Intratracheal administration of mesenchymal stem cell-derived extracellular vesicles reduces lung injuries in a chronic rat model of bronchopulmonary dysplasia.

Early therapeutic effect of intratracheally (IT)-administered extracellular vesicles secreted by mesenchymal stem cells (MSC-EVs) has been demonstrated in a rat model of bronchopulmonary dysplasia (BPD) involving hyperoxia exposure in the first 2 postnatal weeks. The aim of this study was to evaluate the protective effects of IT-administered MSC-EVs in the long term. EVs were produced from MSCs following GMP standards. At birth, rats were distributed in three groups: (a) animals raised in ambient air for 6 weeks (n = 10); and animals exposed to 60% hyperoxia for 2 weeks and to room air for additional 4 weeks and treated with (b) IT-administered saline solution (n = 10), or (c) MSC-EVs (n = 10) on postnatal days 3, 7, 10, and 21. Hyperoxia exposure produced significant decreases in total number of alveoli, total surface area of alveolar air spaces, and proliferation index, together with increases in mean alveolar volume, mean linear intercept and fibrosis percentage; all these morphometric changes were prevented by MSC-EVs treatment. The medial thickness index for <100 µm vessels was higher for hyperoxia-exposed/sham-treated than for normoxia-exposed rats; MSC-EV treatment significantly reduced this index. There were no significant differences in interstitial/alveolar and perivascular F4/8-positive and CD86-positive macrophages. Conversely, hyperoxia exposure reduced CD163-positive macrophages both in interstitial/alveolar and perivascular populations and MSC-EV prevented these hyperoxia-induced reductions. These findings further support that IT-administered EVs could be an effective approach to prevent/treat BPD, ameliorating the impaired alveolarization and pulmonary artery remodeling also in a long-term model. M2 macrophage polarization could play a role through anti-inflammatory and proliferative mechanisms.

Intratracheal administration of clinical-grade mesenchymal stem cell-derived extracellular vesicles reduces lung injury in a rat model of bronchopulmonary dysplasia.

Mesenchymal stem cells (MSCs) prevent the onset of bronchopulmonary dysplasia (BPD) in animal models, an effect that seems to be mediated by their secreted extracellular vesicles (EVs). The aim of this study was to compare the protective effects of intratracheally (IT) administered MSCs versus MSC-EVs in a hyperoxia-induced rat model of BPD. At birth, rats were distributed as follows: animals raised in ambient air for 2 wk ( n = 10), and animals exposed to 60% oxygen for 2 wk and treated with IT-administered physiological solution ( n = 10), MSCs ( n = 10), or MSC-EVs ( n = 10) on postnatal days 3, 7, and 10. The sham-treated hyperoxia-exposed animals showed reductions in total surface area of alveolar air spaces, and total number of alveoli ( Nalv), and an increased mean alveolar volume (Valv). EVs prompted a significant increase in Nalv ( P < 0.01) and a significant decrease in Valv ( P < 0.05) compared with sham-treated animals, whereas MSCs only significantly improved Nalv ( P < 0.05). Small pulmonary vessels of the sham-treated hyperoxia-exposed rats also showed an increase in medial thickness, which only EVs succeeded in preventing significantly ( P < 0.05). In conclusion, both EVs and MSCs reduce hyperoxia-induced damage, with EVs obtaining better results in terms of alveolarization and lung vascularization parameters. This suggests that IT-administered EVs could be an effective approach to BPD treatment.

Diverging Concepts and Novel Perspectives in Regenerative Medicine.

Regenerative medicine has rapidly evolved, due to progress in cell and molecular biology allowing the isolation, characterization, expansion, and engineering of cells as therapeutic tools. Despite past limited success in the clinical translation of several promising preclinical results, this novel field is now entering a phase of renewed confidence and productivity, marked by the commercialization of the first cell therapy products. Ongoing issues in the field include the use of pluripotent vs. somatic and of allogenic vs. autologous stem cells. Moreover, the recognition that several of the observed beneficial effects of cell therapy are not due to integration of the transplanted cells, but rather to paracrine signals released by the exogenous cells, is generating new therapeutic perspectives in the field. Somatic stem cells are outperforming embryonic and induced pluripotent stem cells in clinical applications, mainly because of their more favorable safety profile. Presently, both autologous and allogeneic somatic stem cells seem to be equally safe and effective under several different conditions. Recognition that a number of therapeutic effects of transplanted cells are mediated by paracrine signals, and that such signals can be found in extracellular vesicles isolated from culture media, opens novel therapeutic perspectives in the field of regenerative medicine.

