Biologically Active Sheep Colostrum for Topical Treatment and Skin Care.

Colostrum is gaining popularity in cosmetic products. The present study compared the composition and selected biological properties of colostrum from Polish sheep (colostrum 1) and Swiss sheep (colostrum 2), particularly those that can affect healthy or diseased skin. The antioxidant activity of the colostrums was measured using ABTS and DPPH assays. The effect on the proliferation of human skin fibroblasts, neonatal epidermal keratinocytes, and human diabetic fibroblast (dHF) cells isolated from diabetic foot ulcers was also assayed in vitro by MTT and Presto Blue tests, respectively. The colostrum simulated dHF cell proliferation by up to 115.4%. The highest used concentration of colostrum 1 stimulated normal fibroblast proliferation by 191.2% (24 h) and 222.2% (48 h). Both colostrums inhibited epidermal keratinocyte viability. The influence of the colostrums on the expression of genes related to proliferation (Ki67) and immune response (IL-6, PTGS-2, TSG-6) in dHF cells were compared. Colostrum 1 increased the rate of wound closure (scar test). Analysis of total fat, protein and fatty acid content found the Polish colostrum to be a richer source of fat than the Swiss colostrum, which contained a larger amount of protein. Both colostrums exhibit properties that suggest they could be effective components in cosmetic or medicinal formulations for skin care, especially supporting its regeneration, rejuvenation, and wound healing.

Microbiological Aspects of Pharmaceutical Manufacturing of Adipose-Derived Stem Cell-Based Medicinal Products.

Subcutaneous adipose tissue is an excellent source of mesenchymal stem cells (ADSCs), which can be used in cell therapies as an active substance in advanced therapy medicinal products (ATMPs). Because of the short shelf-life of ATMPs and the time needed to obtain the results of microbiological analysis, the final product is often administered to the patient before sterility is confirmed. Because the tissue used for cell isolation is not sterilized to maintain cell viability, controlling and ensuring microbiological purity at all stages of production is crucial. This study presents the results of monitoring the contamination incidence during ADSC-based ATMP manufacturing over two years. It was found that more than 40% of lipoaspirates were contaminated with thirteen different microorganisms, which were identified as being physiological flora from human skin. Such contamination was successfully eliminated from the final ATMPs through the implementation of additional microbiological monitoring and decontamination steps at various stages of production. Environmental monitoring revealed incidental bacterial or fungal growth, which did not result in any product contamination and was reduced thanks to an effective quality assurance system. To conclude, the tissue used for ADSC-based ATMP manufacturing should be considered contaminated; therefore, good manufacturing practices specific to this type of product must be elaborated and implemented by the manufacturer and the clinic in order to obtain a sterile product.

Secretome of Mesenchymal Stromal Cells as a Possible Innovative Therapeutic Tool in Facial Nerve Injury Treatment.

Facial nerve palsy is a serious neurological condition that strongly affects patient everyday life. Standard treatments provide insufficient improvement and are burdened with the risk of severe complications, e.g., facial synkinesis. Mesenchymal stromal cell-based therapies are a novel and extensively developed field which offers new treatment approaches with promising results in regards to the nervous tissue regeneration. The potential of mesenchymal stromal cells (MSCs) to aid the regeneration of damaged nerves has been demonstrated in several preclinical models, as well as in several clinical trials. However, therapies based on cell transplantation are difficult to standardize in the manner similar to that of routine clinical practices. On the other hand, treatments based on mesenchymal stromal cell secretome harness the proregenerative features of mesenchymal stromal cells but relay on a product with measurable parameters that can be put through standardization procedures and deliver a fully controllable end-product. Utilization of mesenchymal stromal cell secretome allows the controlled dosage and standardization of the components to maximize the therapeutic potential and ensure safety of the end-product.

Allogenic Adipose-Derived Stem Cells in Diabetic Foot Ulcer Treatment: Clinical Effectiveness, Safety, Survival in the Wound Site, and Proteomic Impact.

