Assessment of the Dose-Dependent Effect of Human Platelet Lysate on Wharton's Jelly-Derived Mesenchymal Stem/Stromal Cells Culture for Manufacturing Protocols.

Introduction: Mesenchymal stem/stromal cells (MSCs)-based products have unique characteristics compared to other drugs because of their inherently variable effects depending on culture conditions and microenvironment. In some cases, cells can be produced individually, one batch at a time, for personalized therapy. Therefore, it is very important to optimize both culture conditions and medium composition under Good Manufacturing Practice (GMP) standards. MSCs properties have been exploited as potential cell therapies in regenerative medicine. The main mechanism of their protective and regenerative effect is based on their secretory activity. Simultaneously, their secretome is highly variable and sensitive to any change in environmental conditions. Depending on the type of damage and the target application, it is desirable to enhance the secretion of therapeutic factors. Changes in the modulation of environmental conditions can affect survival, migration ability, and both proliferative and clonogenic potentials. Materials and Methods: This study cultured Wharton's jelly-derived MSCs (WJ-MSCs) in media with varying concentrations of human platelet lysate (hPL). Two groups were created: one with low hPL concentration and another with a high hPL concentration. The effects of these different hPL concentrations were analyzed by assessing mesenchymal phenotype retention, secretory activity, clonogenic potential, proliferation, and migration capabilities. Additionally, the secretion levels of key therapeutic factors, such as Hepatocyte Growth Factor (HGF), Brain-Derived Neurotrophic Factor (BDNF), and Chemokine Ligand 2 (CCL-2), were measured. Results: WJ-MSCs maintained their mesenchymal phenotype regardless of hPL concentration. However, a higher concentration of hPL promoted cell clonogenic potential, proliferation, migration, and increased secretion of therapeutic factors. Conclusion: Adjusting the hPL concentration in the culture medium modulates the response of WJ MSCs and enhances their therapeutic potential. Higher hPL concentration promotes increased secretory activity and improves the regenerative capacity of WJ-MSCs, suggesting a promising strategy to optimize MSC-based therapies.

Stemness properties of SSEA-4+ subpopulation isolated from heterogenous Wharton's jelly mesenchymal stem/stromal cells.

Background: High heterogeneity of mesenchymal stem/stromal cells (MSCs) due to different degrees of differentiation of cell subpopulations poses a considerable challenge in preclinical studies. The cells at a pluripotent-like stage represent a stem cell population of interest for many researchers worldwide, which is worthy of identification, isolation, and functional characterization. In the current study, we asked whether Wharton's jelly-derived MSCs (WJ-MSCs) which express stage-specific embryonic antigen-4 (SSEA-4) can be considered as a pluripotent-like stem cell population. Methods: SSEA-4 expression in different culture conditions was compared and the efficiency of two cell separation methods were assessed: Magnetic Activated Cell Sorting (MACS) and Fluorescence Activated Cell Sorting (FACS). After isolation, SSEA-4+ cells were analyzed for the following parameters: the maintenance of the SSEA-4 antigen expression after cell sorting, stem cell-related gene expression, proliferation potential, clonogenicity, secretome profiling, and the ability to form spheres under 3D culture conditions. Results: FACS allowed for the enrichment of SSEA-4+ cell content in the population that lasted for six passages after sorting. Despite the elevated expression of stemness-related genes, SSEA-4+ cells neither differed in their proliferation and clonogenicity potential from initial and negative populations nor exhibited pluripotent differentiation repertoire. SSEA-4+ cells were observed to form smaller spheroids and exhibited increased survival under 3D conditions. Conclusion: Despite the transient expression of stemness-related genes, our findings could not fully confirm the undifferentiated pluripotent-like nature of the SSEA-4+ WJ-MSC population cultured in vitro.

Advances in Neurorestoratology-Current status and future developments.