Cryopreserved Subcutaneous Adipose Tissue for Fat Graft.

UNLABELLED: Cryopreservation of subcutaneous white adipose tissue (sWAT) avoids multiple surgeries in patients subjected to reconstructive procedure. Fat grafts were performed subcutaneously on 26 mice treated with fresh (13 mice) or cryopreserved (13 mice) human sWAT. Cytofluorometry for CD marker expression of stem cells, differentiation capability, and in vivo survival of fat grafts were evaluated. In vitro analysis evidenced that cryopreservation did not affect the stem potential of samples. In vivo MRI showed that grafts were well preserved in 13 mice treated with fresh sWAT, whereas in 13 animals treated with thawed fat, graft volumes were strongly reduced after 1 week. Ultrastructural studies performed both on fresh and thawed specimens demonstrated that grafts performed with thawed sWAT are able to store lipids more slowly with respect to grafts performed with fresh sWAT and adipocytes maintained a multilocular appearance. Collected data demonstrated that the protocol of cryopreservation could maintain the regenerative capability of the sWAT, but the rate of reabsorption after fat grafting is higher using cryopreserved sWAT. Maintaining the stem potential of sWAT after cryopreservation is a very important aspect for reconstructive and regenerative medicine. The employment of cryopreserved sWAT represents an interesting goal for surgeons. Surely there is the necessity to improve the protocol of cryopreservation. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors http://www.springer.com/00266 .

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.

Extracellular Vesicles From Mesenchymal Umbilical Cord Cells Exert Protection Against Oxidative Stress and Fibrosis in a Rat Model of Bronchopulmonary Dysplasia.

Oxidative stress and fibrosis are important stress responses that characterize bronchopulmonary dysplasia (BPD), a disease for which only a therapy but not a cure has been developed. In this work, we investigated the effects of mesenchymal stromal cells-derived extracellular vesicles (MSC-EVs) on lung and brain compartment in an animal model of hyperoxia-induced BPD. Rat pups were intratracheally injected with MSC-EVs produced by human umbilical cord-derived MSC, following the Good Manufacturing Practice-grade (GMP-grade). After evaluating biodistribution of labelled MSC-EVs in rat pups left in normoxia and hyperoxia, oxidative stress and fibrosis investigation were performed. Oxidative stress protection by MSC-EVs treatment was proved both in lung and in brain. The lung epithelial compartment ameliorated glycosaminoglycan and surfactant protein expression in MSC-EVs-injected rat pups compared to untreated animals. Pups under hyperoxia exhibited a fibrotic phenotype in lungs shown by increased collagen deposition and also expression of profibrotic genes. Both parameters were reduced by treatment with MSC-EVs. We established an in vitro model of fibrosis and another of oxidative stress, and we proved that MSC-EVs suppressed the induction of αSMA, influencing collagen deposition and protecting from the oxidative stress. In conclusion, intratracheal administration of clinical-grade MSC-EVs protect from oxidative stress, improves pulmonary epithelial function, and counteracts the development of fibrosis. In the future, MSC-EVs could represent a new cure to prevent the development of BPD.

Ischemic brain injury: a consortium analysis of key factors involved in mesenchymal stem cell-mediated inflammatory reduction.

Increasing global birth rate, coupled with the aging population surviving into their eighth decade has lead to increased incidence diseases, hitherto designated as rare. Brain related ischemia, at birth, or later in life, during, for example stroke, is increasing in global prevalence. Reactive microglia can contribute to neuronal damage as well as compromising transplantion. One potential treatment strategy is cellular therapy, using mesenchymal stem cells (hMSCs), which possess immunomodulatory and cell repair properties. For effective clinical therapy, mechanisms of action must be understood better. Here multicentre international laboratories assessed this question together investigating application of hMSCs neural involvement, with interest in the role of reactive microglia. Modulation by hMSCs in our in vivo and in vitro study shows they decrease markers of microglial activation (lower ED1 and Iba) and astrogliosis (lower GFAP) following transplantation in an ouabain-induced brain ischemia rat model and in organotypic hippocampal cultures. The anti-inflammatory effect in vitro was demonstrated to be CD200 ligand dependent with ligand expression shown to be increased by IL-4 stimulation. hMSC transplant reduced rat microglial STAT3 gene expression and reduced activation of Y705 phosphorylated STAT3, but STAT3 in the hMSCs themselves was elevated upon grafting. Surprisingly, activity was dependent on heterodimerisation with STAT1 activated by IL-4 and Oncostatin M. Our study paves the way to preclinical stages of a clinical trial with hMSC, and suggests a non-canonical JAK-STAT signaling of unphosphorylated STAT3 in immunomodulatory effects of hMSCs.