Although encouraging results of adipose-derived stem cell (ADSC) use in wound healing are available, the mechanism of action has been studied mainly in vitro and in animals. This work aimed to examine the safety and efficacy of allogenic ADSCs in human diabetic foot ulcer treatment, in combination with the analyses of the wound. Equal groups of 23 participants each received fibrin gel with ADSCs or fibrin gel alone. The clinical effects were assessed at four time points: days 7, 14, 21 and 49. Material collected during debridement from a subset of each group was analyzed for the presence of ADSC donor DNA and proteomic changes. The reduction in wound size was greater at all subsequent visits, significantly on day 21 and 49, and the time to 50% reduction in the wound size was significantly shorter in patients who received ADSCs. Complete healing was achieved at the end of the study in seven patients treated with ADSCs vs. one treated without ADSCs. One week after ADSC application, 34 proteins significantly differentiated the material from both groups, seven of which, i.e., GAPDH, CAT, ACTN1, KRT1, KRT9, SCL4A1, and TPI, positively correlated with the healing rate. We detected ADSC donor DNA up to 21 days after administration. We confirmed ADSC-related improvement in wound healing that correlated with the molecular background, which provides insights into the role of ADSCs in wound healing-a step toward the development of cell-based therapies.

Biobank Personnel - The Key to its Success.

Resources from biobanks and biorepositories, such as human samples, are of increasing interest to specialists in various fields. However, whilst biobanks provide a crucial service, their efficient and effective management can prove challenging. When establishing a biobank many factors should be considered, such as the need for appropriate infrastructure, equipment, financial support, and highly specialised and suitably qualified personnel. The number and qualifications of the necessary personnel depend both on the biobank's size and type - i.e. a biobank that is large and diversified in terms of the stored material should be organised differently to a small biorepository. The core of the biobank should be composed of highly trained personnel that closely co-operate with the general and quality control manager. Due to the large amount of data related to the samples, an IT specialist might be needed. In the case of large population biobanks, personnel responsible for patient recruitment, documentation handling, sample collection and distribution to the biobank would be necessary. Furthermore, staff responsible for the infrastructure are also highly important, as they are the first responders to failures that may be critical for the biobank functioning. Depending on the type and size of the biobank/biorepository, some responsibilities and tasks could potentially be combined. Nevertheless, highly trained personnel with clear and precisely defined duties are the key to the proper functioning of a biobank.

Banking of AT-MSC and its Influence on Their Application to Clinical Procedures.

Processing of MSCs to obtain a therapeutic product consists of two main steps: 1) the in vitro expansion of the cells until an appropriate number of them is obtained, and 2) freezing and storage of the expanded cells. The last step is critical and must be optimized so that after thawing the cells retain all their physiological properties including the secretory function. In this paper, we evaluated physiological parameters of AT-MSC's after a full cycle of their processing, particularly freezing and storing at the liquid nitrogen vapor temperature. Based on the recovered proliferative and secretory capacities of the thawed cells, we have designed the optimal technique for processing of MSCs for clinical applications. In our work, we tried to select the best DMSO-based cryoprotectant mixture on the base of post thawing fully retain their properties. We have demonstrated the effectiveness of the use of DMSO in various configurations of the constituent cryoprotective fluids. We have also shown that AT-MSCs that show control levels in most standard tests (viability, shape, culture behaviour, and proliferative properties) after thawing, may show transient variations in some important physiological properties, such as the level of secreted growth factors. Obtained results let us to indicate how to optimize the AT-MSC preparation process for clinical applications. We suggest that before their clinical application the cells should be cultured for at least one passage to recover their physiological stability and thus assure their optimal therapeutic potential.

Peritoneal fluid stimulates neoplastic transformation of normal HEK 293 cells by high expression of pluripotent genes.

Gynecological cancers constitute a serious problem in the world. Their advanced stages are often characterized by the accumulation of ascites, which leads to spreading of cancer cells outside their primary focus. Despite progress in the treatment, prognoses are still not satisfactory. The main causes of these failures are chemoresistance, metastases and recurrences of the disease, which is influenced by, among others, the microenvironment of cancer cells. This study investigated the effect of the microenvironment, which create ascites derived from patients with ovarian and endometrial cancer to non-gynecological HEK 293 cells. The effect of the gynecological cancer microenvironment on HEK 293 cells behaviour was analysed using RT-PCR, qRT-PCR, Western blotting and functional analysis (invasion assays, hanging drop) methods. Our results suggest that the key genes for the development of cancer can be regulated by epigenetic and hypoxia-inducible factor in dependent manner. It was observed that in vitro microenvironment, which is created by cells originating from patients with gynecological cancer (ovarian cancer, endometrial cancer) is able to generate changes in HEK 293 cells by itself.

Epigenetic Modulation of Stem Cells in Neurodevelopment: The Role of Methylation and Acetylation.

