Human Unrestricted Somatic Stem Cells Research Articles

Restoring Synaptic Function: How Intranasal Delivery of 3D-Cultured hUSSC Exosomes Improve Learning and Memory Deficits in Alzheimer's Disease.

Memory problems are often the first signs of cognitive impairment related to Alzheimer's disease (AD), and stem cells and stem cell-derived exosomes (EXOs) have been studied for their therapeutic potential to improve the disease signs. While many studies have shown the anti-inflammatory and immunomodulatory effects of stem cells and exosomes on improving memory in different AD models, there is still insufficient data to determine how they modulate neural plasticity to enhance spatial memory and learning ability. Therefore, we conducted a study to investigate the effects of exosomes derived from 3D-cultured human Unrestricted Somatic Stem Cells (hUSSCs) on spatial memory and neuroplasticity markers in a sporadic rat model of AD. Using male Wistar rats induced by intracerebral ventricle injection of streptozotocin, we demonstrated that intranasal administration of hUSSC-derived exosomes could decrease Aβ accumulation and improve learning and memory in the Morris water maze test. We also observed an increase in the expression of pre-synaptic and post-synaptic molecules involved in neuronal plasticity, including NMDAR1, integrin β1, synaptophysin, pPKCα, and GAP-43, in the hippocampus. Our findings suggest that intranasal administration of exosomes can ameliorate spatial learning and memory deficits in rats, at least in part, by increasing the expression of neuroplasticity proteins. These results may encourage researchers to further investigate the molecular pathways involved in memory improvement after stem cell and exosome therapy, with the goal of increasing the efficacy and safety of exosome-based treatments for AD.

Influence of Xenogeneic and Alloplastic Carriers for Bone Augmentation on Human Unrestricted Somatic Stem Cells.

Alloplastic and xenogeneic bone grafting materials are frequently used for bone augmentation. The effect of these materials on precursor cells for bone augmentation is yet to be determined. The aim of this study was to ascertain, in vitro, how augmentation materials influence the growth rates and viability of human unrestricted somatic stem cells. The biocompatibility of two xenogeneic and one alloplastic bone graft was tested using human unrestricted somatic stem cells (USSCs). Proliferation, growth, survival and attachment of unrestricted somatic stem cells were monitored after 24 h, 48 h and 7 days. Furthermore, cell shape and morphology were evaluated by SEM. Scaffolds were assessed for their physical properties by Micro-CT imaging. USSCs showed distinct proliferation on the different carriers. Greatest proliferation was observed on the xenogeneic carriers along with improved viability of the cells. Pore sizes of the scaffolds varied significantly, with the xenogeneic materials providing greater pore sizes than the synthetic inorganic material. Unrestricted somatic stem cells in combination with a bovine collagenous bone block seem to be very compatible. A scaffold’s surface morphology, pore size and bioactive characteristics influence the proliferation, attachment and viability of USSCs.

In vitro evaluation of ferutinin on proliferation and osteogenesis differentiation in human unrestricted Somatic stem cells

Osteoporosis is a common disease in post-menopausal women. The increased risk of breast cancer and malignancy with hormone replacement, hampers its wide-usage. Phytoestrogens are known to have selective estrogen receptor modulator activity. The present study aims to determine how ferutinin affects unrestricted human Somatic Stem Cells (USSCs) osteogenic differentiation. The effect of ferutinin on USSCs proliferation was assessed by MTT assay while osteogenesis was evaluated using Alkaline Phosphatase Activity (ALP), calcium deposition and Alizarin Red Staining. Quantitative real-time PCR was applied to examine the expression of bone specific genes such as osteocalcin, Runx2, and BMP-2. Ferutinin (5–15 µg/mL) could positively impact on the proliferation of cells in a dose-dependent manner. Also, ALP enzyme activity and calcium deposition were enhanced in the presence of ferutinin. Based on real-time PCR results, ferutinin could increase the expression of bone marker genes. The pattern of ferutinin effect on gene expression is similar to standard synthetic estrogen, 17-β-estradiol. In the presence of the estrogen activity inhibitor (ICI), the effect of ferutinin on ALP and gene level was diminished. In conclusion, ferutinin may be considered as a potential candidate for the stem cell therapy in osteoporosis.