Neurorestoratology constitutes a novel discipline aimed at the restoration of damaged neural structures and impaired neurological functions. This area of knowledge integrates and compiles all concepts and strategies dealing with the neurorestoration. Although currently, this discipline has already been well recognized by physicians and scientists throughout the world, this article aimed at broadening its knowledge to the academic circle and the public society. Here we shortly introduced why and how Neurorestoratology was born since the fact that the central nervous system (CNS) can be repaired and the subsequent scientific evidence of the neurorestorative mechanisms behind, such as neurostimulation or neuromodulation, neuroprotection, neuroplasticity, neurogenesis, neuroregeneration or axonal regeneration or sprouting, neuroreplacement, loop reconstruction, remyelination, immunoregulation, angiogenesis or revascularization, and others. The scope of this discipline is the improvement of therapeutic approaches for neurological diseases and the development of neurorestorative strategies through the comprehensive efforts of experts in the different areas and all articulated by the associations of Neurorestoratology and its journals. Strikingly, this article additionally explores the "state of art" of the Neurorestoratology field. This includes the development process of the discipline, the achievements and advances of novel neurorestorative treatments, the most efficient procedures exploring and evaluating outcome after the application of pioneer therapies, all the joining of a multidisciplinary expert associations and the specialized journals being more and more impact. We believe that in a near future, this discipline will evolve fast, leading to a general application of cell-based comprehensive neurorestorative treatments to fulfill functional recovery demands for patients with neurological deficits or dysfunctions.

Promising Markers in the Context of Mesenchymal Stem/Stromal Cells Subpopulations with Unique Properties.

The heterogeneity of the mesenchymal stem/stromal cells (MSCs) population poses a challenge to researchers and clinicians, especially those observed at the population level. What is more, the lack of precise evidences regarding MSCs developmental origin even further complicate this issue. As the available evidences indicate several possible pathways of MSCs formation, this diverse origin may be reflected in the unique subsets of cells found within the MSCs population. Such populations differ in specialization degree, proliferation, and immunomodulatory properties or exhibit other additional properties such as increased angiogenesis capacity. In this review article, we attempted to identify such outstanding populations according to the specific surface antigens or intracellular markers. Described groups were characterized depending on their specialization and potential therapeutic application. The reports presented here cover a wide variety of properties found in the recent literature, which is quite scarce for many candidates mentioned in this article. Even though the collected information would allow for better targeting of specific subpopulations in regenerative medicine to increase the effectiveness of MSC-based therapies.

Critical points for optimizing long-term culture and neural differentiation capacity of rodent and human neural stem cells to facilitate translation into clinical settings.

Despite several decades of research on the nature and functional properties of neural stem cells, which brought great advances in regenerative medicine, there is still a plethora of ambiguous protocols and interpretations linked to their applications. Here, we present a whole spectrum of protocol elements that should be standardized in order to obtain viable cell cultures and facilitate their translation into clinical settings. Additionally, this review also presents outstanding limitations and possible problems to be encountered when dealing with protocol optimization. Most importantly, we also outline the critical points that should be considered before starting any experiments utilizing neural stem cells or interpreting their results.

Understanding Intra- and Inter-Species Variability in Neural Stem Cells' Biology Is Key to Their Successful Cryopreservation, Culture, and Propagation.

Although clinical trials on human neural stem cells (hNSCs) have already been implemented in the treatment of neurological diseases and they have demonstrated their therapeutic effects, many questions remain in the field of preclinical research regarding the biology of these cells, their therapeutic properties, and their neurorestorative potential. Unfortunately, scientific reports are inconsistent and much of the NSCs research has been conducted on rodents rather than human cells for ethical reasons or due to insufficient cell material. Therefore, a question arises as to whether or which conclusions drawn on the isolation, cell survival, proliferation, or cell fate observed in vitro in rodent NSCs can be introduced into clinical applications. This paper presents the effects of different spatial, nutritional, and dissociation conditions on NSCs' functional properties, which are highly species-dependent. Our study confirmed that the discrepancies in the available literature on NSCs survival, proliferation, and fate did not only depend on intra-species factors and applied environmental conditions, but they were also affected by significant inter-species variability. Human and rodent NSCs share one feature, i.e., the necessity to be cultured immediately after isolation, which significantly maintains their survival. Additionally, in the absence of experiments on human cells, rat NSCs biology (neurosphere formation potential and neural differentiation stage) seems closer to that of humans rather than mice in response to environmental factors.