Expert Consideration on Regulatory Aspects for Perinatal Derivatives in Clinical Settings.

Perinatal derivatives (PnD) are drawing growing interest among the scientific community as an unrestricted source of multipotent stem cells, secretome, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options, but they require the development of regenerative approaches. With this development, the question of regulation of donation, processing, and distribution has therefore become more important. Within the European Cooperation in Science and Technology (COST) community, we compiled a group of international experts on PnD technologies, who revised and compared existing EU national regulations. Notably, despite clear European directives, each EU Country has developed their own implementation and standard levels for cell- and tissue-based therapies. To enable extended applications of PnD treatments within the EU community and worldwide, harmonization is highly recommended. This paper aims to provide an overview of the various options available to introduce PnD into clinical practice. For this purpose, the different aspects resulting from (1) the type of PnD, (2) the amount of available data, (3) the degree of manipulation, and (4) the intended application and the process toward a possible commercialization will be presented. In the future, it will be important to find a balance between regulatory requirements and the best medical quality of the PnD product.

Perinatal derivatives application: Identifying possibilities for clinical use.

Perinatal derivatives are drawing growing interest among the scientific community as an unrestricted source of multipotent stromal cells, stem cells, cellular soluble mediators, and biological matrices. They are useful for the treatment of diseases that currently have limited or no effective therapeutic options by means of developing regenerative approaches. In this paper, to generate a complete view of the state of the art, a comprehensive 10-years compilation of clinical-trial data with the common denominator of PnD usage has been discussed, including commercialized products. A set of criteria was delineated to challenge the 10-years compilation of clinical trials data. We focused our attention on several aspects including, but not limited to, treated disorders, minimal or substantial manipulation, route of administration, dosage, and frequency of application. Interestingly, a clear correlation of PnD products was observed within conditions, way of administration or dosage, suggesting there is a consolidated clinical practice approach for the use of PnD in medicine. No regulatory aspects could be read from the database since this information is not mandatory for registration. The database will be publicly available for consultation. In summary, the main aims of this position paper are to show possibilities for clinical application of PnD and propose an approach for clinical trial preparation and registration in a uniform and standardized way. For this purpose, a questionnaire was created compiling different sections that are relevant when starting a new clinical trial using PnD. More importantly, we want to bring the attention of the medical community to the perinatal products as a consolidated and efficient alternative for their use as a new standard of care in the clinical practice.

Muscle functional recovery is driven by extracellular vesicles combined with muscle extracellular matrix in a volumetric muscle loss murine model.

Biological scaffolds derived from decellularized tissues are being investigated as a promising approach to repair volumetric muscle losses (VML). Indeed, extracellular matrix (ECM) from decellularized tissues is highly biocompatible and mimics the original tissue. However, the development of fibrosis and the muscle stiffness still represents a major problem. Intercellular signals mediating tissue repair are conveyed via extracellular vesicles (EVs), biologically active nanoparticles secreted by the cells. This work aimed at using muscle ECM and human EVs derived from Wharton Jelly mesenchymal stromal cells (MSC EVs) to boost tissue regeneration in a VML murine model. Mice transplanted with muscle ECM and treated with PBS or MSC EVs were analyzed after 7 and 30 days. Flow cytometry, tissue analysis, qRT-PCR and physiology test were performed. We demonstrated that angiogenesis and myogenesis were enhanced while fibrosis was reduced after EV treatment. Moreover, the inflammation was directed toward tissue repair. M2-like, pro-regenerative macrophages were significantly increased in the MSC EVs treated group compared to control. Strikingly, the histological improvements were associated with enhanced functional recovery. These results suggest that human MSC EVs can be a naturally-derived boost able to ameliorate the efficacy of tissue-specific ECM in muscle regeneration up to the restored tissue function.

Artificial human tissues from cord and cord blood stem cells for multi-organ regenerative medicine: viable alternatives to animal in vitro toxicology.