Synthesis of thermogel modified with biomaterials as carrier for hUSSCs differentiation into cardiac cells: Physicomechanical and biological assessment

One of the major challenges in cardiac cell therapy is cell death and low rate of cardiac differentiation. In this regard, a series of thermosensitive and injectable hydrogels with similar physicomechanical properties of cardiac tissue can be an ideal candidate for delivering human unrestricted somatic stem cells (hUSSCs) and improve the quality of cell therapy. Here, we designed N-isopropylacrylamide/acrylic acid/N-acryloxysuccinimide/2-hydroxyethyl methacrylate-poly lactide (NIPAAm/AAc/NAS/HEMAPLA) hydrogel via ring-opening polymerization for encapsulation of the cells. To improve biological activities, biomaterials like hyaluronic acid (HA), Aloe vera (AV), and silk fibroin (SF) were added with pure hydrogel (Pgel). The modulus of hydrogels was estimated between 68.1 and 74.63 kPa that was similar to the normal cardiac modulus. Moreover, the elastic region increased by adding biomaterials to Pgel. The results of RT-PCR and ICC demonstrated that the most expression of early genes (GATA4, NKX2.5) was related to Pgel/AV/SF group. In contrast, the highest expression of other genes (GJA1, TNNI3, MYH6) was observed in Pgel/HA/SF. The presence of biomaterials in the structure of hydrogel can play a key role in cell fate and the induction of cell differentiation as a factor influencing mechanotransduction. These hydrogels hold an excellent promise to deliver hUSSCs into damaged tissue for cardiac regeneration.

Functional omics analyses reveal only minor effects of microRNAs on human somatic stem cell differentiation

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.

Human unrestricted somatic stem cells ameliorate sepsis-related acute lung injury in mice.

Sepsis and related disorders, especially acute lung injury (ALI), are the most challenging life-threatening diseases in the hospital intensive care unit. Complex pathophysiology, unbalanced immune condition, and high rate of mortality complicate the treatment of sepsis. Recently, cell therapy has been introduced as a promising option to recover the sepsis symptoms. The aim of this study was to investigate the therapeutic potential of human unrestricted somatic stem cells (USSCs) isolated from human umbilical cord blood in the mouse model of ALI. USSCs significantly enhanced the survival rate of mice suffering from ALI and suppressed concentrations of proinflammatory mediators TNF-α, and interleukin (IL)-6, and the level of anti-inflammatory cytokine IL-10. ALI mice injected by USSCs showed notable reduction in lung and liver injury, pulmonary edema, and hepatic enzymes, compared with the control group. These results determined the in vivo immunomodulatory effect of USSCs for recovery of immune balance and reduction of tissue injury in the mouse model of ALI. Therefore, USSCs can be a suitable therapeutic approach to manage sepsis disease through the anti-inflammatory potential.

Unrestricted Somatic Stem Cells Loaded in Nanofibrous Conduit as Potential Candidate for Sciatic Nerve Regeneration.

Motor and sensory recovery following critical size peripheral nerve defects is often incomplete. Although nerve grafting has been proposed as the gold standard, it is associated with several disadvantages. Here we report a novel approach to peripheral nerve repair using Human Unrestricted Somatic Stem Cells (USSC) delivered through an electrospun neural guidance conduit. Conduits were produced from PCL and gelatin blend. Several in vitro methods were utilized to investigate the conduit's physicochemical and biological characteristics. Nerve regeneration was studied across a 10-mm sciatic nerve gap in Wistar rats. For functional analysis, the conduits were seeded with 3 × 104 USSCs and implanted into a 10-mm sciatic nerve defect. After 14weeks, the results of functional recovery analysis and histopathological examinations showed that animals implanted with USSC containing conduits exhibited improved functional and histopathological recovery which was more close to the autograft group compared to other groups. Our results support the potential applicability of USSCs to treat peripheral nerve injury in the clinic.