The Metabolic Changes between Monolayer (2D) and Three-Dimensional (3D) Culture Conditions in Human Mesenchymal Stem/Stromal Cells Derived from Adipose Tissue.

INTRODUCTION: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. RESULTS: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. CONCLUSION: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state.

Deciphering the impact of cerebrospinal fluid on stem cell fate as a new mechanism to enhance clinical therapy development.

Neural stem cells (NSCs) hold a very significant promise as candidates for cell therapy due to their robust neuroprotective and regenerative properties. Preclinical studies using NSCs have shown enough encouraging results to perform deeper investigations into more potential clinical applications. Nevertheless, our knowledge regarding neurogenesis and its underlying mechanisms remains incomplete. To understand them better, it seems necessary to characterize all components of neural stem cell niche and discover their role in physiology and pathology. Using NSCs in vivo brings challenges including limited cell survival and still inadequate integration within host tissue. Identifying overlooked factors that might influence these outcomes becomes pivotal. In this review, we take a deeper examination of the influence of a fundamental element that is present in the brain, the cerebrospinal fluid (CSF), which still remains relatively unexplored. Its role in neurogenesis could be instrumental to help find novel therapeutic solutions for neurological disorders, eventually advancing our knowledge on central nervous system (CNS) regeneration and repair.

Interaction of Neural Stem Cells (NSCs) and Mesenchymal Stem Cells (MSCs) as a Promising Approach in Brain Study and Nerve Regeneration.

Rapid developments in stem cell research in recent years have provided a solid foundation for their use in medicine. Over the last few years, hundreds of clinical trials have been initiated in a wide panel of indications. Disorders and injuries of the nervous system still remain a challenge for the regenerative medicine. Neural stem cells (NSCs) are the optimal cells for the central nervous system restoration as they can differentiate into mature cells and, most importantly, functional neurons and glial cells. However, their application is limited by multiple factors such as difficult access to source material, limited cells number, problematic, long and expensive cultivation in vitro, and ethical considerations. On the other hand, according to the available clinical databases, most of the registered clinical trials involving cell therapies were carried out with the use of mesenchymal stem/stromal/signalling cells (MSCs) obtained from afterbirth or adult human somatic tissues. MSCs are the multipotent cells which can also differentiate into neuron-like and glia-like cells under proper conditions in vitro; however, their main therapeutic effect is more associated with secretory and supportive properties. MSCs, as a natural component of cell niche, affect the environment through immunomodulation as well as through the secretion of the trophic factors. In this review, we discuss various therapeutic strategies and activated mechanisms related to bilateral MSC-NSC interactions, differentiation of MSCs towards the neural cells (subpopulation of crest-derived cells) under the environmental conditions, bioscaffolds, or co-culture with NSCs by recreating the conditions of the neural cell niche.

Critical factors responsible for the therapeutic effect of mesenchymal stem/stromal cells in central nervous system disorders.

Nowadays it is observed that the number of stem-cell based experimental therapies in neurodegenerative disorders is massively increasing. Most of the clinical trials registered to date have been based on autologous mesenchymal stem/stromal cells (MSC) obtained from somatic tissues. In the conducted clinical trials neither serious side effects, nor statistically significant improvement were observed. The lack of statistical significance could result from a relatively small number of patients involved in clinical trials or highly incoherent study protocols. However, most clinical groups describe a trend towards improvement in MSC-treated patients. Hence, the question arises which factors associated with MSC-based therapy may be the key and result in better therapeutic response. In the presented paper, we summarize, in our opinion, the most important factors that could increase the effectiveness of this therapy.

Efficiency assessment of irrigation as an alternative method for improving the regenerative potential of non-healing wounds.