The coordinated development of the nervous system requires fidelity in the expression of specific genes determining the different neural cell phenotypes. Stem cell fate decisions during neurodevelopment are strictly correlated with their epigenetic status. The epigenetic regulatory processes, such as DNA methylation and histone modifications discussed in this review article, may impact both neural stem cell (NSC) self-renewal and differentiation and thus play an important role in neurodevelopment. At the same time, stem cell decisions regarding fate commitment and differentiation are highly dependent on the temporospatial expression of specific genes contingent on the developmental stage of the nervous system. An interplay between the above, as well as basic cell processes, such as transcription regulation, DNA replication, cell cycle regulation and DNA repair therefore determine the accuracy and function of neuronal connections. This may significantly impact embryonic health and development as well as cognitive processes such as neuroplasticity and memory formation later in the adult.

Antibacterial Activity and Cytotoxicity of Silver(I) Complexes of Pyridine and (Benz)Imidazole Derivatives. X-ray Crystal Structure of [Ag(2,6-di(CH2OH)py)2]NO3.

Selected aspects of the biological activity of a series of six nitrate silver(I) complexes with pyridine and (benz)imidazole derivatives were investigated. The present study evaluated the antibacterial activities of the complexes against three Gram-negative strains: Pseudomonas aeruginosa ATCC 15442, Escherichia coli ATCC 25922 and Proteus hauseri ATCC 13315. The results were compared with those of silver nitrate, a silver sulfadiazine drug and appropriate ligands. The most significant antibacterial properties were exerted by silver(I) complexes containing benzimidazole derivatives. The cytotoxic activity of the complexes was examined against B16 (murine melanoma) and 10T1/2 (murine fibroblasts) cells. All of the tested silver(I) compounds were not toxic to fibroblast cells in concentration inhibited cancer cell (B16) viability by 50%, which ranged between 2.44-28.65 µM. The molecular and crystal structure of silver(I) complex of 2,6-di(hydroxymethyl)pyridine was determined by single-crystal X-ray diffraction analysis. The most important features of the crystal packing and intermolecular non-covalent interactions in the Ag(I) complex were quantified via Hirshfeld surface analysis.

Low oxygen atmosphere facilitates proliferation and maintains undifferentiated state of umbilical cord mesenchymal stem cells in an hypoxia inducible factor-dependent manner.

BACKGROUND AIMS: As we approach the era of mesenchymal stem cell (MSC) application in the medical clinic, the standarization of their culture conditions are of the particular importance. We re-evaluated the influences of oxygens concentration on proliferation, stemness and differentiation of human umbilical cord Wharton Jelly-derived MSCs (WJ-MSCs). METHODS: Primary cultures growing in 21% oxygen were either transferred into 5% O2 or continued to grow under standard 21% oxygen conditions. Cell expansion was estimated by WST1/enzyme-linked immunosorbent assay or cell counting. After 2 or 4 weeks of culture, cell phenotypes were evaluated using microscopic, immunocytochemical, fluorescence-activated cell-sorting and molecular methods. Genes and proteins typical of mesenchymal cells, committed neural cells or more primitive stem/progenitors (Oct4A, Nanog, Rex1, Sox2) and hypoxia inducible factor (HIF)-1α-3α were evaluated. RESULTS: Lowering O2 concentration from 21% to the physiologically relevant 5% level substantially affected cell characteristics, with induction of stemness-related-transcription-factor and stimulation of cell proliferative capacity, with increased colony-forming unit fibroblasts (CFU-F) centers exerting OCT4A, NANOG and HIF-1α and HIF-2α immunoreactivity. Moreover, the spontaneous and time-dependent ability of WJ-MSCs to differentiate into neural lineage under 21% O2 culture was blocked in the reduced oxygen condition. Importantly, treatment with trichostatin A (TSA, a histone deacetylase inhibitor) suppressed HIF-1α and HIF-2α expression, in addition to blockading the cellular effects of reduced oxygen concentration. CONCLUSIONS: A physiologically relevant microenvironment of 5% O2 rejuvenates WJ-MSC culture toward less-differentiated, more primitive and faster-growing phenotypes with involvement of HIF-1α and HIF-2α-mediated and TSA-sensitive chromatin modification mechanisms. These observations add to the understanding of MSC responses to defined culture conditions, which is the most critical issue for adult stem cells translational applications.

Induced pluripotent stem cells (iPSc) for gene therapy.