Unrestricted somatic stem cells, as a novel feeder layer: Ex vivo culture of human limbal stem cells.

Ex vivo culture of limbal stem cells (LSCs) is a current promising approach for reconstruction of the ocular surface. In this context, 3T3 feeder layer cells (mouse embryo fibroblast) are generally utilized to maintain and expand LSCs. The aim of this study is to develop a novel culture method (animal-derived products free) to expand LSCs, using umbilical cord derived human unrestricted somatic stem cells (hUSSCs) instead of 3T3 cell with an emphasis on maintaining of the Stemness in LSCs. Using flow-cytometer, isolated hUSSCs were characterized for CD105, CD90, CD166, CD34, CD45, CD31 cell surface markers and their differentiation capability into adipogenic as well as osteogenic lineages were evaluated. In addition to colony-forming efficiency (CFE), epithelial lineage differentiation and karyotyping, LSC properties were evaluated for ABCG2, ΔNP63-α, CK19, CK3, and CK12 mRNA and protein expressions using quantitative RT-PCR (qRT-PCR) and immunocytochemistry, when these cells were co-cultured with hUSSCs (in comparison with 3T3 feeder layer). LSCs, co-cultured with hUSSCs, showed normal karyotype (46, XX), while they could efficiently form colony (86 ± 3) and display up-regulation of the genes associated with stemness and down-regulation of corneal epithelial differentiation genes. Consistent with 3T3 feeder cells, hUSSCs with spindle-shaped morphology and quick splitting up properties had ability to preserve the stem like-cell phenotype of LSCs. These findings were confirmed by qRT-PCR and flow-cytometer. Findings of present study suggest hUSSCs as a promising alternative method for 3T3 feeder layer cells, to preserve growth and stemness of LSCs ex vivo culture.

Human unrestricted somatic stem cells loaded in nanofibrous PCL scaffold and their healing effect on skin defects

Unrestricted somatic stem cells (USSCs) loaded in nanofibrous polycaprolactone (PCL) scaffolds can be used for skin regeneration when grafted onto full-thickness skin defects of rats. Nanofibrous PCL scaffolds were designed by the electrospinning method and crosslinked with laminin protein. Afterwards, the scaffolds were evaluated by scanning electron microscopy, and physical and mechanical assays. In this study, nanofibrous PCL scaffolds loaded with USSCs were grafted onto the skin defects. The wounds were subsequently investigated 21 days after grafting. Results of mechanical and physical analyses showed good resilience and compliance to movement as a skin graft. In animal models; study samples exhibited the most pronounced effect on wound closure, with statistically significant improvement in wound healing being seen at 21 days post-operatively. Histological examinations of healed wounds from all samples showed a thin epidermis plus recovered skin appendages in the dermal layer for samples with cell. Thus, the graft of nanofibrous PCL scaffolds loaded with USSC showed better results during the healing process of skin defects in rat models.

Mir-302 cluster exhibits tumor suppressor properties on human unrestricted somatic stem cells