The application of mesenchymal stem/stromal cells (MSC) in regenerative medicine offers hope for the effective treatment of incurable or difficult-to-heal diseases. However, it requires the development of unified protocols for both safe and efficient cell acquisition and clinical usage. The therapeutic effect of fat grafts (containing stem cells) in non-healing wounds has been discussed in previous studies, although the application requires local or general anaesthesia. The treatment of MSC derived from adipose tissue (ASC) could be a less invasive method, and efficient delivery could lead to more favourable outcomes, which should encourage clinicians to use such therapeutic approaches more frequently. Therefore, the aim of this study was to optimise the methods of ASC isolation, culture and administration while maintaining their high survival, proliferation and colonisation potential. The ASC were isolated by an enzymatic method and were characterised according to International Society for Cellular Therapy and International Federation for Adipose Therapeutics and Science guidelines. To assess the opportunity to obtain a sufficient number of cells for transplantation, long-term cell cultures in two oxygen concentrations (5% vs. 21%) were conducted. For these cultures, the population doubling time, the cumulative time for cell population doublings and the rate of cell senescence were estimated. In a developed and pre-defined protocol, ASC can be efficiently cultured at physiological oxygen concentrations (5%), which leads to faster proliferation and slower cell senescence. Subsequently, to select the optimal and minimally invasive methods of ASC transplantation, direct cell application with an irrigator or with skin dressings was analysed. Our results confirmed that both the presented methods of cell application allow for the safe delivery of isolated ASC into wounds without losing their vitality. Cells propagated in the described conditions and applied in non-invasive cell application (with an irrigation system and dressings) to treat chronic wounds can be a potential alternative or supplement to more invasive clinical approaches.

Evaluation of the Optimal Manufacturing Protocols and Therapeutic Properties of Mesenchymal Stem/Stromal Cells Derived from Wharton's Jelly.

Wharton's jelly (WJ) from the umbilical cord (UC) is a good source of mesenchymal stem/stromal cells (MSCs), which can be isolated and used in therapy. Current knowledge shows that even small changes in the cell environment may result in obtaining a subpopulation of cells with different therapeutic properties. For this reason, the conditions of UC transportation, cell isolation, and cultivation and the banking of cells destined for clinical use should be unified and optimized. In this project, we tried various protocols for cell vs. bioptat isolation, banking, and transport in order to determine the most optimal. The most efficient isolation method of WJ-MSCs was chopping the whole umbilical matrix with a scalpel after vessel and lining membrane removal. The optimal solution for short term cell transportation was a multi-electrolyte fluid without glucose. Considering the use of WJ-MSCs in cell therapies, it was important to investigate the soluble secretome of both WJ bioptats and WJ-MSCs. WJ-MSCs secreted higher levels of cytokines and chemokines than WJ bioptats. WJ-MSCs secreted HGF, CCL2, ICAM-1, BDNF, and VEGF. Since these cells might be used in treating neurodegenerative disorders, we investigated the impact of cerebrospinal fluid (CSF) on WJ-MSCs' features. In the presence of CSF, the cells expressed consecutive neural markers both at the protein and gene level: nestin, ß-III-tubulin, S-100-ß, GFAP, and doublecortin. Based on the obtained results, a protocol for manufacturing an advanced-therapy medicinal product was composed.

Biochemical aspirin resistance in acute stroke patients and its association with clinical factors: a prospective pilot study.

INTRODUCTION: Aspirin is still widely used in treatment and prevention of cardiovascular diseases. To predict which patients cannot benefit from aspirin due to aspirin resistance remains a great clinical challenge. MATERIAL AND METHODS: Fifty one acute stroke/transient ischemic attack (TIA) patients (ASG) with a history of regular aspirin intake for the previous 7 days or more were included to the study within 24 hours of symptoms onset. Twenty nine patients admitted to our department for other reasons were the controls (CG). Each patient underwent routine blood tests (white blood cells, platelets, total cholesterol, C-reactive protein) and additional blood test: glycated haemoglobin (HbA1c), insulin, and N-terminal prohormone of brain natriuretic peptide (NT-proBNP). Biochemical aspirin resistance was measured using the VerifyNow Aspirin platelet function analyzer. RESULTS: There were 9 aspirin resistance patients in ASG (17.5%) and 3 in CG (10.3%) (p = 0.38). There were no differences in either age or gender between those groups. Twelve aspirin-resistant patients differed from aspirin nonresistant patients in age, NT-proBNP and total cholesterol levels (univariate model, p = 0.004, 0.04, 0.02, respectively). In a multivariate model patients aged 76 years and more would likely to be aspirin resistant with odds ratio = 9 (95% confidence interval: 1-78). CONCLUSIONS: Patients aged 76 and more can be more likely aspirin resistant than younger patients. We believe that especially in the elderly with congestive heart failure there is a strong need for further investigations in this field, including searching for alternative antiplatelet therapies.

