear separation from native USSC thereby shifting from a proliferating to a metabolic phenotype. Bioinformatic integration of up- and downregulated miRNAs and proteins initially implied a strong impact of the miRNome on the XXL-USSC proteome. However, quantitative proteome analysis of the miRNA contribution on the final proteome after ectopic overexpression of downregulated miR-27a-5p and miR-221-5p or inhibition of upregulated miR-34a-5p, respectively, followed by RA-induction revealed only minor proportions of differentially abundant proteins. In addition, only small overlaps of these regulated proteins with inversely abundant proteins in non-transfected RA-treated USSC were observed. Hence, mRNA transcription rather than miRNA-mediated regulation is the driving force for protein regulation upon RA-incubation, strongly suggesting that miRNAs are fine-tuning regulators rather than active primary switches during RA-induction of USSC.

Background In multiple sclerosis loss of myelin and oligodendrocytes impairs saltatory signal transduction and leads to neuronal loss and functional deficits. Limited capacity of oligodendroglial precursor cells to differentiate into mature cells is the main reason for inefficient myelin repair in the central nervous system. Drug repurposing constitutes a powerful approach for identification of pharmacological compounds promoting this process. Methods A phenotypic compound screening using the subcellular distribution of a potent inhibitor of oligodendroglial cell differentiation, namely p57kip2, as differentiation competence marker was conducted. Hit compounds were validated in terms of their impact on developmental cell differentiation and myelination using both rat and human primary cell cultures and organotypic cerebellar slice cultures, respectively. Their effect on spontaneous remyelination was then investigated following cuprizone-mediated demyelination of the corpus callosum. Findings A number of novel small molecules able to promote oligodendroglial cell differentiation were identified and a subset was found to foster human oligodendrogenesis as well as myelination ex vivo. Among them the steroid danazol and the anthelminthic parbendazole were found to increase myelin repair. Interpretation We provide evidence that early cellular processes involved in differentiation decisions are applicable for the identification of regeneration promoting drugs and we suggest danazol and parbendazole as potent therapeutic candidates for demyelinating diseases. Funding This work was supported by the Jurgen Manchot Foundation, Dusseldorf; Research Commission of the Medical Faculty of Heinrich-Heine-University Dusseldorf; Christiane and Claudia Hempel Foundation; Stifterverband/Novartisstiftung; James and Elisabeth Cloppenburg, Peek and Cloppenburg Dusseldorf Stiftung and International Progressive MS Alliance (BRAVEinMS).

Proteins are released from cells by different secretory pathways. The secretory pathway via the ER-Golgi route - realized by a signal sequence - is referred to as "classical secretion". In contrast, alternative secretory pathways were summarized as "unconventional protein secretion". Until now, unconventional protein secretion was lacking attention due to the absence of detailed mechanistic insight and limited experimental access. However, there is a growing number of experimental data showing that a large proportion of secreted proteins is released by these alternative routes. Secretomics - the analysis of all secreted proteins of a cell population - offers the opportunity to gain more functional insight into unconventional protein secretion. Several pitfalls in secretome analysis starting with the analyzed cell model and sample preparation to data analysis have to be considered for detailed characterization of the secretome. Here, we highlight the investigation of secretomes by quantitative LC-MS/MS analysis and discuss pitfalls and opportunities in the characterization of unconventionally secreted proteins by secretome analysis.

Recently, it has been shown that human unrestricted somatic stem cells (USSCs) from umbilical cord blood represent pluripotent, neonatal, nonhematopoietic stem cells with the potential to differentiate into the neural lineage. However, molecular and functional characterization of the neural phenotype and evaluation of the degree of maturity of the resulting cells are still lacking. In this study, we addressed the question of neuronal differentiation and maturation induced by a defined composition of growth and differentiation factors (XXL medium). We demonstrated the expression of different neuronal markers and their enrichment in USSC cultures during XXL medium incubation. Furthermore, we showed enrichment of USSCs expressing tyrosine hydroxylase (TH), an enzyme specific for dopaminergic neurons and other catecholamine-producing neurons, accompanied by induction of Nurr1, a factor regulating dopaminergic neurogenesis. The functionality of USSCs has been analyzed by patch-clamp recordings and high-performance liquid chromatography (HPLC). Voltage-gated sodium-channels could be identified in laminin-predifferentiated USSCs. In addition, HPLC analysis revealed synthesis and release of the neurotransmitter dopamine by USSC-derived cells, thus correlating well with the detection of TH transcripts and protein. This study provides novel insight into the potential of unrestricted somatic stem cells from human umbilical cord blood to acquire a neuronal phenotype and function.