Many studies have reported that miR-302-367 cluster acts in different ways in various cell types. For instance, this cluster is shown to have a potential role in stemness regulation in embryonic stem cells (ESCs). On the other hand, this cluster inhibits the tumorigenicity of human pluripotent stem cells by coordinated suppression of CDK2 and CDK4/6 cell cycle pathways. Indeed, this cluster has a significant posttranscriptional impact on cell cycle progression. Previous reports have shown the participation of miR-302-367 cluster in cell cycle regulation of hESCs, MCF7, HepG2, and Teta-2 embryonal teratocarcinoma cells, but its effect on unrestricted somatic stem cells (USSCs) as a new source of human somatic stem cells from the umbilical cord blood remains to be elucidated. Therefore, in this study, we aimed to investigate the effect of miR-302-367 cluster on cell proliferation by MTT assay, cell cycle analysis, and colony formation assay. In addition, the expression of candidate cell cycle regulatory performance and tumor suppressor genes was determined. In this study, for the first time, we found that miR-302-367 cluster not only did not reprogram human USSCs into a pluripotent ESC-like state, but also inhibited the proliferation of human USSCs. Moreover, analyzing the cell cycle curve revealed a significant apoptotic phase upon viral introduction of miR-302-367. Our gene expression study revealed the overexpression of candidate genes after transduction of USSCs with miR-302-367 cluster. In conclusion, the controversial role of miR-302-367 in different cell types may provide better understanding for its role in stemness level and its antitumorigenicity potential in different contexts.

Unrestricted somatic stem cells as vehicle for nerve growth factor gene transfer

Objective: Nerve growth factor (NGF), a member of the neurotrophic factor family, plays a critical role in the maintenance and regeneration of different types of neurons. To overcome drastic challenges in the peripheral delivery of NGF, transplantation of NGF secreting stem cells to the target site of an injury may be an effective procedure. Unrestricted somatic stem cells (USSCs), a subtype of umbilical cord blood (UCB) stem cells, have shown promise for gene therapy purposes, and proper results have been observed from transplantation experiments in neurodegenerative disorders. Based on the considerable potential of USSCs for gene delivery applications, the goal of the current study was to establish a betaNGF gene containing USSCs, which is able to secrete functional recombinant betaNGF protein.Methods: Unrestricted somatic stem cells were isolated from UCB and were cultured in a DMEM medium. The betaNGF gene was cloned in the EFalpha-promoting lentiviral vector, and virus production was performed as a third generation lentivirus packaging system. Enzyme-linked immunosorbent assay, real-time PCR, co-culture, MTT, and immunocytochemistry assays were performed to evaluate the genetically engineered USSCs.Results: Overexpression of betaNGF gene in human USSCs created a USSC line that is able to secrete high amounts of functional betaNGF protein. betaNGF-secreting USSCs showed a high rate of viability along with acceptable immunological and morphological properties for transplantation into the nervous system.Discussion: Long-term expression of functional betaNGF, high viability of betaNGF producing USSCs, and expression of primary neuronal markers suggest that USSCs may be useful to deliver betaNGF into targeted sites of the nervous system in neurodegenerative disorders.

Xenotransplantation of human unrestricted somatic stem cells in a pig model of acute myocardial infarction

Stem cell therapy may help restore cardiac function after acute myocardial infarction (AMI), but the optimal therapeutic cell type has not been identified. We examined the effects of CD34-/CD45- human unrestricted somatic stem cells (USSCs) in pigs (n=30) with an AMI created by a 90-min occlusion of the left anterior descending coronary artery. Pigs were randomly assigned to receive either USSCs (302±23×10(6) cells) or phosphate-buffered saline via 15 NOGA-guided transendocardial injections 10days after AMI. Cyclosporine A (10mg/kg orally, twice a day) was started in all pigs 3days before control or cell treatment. Cardiac function was assessed by echocardiography before injection and at 4 and 8weeks after treatment. Serum titers for pig IgG antibodies against USSCs were also measured at these time points and before AMI. Compared with control pigs, USSC-treated pigs showed no significant differences in any of the functional parameters examined. USSC-treated pigs showed variable increases in anti-USSC IgG antibody titers in the blood and chronic inflammatory infiltrates at the cell injection sites. Immunohistochemical studies of the injection sites using human anti-mitochondrial antibodies failed to detect implanted USSCs. We conclude that human USSCs did not improve cardiac function in a pig model of AMI. Cell transplantation in a xenogeneic setting may obscure the benefits of stem cell therapy.