The generation of autologous pluripotent stem cells by reprogramming and then their differentiation into any cell type is a very attractive prospect for biomedicine. Additionally, if it were possible to repair malfunctioning genes it would mean that there is hope for patients suffering from incurable diseases in which conventional treatment does not give satisfactory results. Data from animal models are promising. But there are still issues that must be solved, such as the low efficiency of the derivation of induced pluripotent cells, and most importantly, making sure that the techniques used both for reprogramming cells, as well as for gene therapy are safe.

Epigenetic and molecular signature of human umbilical cord blood-derived neural stem cell (HUCB-NSC) neuronal differentiation.

In the context of cell therapy, the epigenetic status of core stemness transcription factor (STF) genes regulating the cell proliferation/differentiation program is of primary interest. Our results confirmed that in vitro differentiation of the umbilicalcord-blood-derived-neural-stem-cells (HUCB-NSC) coincides with the progressive down-regulation of Oct3/4 and Nanog gene expression. Consistently and in parallel with the repression of gene transcription, a substantial increase in the mosaic cytosine methylation CpG dinucleotide was observed in the promoter regions of these STF genes. However none of the histone-H3 post-translational-modifications (PTM) known to be associated with transcriptionally active genes (H3Ac and H3K4me3) or repressed genes (H3K9me3 and H3K27me3) seemed to vary in relation to the progression of cell differentiation and down-regulation of STF genes. This indicates an uncoupling between STF gene expression and above mentioned histone PTMs. In contrast, the overall methylation of nuclear chromatin at repressive histone H3K9me3 was significantly higher than H3K4 trimetylation in expanding HUCB-NSC cultures and then increases through the progression of cell differentiation. These observations suggest different epigenetic programs of gene repression realized in the cell nuclei of differentiating HUCB-NSC cultures with uneven involvement of the repressive histone PTMs.

Treatment with small molecules is an important milestone towards the induction of pluripotency in neural stem cells derived from human cord blood.

Standardization of methods for obtaining iPS cells from the human somatic cells and then their successful differentiation are important in the context of their possible application in personalized cell therapy and the development of toxicological and pharmacological tests. In the present study, the influence of the small molecules representing epigenetic modulators (histone deacetylase inhibitor Trichostatin A and DNA methyltransferase inhibitor RG-108) on the process of reverting neural progenitors from HUCB-NSC (Human Umbilical Cord Blood Neural Stem Cell) line to the pluripotent state was tested. The experiments were conducted in low oxygen tension, in three different experimental layouts: (1) in the presence of reprogramming/recombinant polyarginine-tailed proteins; (2) with recombinant proteins and small molecules; (3) only in the presence of small molecules. We wanted to find out, whether it will be possible to induce pluripotent state of neural stem cells only by epigenetic modulators. Our results revealed that the inhibitors of DNA methylation and histone deacetylation used along with 5 percent oxygen tension can only transiently induce or elevate some pluripotency genes in neural progenitors with different pattern, but were not sufficient for stable reprogramming. The iPS cells from neural progenitor cells of HUCBNSC were obtained only when TSA, RG-108 and reprogramming proteins have been applied simultaneously. These cells were tested for the expression of the selected pluripotency genes and in functional assays to prove their pluripotency stage. The obtained data show that the small molecules in conjunction with reprogramming factors are the potent tools in cell reprogramming.

Influence of low oxygen tensions on expression of pluripotency genes in stem cells.

The stem cells are characterized by self-renewal ability and potential to differentiate into other cell types of the body. They are residuing in defined microenvironments - "stem cell niches". The embryonic stem cells (ESC) are derived from embryos which exist in 3-5 percent oxygen condition. This environment is physiologically normal not only for ES cells but also for many other types of stem cells including neural stem cells (NSC). These observations suggest that low oxygen condition plays a very important role in the maintenance of cell stemness. Pluripotency is regulated by the family of hypoxia inducible factors (HIFs), which are dependent on oxygen tensions. HIF-2α is an upstream regulator of Oct4, which is one of the main transcription factors used to generate the first induced pluripotent stem cells (iPSCs). It has been shown that knock-down of HIF-2α but not HIF-1α, leads to a decrease in the expression of Oct4, Nanog and Sox2, which are important stem cells markers. The structure of hypoxia inducible factors as well as their behavior in hypoxia and normoxia was described. Therefore optimization of oxygen concentration seems to be crucial from the stem cell transplantation as well as iPS transplantation standpoint. Although many experiments with cell culture under low oxygen condition were performed, there is still much that is unknown. This short review presents some aspects on important issue of hypoxia induced regulation of stemness.