Scaphoid bones have a high prevalence for non‐union. Even with adequate treatment, bone regeneration may not occur in certain instances. Although this condition is well described, the molecular pathology of scaphoid non‐unions is still poorly defined. In this study, gene expression of osteogenic and angiogenic growth and transcription factors as well as inflammatory mediators were analysed in human scaphoid non‐unions and intraindividually compared to adjacent autologous cancellous bone from the distal radius. In addition, histology and immunohistochemical stainings were performed to verify qRT‐PCR data. Gene expression analysis revealed a significant up‐regulation of RANKL, ALP, CYCLIN D1, MMP‐13, OPG, NFATc1, TGF‐β and WNT5A in scaphoid non‐unions. Interestingly, RANKL and NFATc1, both markers for osteoclastogenesis, were significantly induced in non‐unions. Moreover, WNT5A was highly up‐regulated in all non‐union samples. TRAP staining confirmed the observation of induced osteoclastogenesis in non‐unions. With respect to genes related to osteogenesis, alkaline phosphatase was significantly up‐regulated in scaphoid non‐unions. No differences were detectable for other osteogenic genes such as RUNX‐2 or BMP‐2. Importantly, we did not detect differences in angiogenesis between scaphoid non‐unions and controls in both gene expression and immunohistochemistry. Summarized, our data indicate increased osteoclast activity in scaphoid non‐unions possibly as a result of the alterations in RANKL, TGF‐β and WNT5A expression levels. These data increase our understanding for the reduced bone regeneration capacity present in scaphoid non‐unions and may translate into the identification of new therapeutic targets to avoid secondary damages and prevent occurrence of non‐unions to scaphoid bones.

Stem cell transplantation is a promising therapeutic strategy to enhance axonal regeneration after spinal cord injury. Unrestricted somatic stem cells (USSC) isolated from human umbilical cord blood is an attractive stem cell population available at GMP grade without any ethical concerns. It has been shown that USSC transplantation into acute injured rat spinal cords leads to axonal regrowth and significant locomotor recovery, yet lacking cell replacement. Instead, USSC secrete trophic factors enhancing neurite growth of primary cortical neurons in vitro. Here, we applied a functional secretome approach characterizing proteins secreted by USSC for the first time and validated candidate neurite growth promoting factors using primary cortical neurons in vitro. By mass spectrometric analysis and exhaustive bioinformatic interrogation we identified 1156 proteins representing the secretome of USSC. Using Gene Ontology we revealed that USSC secretome contains proteins involved in a number of relevant biological processes of nerve regeneration such as cell adhesion, cell motion, blood vessel formation, cytoskeleton organization and extracellular matrix organization. We found for instance that 31 well-known neurite growth promoting factors like, e.g. neuronal growth regulator 1, NDNF, SPARC, and PEDF span the whole abundance range of USSC secretome. By the means of primary cortical neurons in vitro assays we verified SPARC and PEDF as significantly involved in USSC mediated neurite growth and therewith underline their role in improved locomotor recovery after transplantation. From our data we are convinced that USSC are a valuable tool in regenerative medicine as USSC's secretome contains a comprehensive network of trophic factors supporting nerve regeneration not only by a single process but also maintained its regenerative phenotype by a multitude of relevant biological processes.

Transplanted stem cells provide beneficial effects on regeneration/recovery after spinal cord injury (SCI) by the release of growth-promoting factors, increased tissue preservation, and provision of a permissive environment for axon regeneration. A rise in chemokine stromal cell-derived factor 1 (SDF-1/CXCL12) expression levels in central nervous system (CNS) injury sites has been shown to play a central role in recruiting transplanted stem cells. Although technically more challenging, it has been shown that after SCI few endogenous stem cells are recruited via SDF-1/CXCR4 signaling. Evidence is accumulating that increasing SDF-1 levels at the injury site (e.g., by exogenous application or transfection methods) further enhances stem cell recruitment. Moreover, SDF-1 might, in addition to migration, also influence survival, proliferation, differentiation, and cytokine secretion of stem cells. Here, we discuss the experimental data available on the role of SDF-1 in stem and progenitor cell biology following CNS injury and suggest strategies for how manipulation of the SDF-1 system could facilitate stem cell-based therapeutic approaches in SCI. In addition, we discuss challenges such as how to circumvent off-target effects in order to facilitate the transfer of SDF-1 to the clinic.