Portal Application of Human Unrestricted Somatic Stem Cells to Support Hepatic Regeneration after Portal Embolization and Tumor Surgery

Insufficient liver remnant volume still precludes patients with potentially resectable tumors from curative surgery. Clinically, it has been demonstrated that transplanted adult stem cells promote liver regeneration. However, the mechanisms of the observed functional improvements are unknown. The aim of our study was to evaluate the impact of transplanted human multipotent cord blood-derived unrestricted somatic stem cells (USSC) on liver regeneration and identify the underlying mechanisms in an ovine model. We performed partial embolization of the right liver lobe and grafted USSC in the portal venous system of the left liver lobe. After 4 weeks, livers were explanted and analyzed for differentiation of USSC into hepatocytes by histopathologic examination and for fusion of USSC with recipient hepatocytes by single-cell polymerase chain reaction. The studies revealed that transplanted USSC differentiate into hepatocytes and produce human albumin. No ovine DNA was found in the hepatocytes with a human phenotype. Transplantation of USSC enhances the number of viable hepatocytes in liver disease by differentiation and opens new therapeutic perspectives.

Down-regulation of miRNA-221 triggers osteogenic differentiation in human stem cells

Despite interesting in silico evidence, the specific role of mir-221 in osteogenesis has not been studied. We evaluated the osteogenic induction of transient-transfected anti-mir-221 in human unrestricted somatic stem cells and human mesenchymal stem cells both transcriptionally and translationally. In transfected unrestricted somatic stem cells, transcriptions of some osteogenic markers were twice that of the control and translations of osteopontin and osteocalcin were increased from 9 to 39 % and from 0 to 21 %, respectively. Up-regulation of transcribed osteogenic markers in transfected mesenchymal stem cells were 50 times greater than controls while no significant change in translations were observed. Prior to these analyses, the authenticity of stem cells, their osteogenic differentiation and transfection efficiency were verified. Transient modulation of mir-221 therefore suggests a mechanism for rapid induction of osteogenesis as a useful strategy for cell-based therapy.

A comparative study on nonviral genetic modifications in cord blood and bone marrow mesenchymal stem cells.

The focus of both clinical and basic studies on stem cells is increasing due to their potentials in regenerative medicine and cell-based therapies. Recently stem cells have been genetically modified to enhance an existing character in or to bring a new property to them. However, accomplishment of declared goals requires detailed knowledge about their molecular characteristics which could be achieved by genetic modifications mostly through nonviral transfection strategies. Capable of differentiating into multiple cells, human unrestricted somatic stem cells (hUSSCs) and human mesenchymal stem cells (hMSCs) seem to be suitable candidates for transfection approaches. Involvement of microRNAs (miRNAs) in many biological processes makes their transfection evaluation valuable. Herein we investigated the efficacy and toxicity of four typically used transfection reagents (Arrest-In, Lipofectamine 2000, Oligofectamine and HiPerfect) systematically to deliver fluorescent labeled-miRNA and Green Fluorescent Protein (GFP) expressing plasmid into hUSSCs and hMSCs. The authenticity of stem cells was verified by differentiation experiments along with flow cytometry of surface markers. Our study revealed that stemness properties of these stem cells were not affected by transient transfection. Moreover the ratios of cell viability and transfection efficiency in both analyzed stem cells were reversed. Considering cell viability, the highest fraction of GFP-expressing cells was obtained using Oligofectamine (~50%) while the highest transfection rate of miRNA was achieved by Lipofectamine 2000 (~90%). Moreover dependency of hMSCs to size of transfected nucleic acid and time-dependency of Oligofectamine and their affection on the yield of transfection were observed. Cytotoxicity assessments also showed that hUSSCs are sensitive to HiPerFect. In addition cells treated by Lipofectamine showed morphological changes. Representing the efficient nucleic acid transfection, our research facilitates comprehensive genetic modification of stem cells and demonstrates powerful approaches to understand stem cell molecular regulation mechanisms, which eventually improves nonviral cell-mediated gene therapy.