New medicinal products and procedures must meet very strict safety criteria before being applied for use in humans. The laboratory procedures involved require the use of large numbers of animals each year. Furthermore, such investigations do not always give an accurate translation to the human setting. Here, we propose a viable alternative to animal testing, which uses novel technology featuring human cord and cord blood stem cells. With over 130 million children born each year, cord and cord blood remains the most widely available alternative to the use of animals or cadaveric human tissues for in vitro toxicology.

Defined serum-free culturing conditions for neural tissue engineering of human cord blood stem cells.

Taking tissue engineering applications into clinical trials requires the development of efficient and safe protocols incorporated with effective 3-dimensional cell culturing and differentiation systems in order to develop transplantable tissues that may offer a life-line for patients in the future. Cord blood, which is perhaps the most abundant world stem cell source, has shown previously practical and ethical advantages over other stem cells sources in many research and clinical applications including regenerative medicine. We previously developed a three-step protocol for isolation, expansion and sequential neuronal differentiation of cord blood pluripotent stem cells (characterized with our unique triple immunocytochemisty scheme for Oct-4, Sox-2 and Nanog) in defined serum-free culturing conditions. In this study we incorporated this protocol with 3-dimensional culturing systems which produced artificial neuronal tissues expressing Nestin, NF-200, TUJ1, PSD-95 and NeuN. We showed that cord blood pluripotent stem cells are a potential and promising candidate for future neural tissue engineering and regenerative medicine.

Transplantation of a novel human cord blood-derived neural-like stem cell line in a rat model of cortical infarct.

Umbilical cord blood can be a rich source of stem/progenitor cells, not only for hematopoetic but also for other tissue-specific lineages. Recently, we have developed a novel, self-renewed neural-like stem cell line named HUCB-NSC from human cord blood. To test if HUCB-NSCs can supply brain in need of regeneration, we injected these cells into immunosuppressed intact rat forebrain and to animals suffering from a photothrombotic cortical lesion at 48 h after injury. The survival, migration, and differentiation of the transplanted HUCB-NSCs were measured at 7 and 30 days post-transplantation by immunohistochemical methods. Results show survival and extensive migration of transplanted neural-like progenitors into damaged brain cortex during the first week of post-stroke recovery. The donor cells accumulated mainly in peri-infarct area and then differentiated showing a strong co-expression of neuronal (NF-200) but only moderate of astrocytic (GFAP) cell markers. However, the paucity of HUCB-NSCs detected within post-ischemic rat brain at the end of a 1 month period, as well as acute rejection of grafted cells by intact, yet cyclosporin A (CsA) immunosuppressed, rat brain tissue, suggests development of a severe adverse host reaction to the presence of alien donor cells and an urgent need for further study of the immunological response evoked by xenotransplantations of human cord blood-derived cells in animal experimental models.

Early appearance of stem/progenitor cells with neural-like characteristics in human cord blood mononuclear fraction cultured in vitro.

OBJECTIVE: The exposure of human umbilical cord blood mononuclear cells devoid of hematopoietic stem cells (HUCB-MNCsCD34-) to defined culture condition promotes their conversion into neural lineage. We have asked the question if observed fate change of HUCB-MNCsCD34- results from direct conversion of hematopoietic precursors into neural-like phenotypes due to expression of overlapping genetic program or, alternatively, these neural phenotypes arise from sequential differentiation of more primitive progenitors (embryonic-like cells) preexisting in HUCB-MNCsCD34- fraction. MATERIALS AND METHODS: HUCB-MNCs negatively selected for CD34 antigens were cultured in vitro up to 14 days. Changes in stem/neural cell genes and proteins were successively evaluated during this period and after evoked neuronal differentiation of cells in the presence of RA or BDNF or cocultured with neonatal rat brain astrocytes. RESULTS: Freshly isolated HUCB-MNCsCD34- expressed pluripotent cell markers: Oct3/4, Sox2, and Rex1 genes. During 24 hours of culture the frequency of Oct3/4 immunopositive cells increased markedly with parallel enlargement of "side population" and CD133+ cell appearance. Concomitantly, cultured cells start to form aggregates and express pro-neural genes, i.e., enhanced Sox2, OTX1, Nestin, GFAP, and NF-200. During the next days of culture immunoreactions for beta-tubulin III, MAP2, GFAP, S100beta, Doublecortin, and GalC were induced with reciprocal lowering of stem cell gene and protein markers. At this stage cells successively adhered to the bottom, dispersed, and decreased proliferation rate (Ki67 expression). Additional treatments with neuromorphogenes or coculturing with rat brain primary culture induced further differentiation of these neural precursors toward more advanced neuronal phenotypes. CONCLUSIONS: HUCB-MNCs(CD34-) fraction contains embryonic-like stem/progenitor cells which increase rapidly but transiently in culture, then differentiate spontaneously after cell aggregate adhesion toward neural lineage. Neurally promoted cells from 10-14 DIV culture acquire three main neural-like phenotypes, i.e., neurons, astrocytes, and oligodendrocytes. In this respect they are promising candidates for experimental treatment of neuronal injury; however, the final proof for conversion of HUCB cells to neural cells can be obtained through transplantation experiments.