Neurogenic and Neuroprotective Potential of Stem/Stromal Cells Derived from Adipose Tissue.

Currently, the number of stem-cell based experimental therapies in neurological injuries and neurodegenerative disorders has been massively increasing. Despite the fact that we still have not obtained strong evidence of mesenchymal stem/stromal cells' neurogenic effectiveness in vivo, research may need to focus on more appropriate sources that result in more therapeutically promising cell populations. In this study, we used dedifferentiated fat cells (DFAT) that are proven to demonstrate more pluripotent abilities in comparison with standard adipose stromal cells (ASCs). We used the ceiling culture method to establish DFAT cells and to optimize culture conditions with the use of a physioxic environment (5% O2). We also performed neural differentiation tests and assessed the neurogenic and neuroprotective capability of both DFAT cells and ASCs. Our results show that DFAT cells may have a better ability to differentiate into oligodendrocytes, astrocytes, and neuron-like cells, both in culture supplemented with N21 and in co-culture with oxygen-glucose-deprived (OGD) hippocampal organotypic slice culture (OHC) in comparison with ASCs. Results also show that DFAT cells have a different secretory profile than ASCs after contact with injured tissue. In conclusion, DFAT cells constitute a distinct subpopulation and may be an alternative source in cell therapy for the treatment of nervous system disorders.

Assessment of the Neuroprotective and Stemness Properties of Human Wharton's Jelly-Derived Mesenchymal Stem Cells under Variable (5% vs. 21%) Aerobic Conditions.

To optimise the culture conditions for human Wharton's jelly-derived mesenchymal stem cells (hWJ-MSCs) intended for clinical use, we investigated ten different properties of these cells cultured under 21% (atmospheric) and 5% (physiological normoxia) oxygen concentrations. The obtained results indicate that 5% O2 has beneficial effects on the proliferation rate, clonogenicity, and slowdown of senescence of hWJ-MSCs; however, the oxygen level did not have an influence on the cell morphology, immunophenotype, or neuroprotective effect of the hWJ-MSCs. Nonetheless, the potential to differentiate into adipocytes, osteocytes, and chondrocytes was comparable under both oxygen conditions. However, spontaneous differentiation of hWJ-MSCs into neuronal lineages was observed and enhanced under atmospheric oxygen conditions. The cells relied more on mitochondrial respiration than glycolysis, regardless of the oxygen conditions. Based on these results, we can conclude that hWJ-MSCs could be effectively cultured and prepared under both oxygen conditions for cell-based therapy. However, the 5% oxygen level seemed to create a more balanced and appropriate environment for hWJ-MSCs.

Effect of Long-Term 3D Spheroid Culture on WJ-MSC.

The aim of our work was to develop a protocol enabling a derivation of mesenchymal stem/stromal cell (MSC) subpopulation with increased expression of pluripotent and neural genes. For this purpose we used a 3D spheroid culture system optimal for neural stem cells propagation. Although 2D culture conditions are typical and characteristic for MSC, under special treatment these cells can be cultured for a short time in 3D conditions. We examined the effects of prolonged 3D spheroid culture on MSC in hope to select cells with primitive features. Wharton Jelly derived MSC (WJ-MSC) were cultured in 3D neurosphere induction medium for about 20 days in vitro. Then, cells were transported to 2D conditions and confront to the initial population and population constantly cultured in 2D. 3D spheroids culture of WJ-MSC resulted in increased senescence, decreased stemness and proliferation. However long-termed 3D spheroid culture allowed for selection of cells exhibiting increased expression of early neural and SSEA4 markers what might indicate the survival of cell subpopulation with unique features.