Effective combination of aligned nanocomposite nanofibers and human unrestricted somatic stem cells for bone tissue engineering

Bioartificial bone tissue engineering is an increasingly popular technique to solve bone defect challenges. This study aimed to investigate the interactions between matrix composition and appropriate cell type, focusing on hydroxyapatite (HA), to achieve a more effective combination for bone regeneration. Human unrestricted somatic stem cells (USSCs) were isolated from placental cord blood. The cellular and molecular events during the osteo-induction of USSCs were evaluated for 21 d under the following conditions: (1) in basal culture, (2) supplemented with hydroxyapatite nanoparticle (nHA) suspension, and (3) seeded on electrospun aligned nanofibrous poly-ɛ-caprolactone/poly-L-lactic acid/nHA (PCL/PLLA/nHA) scaffolds. The scaffolds were characterized using scanning electron microscope (SEM), fourier transform infrared spectroscopy (FTIR) and tensile test. Maintenance of USSCs for 21 d in basal or osteogenic culture resulted in significant increase in osteoblast differentiation. With nHA suspension, even soluble osteo-inductive additives were ineffective, probably due to induced apoptosis of the cells. In contrast to the hindrance of proliferation by nHA suspension, the scaffolds improved cell growth. The scaffolds mimic the nanostructure of natural bone matrix with the combination of PLLA/PCL (organic phase) and HA (inorganic phase) offering a favorable surface topography, which was demonstrated to possess suitable properties for supporting USSCs. Quantitative measurement of osteogenic markers, enzymatic activity and mineralization indicated that the scaffolds did not disturb, but enhanced the osteogenic potential of USSCs. Moreover, the alignment of the fibers led to cell orientation during cell growth. The results demonstrated the synergism of PCL/PLLA/nHA nanofibrous scaffolds and USSCs in the augmentation of osteogenic differentiation. Thus, nHA grafted into PCL/PLLA scaffolds can be a suitable choice for bone tissue regeneration.

A comparison between osteogenic differentiation of human unrestricted somatic stem cells and mesenchymal stem cells from bone marrow and adipose tissue

To evaluate the potential of three stem cells for cell therapy and tissue engineering applications, the biological behavior and osteogenic capacity of the newly introduced cord-blood-derived, unrestricted somatic stem cells (USSC) were compared with those of mesenchymal stem cells isolated from bone marrow (BM-MSC) and adipose tissue (AT-MSC). There was no significant difference between the rates of proliferation of the three stem cells. During osteogenic differentiation, alkaline phosphatase (ALP) activity peaked on day 7 in USSC compared to BM-MSC which showed the maximum value of ALP activity on day 14. However, BM-MSC had the highest ALP activity and mineralization during osteogenic induction. In addition, AT-MSC showed the lowest capacity for mineralization during differentiation and had the lowest ALP activity on days 7 and 14. Although AT-MSC expressed higher levels of collagen type I, osteonectin and BMP-2 in undifferentiated state, but these genes were expressed higher in BM-MSC during differentiation. BM-MSC also expressed higher levels of ALP, osteocalcin and Runx2 during induction. Taking together, BM-MSC showed the highest capacity for osteogenic differentiation and hold promising potential for bone tissue engineering and cell therapy applications.