Stem cell therapy is a potential treatment for spinal cord injury and different stem cell types have been grafted into animal models and humans suffering from spinal trauma. Due to inconsistent results, it is still an important and clinically relevant question which stem cell type will prove to be therapeutically effective. Thus far, stem cells of human sources grafted into spinal cord mostly included barely defined heterogeneous mesenchymal stem cell populations derived from bone marrow or umbilical cord blood. Here, we have transplanted a well-defined unrestricted somatic stem cell isolated from human umbilical cord blood into an acute traumatic spinal cord injury of adult immune suppressed rat. Grafting of unrestricted somatic stem cells into the vicinity of a dorsal hemisection injury at thoracic level eight resulted in hepatocyte growth factor-directed migration and accumulation within the lesion area, reduction in lesion size and augmented tissue sparing, enhanced axon regrowth and significant functional locomotor improvement as revealed by three behavioural tasks (open field Basso-Beattie-Bresnahan locomotor score, horizontal ladder walking test and CatWalk gait analysis). To accomplish the beneficial effects, neither neural differentiation nor long-lasting persistence of the grafted human stem cells appears to be required. The secretion of neurite outgrowth-promoting factors in vitro further suggests a paracrine function of unrestricted somatic stem cells in spinal cord injury. Given the highly supportive functional characteristics in spinal cord injury, production in virtually unlimited quantities at GMP grade and lack of ethical concerns, unrestricted somatic stem cells appear to be a highly suitable human stem cell source for clinical application in central nervous system injuries.

Unrestricted somatic stem cells (USSCs) represent an intrinsically multipotent CD45-negative fetal population from human cord blood. They show differentiation into neuronal cells of a dopaminergic phenotype, which express neuronal markers such as synaptophysin, neuronal-specific nuclear protein, and neurofilament and release the neurotransmitter dopamine accompanied by expression of dopaminergic key factors tyrosine hydroxylase and Nurr1 (NR4A2). MicroRNA expression analysis highlighted their importance in neural development but their specific functions remain poorly understood. Here, downregulation of a set of 18 microRNAs during neuronal lineage differentiation of unrestricted somatic stem cells, including members of the miR-17-92 family and additional microRNAs such as miR-130a, -138, -218, and -335 as well as their target genes, is described. In silico target gene predictions for this microRNA group uncovered a large set of proteins involved in neuronal differentiation and having a strong impact on differentiation-related pathways such as axon guidance and TGFβ, WNT, and MAPK signaling. Experimental target validations confirmed approximately 35% of predictions tested and revealed a group of proteins with specific impact on neuronal differentiation and function including neurobeachin, neurogenic differentiation 1, cysteine-rich motor neuron protein 1, neuropentraxin 1, and others. These proteins are combined targets for several subgroups from the set of 18 downregulated microRNAs. This finding was further supported by the observed upregulation of a significant amount of predicted and validated target genes based on Illumina Beadstudio microarray data. Confirming the functional relationship of a limited panel of microRNAs and predicted target proteins reveals a clear network-like impact of the group of 18 downregulated microRNAs on proteins involved in neuronal development and function.

Background MicroRNAs are short (∼22 nt) non-coding regulatory RNAs that control gene expression at the post-transcriptional level. Here the functional impact of microRNAs on cell cycle arrest during neuronal lineage differentiation of unrestricted somatic stem cells from human cord blood (USSC) was analyzed. Methodology/Principal Findings Expression profiling revealed downregulation of microRNAs miR-17, -20a, and -106b in USSC differentiated into neuronal lineage but not in USSC differentiated into osteogenic lineage. Transfection experiments followed by Ki67 immunostainings demonstrated that each of these microRNAs was able to promote proliferation of native USSC and to prevent in part cell cycle arrest during neuronal lineage differentiation of USSC. Bioinformatic target gene predictions followed by experimental target gene validations revealed that miR-17, -20a, and -106b act in a common manner by downregulating an overlapping set of target genes mostly involved in regulation and execution of G1/S transition. Pro-proliferative target genes cyclinD1 (CCND1) and E2F1 as well as anti-proliferative targets CDKN1A (p21), PTEN, RB1, RBL1 (p107), RBL2 (p130) were shown as common targets for miR-17, -20a, and -106b. Furthermore, these microRNAs also downregulate WEE1 which is involved in G2/M transition. Most strikingly, miR-17, -20a, and -106b were found to promote cell proliferation by increasing the intracellular activity of E2F transcription factors, despite the fact that miR-17, -20a, and -106b directly target the transcripts that encode for this protein family. Conclusions/Significance Mir-17, -20a, and -106b downregulate a common set of pro- and anti-proliferative target genes to impact cell cycle progression of USSC and increase intracellular activity of E2F transcription factors to govern G1/S transition.