The Effect of Proinflammatory Cytokines on the Proliferation, Migration and Secretory Activity of Mesenchymal Stem/Stromal Cells (WJ-MSCs) under 5% O2 and 21% O2 Culture Conditions.

Treatment with Mesenchymal Stem/Stromal Cells (MSCs) in clinical trials is becoming one of the most-popular and fast-developing branches of modern regenerative medicine, as it is still in an experimental phase. The cross-section of diseases to which these cells are applied is very wide, ranging from degenerative diseases, through autoimmune processes and to acute inflammatory diseases, e.g., viral infections. Indeed, now that first clinical trials applying MSCs against COVID-19 have started, important questions concern not only the therapeutic properties of MSCs, but also the changes that might occur in the cell features as a response to the "cytokine storm" present in the acute phase of an infection and capable of posing a risk to a patient. The aim of our study was thus to assess changes potentially occurring in the biology of MSCs in the active inflammatory environment, e.g., in regards to the cell cycle, cell migration and secretory capacity. The study using MSCs derived from Wharton's jelly (WJ-MSCs) was conducted under two aerobic conditions: 21% O2 vs. 5% O2, since oxygen concentration is one of the key factors in inflammation. Under both oxygen conditions cells were exposed to proinflammatory cytokines involved significantly in acute inflammation, i.e., IFNγ, TNFα and IL-1ß at different concentrations. Regardless of the aerobic conditions, WJ-MSCs in the inflammatory environment do not lose features typical for mesenchymal cells, and their proliferation dynamic remains unchanged. Sudden fluctuations in proliferation, the early indicator of potential genetic disturbance, were not observed, while the cells' migration activity increased. The presence of pro-inflammatory factors was also found to increase the secretion of such anti-inflammatory cytokines as IL-4 and IL-10. It is concluded that the inflammatory milieu in vitro does not cause phenotype changes or give rise to proliferation disruption of WJ-MSCs, and nor does it inhibit the secretory properties providing for their use against acute inflammation.

Secondary release of the peripheral nerve with autologous fat derivates benefits for functional and sensory recovery.

The reconstruction of nerve continuity after traumatic nerve injury is the gold standard in hand surgery. Immediate, tension-free, end-to-end nerve suture ensures the best prognosis. The recovery is mostly promising; however, in a few cases, insufficient outcomes in motor or sensory function are observed. Intra- and extra-fascicular scarring accompanies the nerve regeneration process and limits final outcomes. Secondary nerve release in those cases is recommended. Unfortunately, scarring recurrence cannot be eliminated after secondary revision and neurolysis. The supportive influences of mesenchymal stem cells in the process of nerve regeneration were observed in many preclinical studies. However, a limited number of studies in humans have analyzed the clinical usage of mesenchymal stem cells in peripheral nerve reconstruction and revisions. The objective of this study was to evaluate the effects of undifferentiated adipose-derived stromal/stem cell injection during a last-chance surgery (neurolysis, nerve release) on a previously reconstructed nerve. Three patients (one female, two males; mean age 59 ± 4.5 years at the time of injury), who experienced failure of reconstructions of median and ulnar nerves, were included in this study. During the revision surgery, nerve fascicles were released, and adipose-derived stromal/stem cells were administered through microinjections along the fascicles and around the adjacent tissues after external neurolysis. During 36 months of follow-up, patients noticed gradual signs of sensory and in consequence functional recovery. No adverse effects were observed. Simultaneous nerve release with adipose-derived stromal/stem cells support is a promising method in patients who need secondary nerve release after nerve reconstruction. This method can constitute an alternative procedure in patients experiencing recovery failure and allow improvement in cases of limited nerve regeneration. The study protocol was approved by the Institutional Review Board (IRB) at the Centre of Postgraduate Medical Education (No. 62/PB/2016) on September 14, 2016.