Comparison of Ectopic Bone Formation of Embryonic Stem Cells and Cord Blood Stem CellsIn Vivo

Cell-based reconstruction therapies promise new therapeutic opportunities for bone regeneration. Unrestricted somatic stem cells (USSC) from cord blood and embryonic stem cells (ESCs) can be differentiated into osteogenic cells. The purpose of this in vivo study was to compare their ability to induce ectopic bone formation in vivo. Human USSCs and murine ESCs were cultured as both monolayer cultures and micromasses and seeded on insoluble collagenous bone matrix (ICBM). One week and 1, 2, and 3 months after implanting the constructs in immune-deficient rats, computed tomography scans were performed to detect any calcification. Subsequently, the implanted constructs were examined histologically. The radiological examination showed a steep increase in the mineralized bone-like tissue in the USSC groups. This increase can be considered as statistically significant compared to the basic value. Moreover, the volume and the calcium portion measured by computed tomography scans were about 10 times higher than in the ESC group. The volume of mineralization in the ESC group increased to a much smaller extent over the course of time, and the control group (ICBM without cells) showed almost no alterations during the study. The histological examinations parallel the radiological findings. Cord blood stem cells in combination with ICBM-induced ectopic bone formation in vivo are stronger than ESCs.

MicroRNA hsa-miR-135b Regulates Mineralization in Osteogenic Differentiation of Human Unrestricted Somatic Stem Cells

Unrestricted somatic stem cells (USSCs) have been recently identified in human umbilical cord blood and have been shown to differentiate into lineages representing all 3 germ layers. To characterize microRNAs that may regulate osteogenic differentiation of USSCs, we carried out expression analysis for 157 microRNAs using quantitative RT-PCR before and after osteogenic induction (t = 0.5, 24, 72, 168, 216 h). Three microRNAs, hsa-miR-135b, hsa-miR-224, and hsa-miR-31, were consistently down-regulated during osteogenesis of USSC line 1. Hsa-miR-135b was shown to be the most profoundly down-regulated in osteogenesis of USSC line 1 and further confirmed to be down-regulated in the osteogenic differentiation of 2 additional USSC lines. Function of hsa-miR-135b in osteogenesis of USSCs was examined by retroviral overexpression, which resulted in an evident decreased mineralization, indicating that hsa-miR-135b down-regulation is functionally important for full osteogenic differentiation of USSCs. MicroRNAs have been shown to regulate negatively expression of their target gene(s). To identify putative targets of hsa-miR-135b, we performed cDNA microarray expression analysis. We selected in total 10 transcripts that were down-regulated (>or=2-fold) in response to hsa-miR-135b overexpression at day 7 and day 9 of osteogenic differentiation. The function of most of these targets in human osteogenesis is unknown and requires further investigation. Markedly, quantitative RT-PCR data showed decreased expression of osteogenic markers IBSP and Osterix, both known to be involved in bone mineralization, in osteogenesis of USSCs that overexpress hsa-miR-135b. This finding suggests that hsa-miR-135b may control osteoblastic differentiation of USSCs by regulating expression of bone-related genes.

Biocompatibility of Osteogenic Predifferentiated Human Cord Blood Stem Cells with Biomaterials and the Influence of the Biomaterial on the Process of Differentiation

Modern cell-based bone reconstruction therapies offer new therapeutic opportunities and tissue engineering represents a more biological-oriented approach to heal bone defects of the skeleton. Human unrestricted somatic stem cells (USSCs) derived form umbilical cord blood offer new promising aspects e.g., can differentiate into osteogenetic cells. Furthermore these cells have fewer ethical and legal restrictions compared to embryonic stem cells (ESCs). The purpose of this study was to evaluate the compatibility of osteogenic pre-differentiated USSCs with various biomaterials and to address the question, whether biomaterials influence the process of differentiation of the USSCs. After osteogenic differentiation with DAG USSCs were cultivated with various biomaterials. To asses the biocompatibility of USSCs the attachment and the proliferation of the cells on the biomaterial were measured by a CyQUANT(®) assay, the morphology was analyzed by scanning electron microscopy and the influence of the gene expression was analyzed by real time PCR. Our results provide evidence that insoluble collagenous bone matrix followed by β-tricalciumphosphate is highly suitable for bone tissue engineering regarding cell attachment and proliferation. The gene expression analysis indicates that biomaterials influence the gene expression of USSCs. These results are in concordance with our previous study with ESCs.