Neural stem-like cell line derived from a nonhematopoietic population of human umbilical cord blood.

The ability of stem and progenitor cells to proliferate and differentiate into other lineages is widely viewed as a characteristic of stem cells. Previously, we have reported that cells from a CD34(-) (nonhematopoietic) adherent subpopulation of human cord blood can acquire a feature of multipotential neural progenitors in vitro. In the present study, using these cord blood-derived stem cells, we have established a clonal cell line termed HUCB-NSCs (human umbilical cord blood-neural stem cells) that expresses several neural antigens and has been grown in culture for more than 60 passages. During this time, HUCB-NSCs retained their growth rate, the ability to differentiate into neuronal-, astrocyte-, and oligodendrocyte-like cells and displayed a stable karyotype. DNA microarray analysis of HUCB-NSCs revealed enhanced expression of selected genes encoding putative stem and progenitor cell markers when compared to other mononuclear cells. dBcAMP-induced HUCBNSCs were further differentiated into more advanced neuronal cells. This is the first report of the establishment and characterization of a nontransformed HUCB-NSC line that can be grown continuously in a monolayer culture and induced to terminal differentiation. These cells should further our understanding of the regulatory mechanisms involved in NSC self-renewal and differentiation.

Quick and effective method of bone marrow mesenchymal stem cell extraction.

BACKGROUND: Mesenchymal stem cells (MSCs) are currently exploited in numerous clinical trials to investigate their potential in immune regulation, hematopoesis or tissue regeneration. The most common source of MSCs for clinical use is human bone marrow. To generate sufficient numbers of cells relevant to clinical use in most cases the high volumes (20-50 ml) of bone marrow aspirates are taken. METHODS: In this pilot study, 8 healthy bone marrow donors were included. Two different MSC extraction methods were evaluated: MSCs extraction from 60 ml of bone marrow using density gradient and MSCs extraction from 6 ml using red blood cell (RBC) lysis. RESULTS: Our results showed that after RBC lysis the efficient amount of human MSCs can be isolated from 10 times less bone marrow volume (6 ml). Moreover, using small volume of bone marrow the adequate therapeutical dose of MSCs could be achieved during similar period of time (3-4 weeks). In conclusion, we have shown that MSCs isolation using RBC lysis is an effective and more advantageous method in comparison to standard MSCs isolation using density-gradient. Using RBC lysis from small volume of bone marrow the same amount of MSCs were obtained as usually using large volume and density-gradient.

Encapsulation of mesenchymal stem cells by bioscaffolds protects cell survival and attenuates neuroinflammatory reaction in injured brain tissue after transplantation.

Since the brain is naturally inefficient in regenerating functional tissue after injury or disease, novel restorative strategies including stem cell transplantation and tissue engineering have to be considered. We have investigated the use of such strategies in order to achieve better functional repair outcomes. One of the fundamental challenges of successful transplantation is the delivery of cells to the injured site while maintaining cell viability. Classical cell delivery methods of intravenous or intraparenchymal injections are plagued by low engraftment and poor survival of transplanted stem cells. Novel implantable devices such as 3D bioactive scaffolds can provide the physical and metabolic support required for successful progenitor cell engraftment, proliferation, and maturation. In this study, we performed in situ analysis of laminin-linked dextran and gelatin macroporous scaffolds. We revealed the protective action of gelatin-laminin (GL) scaffolds seeded with mesenchymal stem cells derived from donated human Wharton's jelly (hUCMSCs) against neuroinflammatory reactions of injured mammalian brain tissue. These bioscaffolds have been implanted into (i) intact and (ii) ischemic rat hippocampal organotypic slices and into the striatum of (iii) normal and (iv) focally injured brains of adult Wistar rats. We found that transplantation of hUCMSCs encapsulated in GL scaffolds had a significant impact on the prevention of glial scar formation (low glial acidic fibrillary protein) and in the reduction of neuroinflammation (low interleukin-6 and the microglial markers ED1 and Iba1) in the recipient tissue. Moreover, implantation of hUCMSCs encapsulated within GL scaffolds induced matrix metalloproteinase-2 and -9 proteolytic activities in the surrounding brain tissue. This facilitated scaffold biodegradation while leaving the remaining grafted hUCMSCs untouched. In conclusion, transplanting GL scaffolds preseeded with hUCMSCs into mammalian brain tissue escaped the host's immune system and protected neural tissue from neuroinflammatory injury. This manuscript is published as part of the International Association of Neurorestoratology (IANR) supplement issue of Cell Transplantation.