Biomimetic microenvironmental preconditioning enhance neuroprotective properties of human mesenchymal stem cells derived from Wharton's Jelly (WJ-MSCs).

Tuning stem cells microenvironment in vitro may influence their regenerative properties. In this study Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) were encapsulated in 3D hydrogels derived from human fibrin (FB) or platelet lysate (PL) and the oxygen level was adjusted to physiological normoxia (5% O2). The influence of the type of the scaffold and physiological normoxia conditions was tested on the WJ-MSCs' survivability, proliferation, migratory potential, the level of expression of selected trophic factors, cytokines, and neural markers. Encapsulated WJ-MSCs revealed high survivability, stable proliferation rate, and ability to migrate out of the hydrogel and the up-regulated expression of all tested factors, as well as the increased expression of neural differentiation markers. Physiological normoxia stimulated proliferation of encapsulated WJ-MSCs and significantly enhanced their neuronal, but not glial, differentiation. Ex vivo studies with indirect co-culture of organotypic hippocampal slices and cell-hydrogel bio-constructs revealed strong neuroprotective effect of WJ-MSCs against neuronal death in the CA1 region of the rat hippocampus. This effect was potentiated further by FB scaffolds under 5% O2 conditions. Our results indicating significant effect of oxygen and 3D cytoarchitecture suggest the urgent need for further optimization of the microenvironmental conditions to improve therapeutical competence of the WJ-MSCs population.

Bone Defect Repair Using a Bone Substitute Supported by Mesenchymal Stem Cells Derived from the Umbilical Cord.

OBJECTIVE: Bone defects or atrophy may arise as a consequence of injury, inflammation of various etiologies, and neoplastic or traumatic processes or as a result of surgical procedures. Sometimes the regeneration process of bone loss is impaired, significantly slowed down, or does not occur, e.g., in congenital defects. For the bone defect reconstruction, a piece of the removed bone from ala of ilium or bone transplantation from a decedent is used. Replacement of the autologous or allogenic source of the bone-by-bone substitute could reduce the number of surgeries and time in the pharmacological coma during the reconstruction of the bone defect. Application of mesenchymal stem cells in the reconstruction surgery may have positive influence on tissue regeneration by secretion of angiogenic factors, recruitment of other MSCs, or differentiation into osteoblasts. Materials and Methods. Mesenchymal stem cells derived from the umbilical cord (Wharton's jelly (WJ-MSC)) were cultured in GMP-grade DMEM low glucose supplemented with heparin, 10% platelet lysate, glucose, and antibiotics. In vitro WJ-MSCs were seeded on the bone substitute Bio-Oss Collagen® and cultured in the StemPro® Osteogenesis Differentiation Kit. During the culture on the 1st, 7th, 14th, and 21st day (day in vitro (DIV)), we analyzed viability (confocal microscopy) and adhesion capability (electron microscopy) of WJ-MSC on Bio-Oss scaffolds, gene expression (qPCR), and secretion of proteins (Luminex). In vivo Bio-Oss® scaffolds with WJ-MSC were transplanted to trepanation holes in the cranium to obtain their overgrowth. The computed tomography was performed 7, 14, and 21 days after surgery to assess the regeneration. RESULTS: The Bio-Oss® scaffold provides a favourable environment for WJ-MSC survival. WJ-MSCs in osteodifferentiation medium are able to attach and proliferate on Bio-Oss® scaffolds. Results obtained from qPCR and Luminex® indicate that WJ-MSCs possess the ability to differentiate into osteoblast-like cells and may induce osteoclastogenesis, angiogenesis, and mobilization of host MSCs. In animal studies, WJ-MSCs seeded on Bio-Oss® increased the scaffold integration with host bone and changed their morphology to osteoblast-like cells. CONCLUSIONS: The presented construct consisted of Bio-Oss®, the scaffold with high flexibility and plasticity, approved for clinical use with seeded immunologically privileged WJ-MSC which may be considered reconstructive therapy in bone defects.