Neurogenic properties and a clinical relevance of multipotent stem cells derived from cord blood samples stored in the biobanks.

Several innovative therapies with human umbilical cord blood stem cells (SCs) are currently developing to treat central nervous system (CNS) diseases. It has been shown that cord blood contains multipotent lineage-negative (LinNEG) SCs capable of neuronal differentiation. Clinically useful cord blood samples are stored in different biobanks worldwide, but the content and neurogenic properties of LinNEG cells are unknown. Here we have compared 5 major methods of blood processing: Sepax, Hetastarch, plasma depletion, Prepacyte-SC, and density gradient. We showed that Sepax-processed blood units contained 10-fold higher number of LinNEG cells after cryopreservation in comparison to all other methods. We showed in this study that multipotent SCs derived from fresh and frozen cord blood samples could be efficiently induced in defined serum-free medium toward neuronal progenitors (NF200+, Ki67+). During neuronal differentiation, the multipotent SCs underwent precise sequential changes at the molecular and cellular levels: Oct4 and Sox2 downregulation and Ngn1, NeuN, and PSD95 upregulation, similar to neurogenesis process in vivo. We expect that data presented here will be valuable for clinicians, researchers, biobanks, and patients and will contribute for better efficacy of future clinical trials in regeneration of CNS.

In vitro modelling of cortical neurogenesis by sequential induction of human umbilical cord blood stem cells.

Neurogenesis of excitatory neurons in the developing human cerebral neocortex is a complex and dynamic set of processes and the exact mechanisms controlling the specification of human neocortical neuron subtypes are poorly understood due to lack of relevant cell models available. It has been shown that the transcription factors Pax6, Tbr2 and Tbr1, which are sequentially expressed in the rodent neocortex, regulate and define corticogenesis of glutamatergic neocortical neurons. In humans the homologues of these genes are generally expressed in a similar pattern, but with some differences. In this study, we used purified human umbilical cord blood stem cells, expressing pluripotency marker genes (OCT4, SOX2 and NANOG), to model human neocortical neurogenesis in vitro. We analyzed the expression patterns of PAX6, TBR2 and TBR1, at both protein and mRNA levels, throughout the 24 days of a sequential neuronal induction protocol. Their expression patterns correlated with those found in the developing human neocortex where they define different developmental stages of neocortical neurons. The derived cord blood neuron-like cells expressed a number of neuronal markers. They also expressed components of glutamatergic neurotransmission including glutamate receptor subunits and transporters, and generated calcium influxes upon stimulation with glutamate. Thus we have demonstrated that it is possible to model neocortical neurogenesis using cord blood stem cells in vitro. This may allow detailed analysis of the molecular mechanisms regulating neocortical neuronal specification, thus aiding the development of potential therapeutic tools for diseases and injuries of the cerebral cortex.

miRNAs stem cell reprogramming for neuronal induction and differentiation.

Mimicking the natural brain environment during neurogenesis represents the main challenge for efficient in vitro neuronal differentiation of stem cells. The discovery of miRNAs opens new possibilities in terms of modulation of stem cells lineage commitment and differentiation. Many studies demonstrated that in vitro transient overexpression or inhibition of brain-specific miRNAs in stem cells significantly directed differentiation along neuronal cell lineages. Modulating miRNA expression offers new pathways for post-transcriptional gene regulation and stem cell commitment. Neurotrophins and neuropoietins signaling pathways are the main field of investigation for neuronal commitment, differentiation, and maturation. This review will highlight examples of crosstalk between stem-cell-specific and brain-specific signaling pathways and key miRNA candidates for neuronal commitment. Recent progress on understanding miRNAs genetic networks offers promising prospects for their increasing application in the development of new cellular therapies in humans.