High Hopes for the Biofabrication of Articular Cartilage-What Lies beyond the Horizon of Tissue Engineering and 3D Bioprinting?

Technologies and biomaterials for 3D bioprinting have been developing extremely quickly in the past decade as they hold great potential in tissue engineering. This, together with the possibility to differentiate stem cells of different origin into any cell type, raises the hopes in regenerative medicine once again after the initial breakthrough with stem cells in the 1980s. Nevertheless, three decades of 3D bioprinting experiments have shown that the production of functional tissues would take a longer time than anticipated. Cartilage, one of the simplest tissues in the body, consists of only one cell type. It is not vascularised and innervated and does not have lymphatic vessels either, which makes it a perfect target tissue for successful implantation. The tremendous amount of work since the beginning of this century, combining the efforts of bioengineers, material scientists, biologists, and physicians, has culminated in multiple proof-of-concept constructs that have been implanted in animals. However, there is no single reproducible, standardised, widely accessible and accepted strategy that can be readily applied in the clinic. In this review, we focus on the current progress in the field of the 3D biofabrication of articular cartilage and critically assess failures and future challenges.

World's First Long-Term Colorectal Cancer Model by 3D Bioprinting as a Mechanism for Screening Oncolytic Viruses.

Long-term modelization of cancer as it changes in the human body is a difficult goal, particularly when designing and testing new therapeutic strategies. This becomes even more difficult with metastasis modeling to show chemotherapeutic molecule delivery directly to tumoral cells. Advanced therapeutics, including oncolytic viruses, antibody-based and cell-based therapies are increasing. The question is, are screening tests also evolving? Next-generation therapeutics need equally advanced screening tests, which whilst difficult to achieve, are the goal of our work here, creating models of micro- and macrotumors using 3D bioprinting. We developed advanced colorectal cancer tumor processing techniques to provide options for cellular expansion, microtumor printing, and long-term models, which allow for the evaluation of the kinetics of penetration testing, therapeutic success, targeted therapies, and personalized medicine. We describe how we tested tumors from a primary colorectal patient and, applying 3D bioprinting, matured long-term models for oncolytic metastatic screening. Three-dimensional microtumors were kept alive for the longest time ever recorded in vitro, allowing longitudinal studies, screening of oncolytic viruses and realistic modelization of colorectal cancer. These 3D bioprinted models were maintained for around 6 months and were able to demonstrate the effective delivery of a product to the tumoral environment and represent a step forward in therapeutic screening.

The World's First Acne Dysbiosis-like Model of Human 3D Ex Vivo Sebaceous Gland Colonized with Cutibacterium acnes and Staphylococcus epidermidis.

Acne-prone skin is associated with dysbiosis involving Cutibacterium acnes (C. acnes) and Staphylococcus epidermidis (S. epidermidis) causing increased seborrhea in sebaceous glands (SG) and inflammation. Human primary sebocytes were cultivated using 1.106 UFC/mL C. acnes Type IA (facial acne, ATCC6919) and/or 1.105 UFC/mL S. epidermidis (unknown origin, ATCC12228) for 48 h in our SEB4GLN-optimized media without antibiotics. Bacteria and sebocytes were enumerated and assessed to determine their viability. Lipid production was imaged and quantified via Nile Red staining. SG with hair follicles were microdissected from healthy skin and cultured using 1.105 UFC/mL C. acnes Type 1A and/or 1.104 UFC/mL S. epidermidis (wild-type facial skin strain) through prior fixation and immunostaining for MC5R, C. acnes and nuclei (DAPI) via Z-stack confocal microscopy bioimaging (Leica SP5X & FIJI software, Version 2.9.0). C. acnes growth was not impacted when co-cultivated with sebocytes (2D) or SG (3D) models. Phylotype IA stimulated sebocyte lipid production, which had no impact on viability. The S. epidermidis reference strain overproliferated, inducing sebocyte mortality. For 3D SG model, culture conditions were optimized using a wild-type facial skin strain at a lower concentration, 1:10 ratio to C. acnes, reduced contact time, sequential inoculation and rinsing step. Bioimaging revealed strong C. acnes labeling in the active areas of the pilosebaceous unit. S. epidermidis formed biofilm, which was distributed across the SG via non-specific fluorescence imaging. We developed an innovative model of a sebaceous gland that mimics acne-prone skin with lipid overproduction and virulent phylotype IA C. acnes inoculation.

Topical Administration of Lactiplantibacillus plantarum (SkinDuoTM) Serum Improves Anti-Acne Properties.

The tailoring of the skin microbiome is challenging and is a research hotspot in the pathogenesis of immune-mediated inflammatory skin diseases such as acne. Commonly encountered preservatives used as functional ingredients have an impact on the skin microbiota and are known to inhibit the survival of skin commensal bacteria. The selected species is Lactiplantibacillus plantarum, formulated with natural enhancers for topical use (SkinDuoTM). Ex vivo human skin models were used as a test system to assess the strain viability which was then validated on healthy volunteers. SkinDuoTM showed increased viability over time for in vitro skin models and a stable viability of over 50% on healthy skin. The strain was tested on human primary sebocytes obtained from sebaceous gland rich areas of facial skin and inoculated with the most abundant bacteria from the skin microbiota. Results on human ex vivo sebaceous gland models with the virulent phylotype of Cutibacterium acnes and Staphylococcus epidermidis present a significant reduction in viability, lipid production, and anti-inflammatory markers. We have developed an innovative anti-acne serum with L. plantarum that mimics the over-production of lipids, anti-inflammatory properties, and improves acne-disease skin models. Based on these results, we suggest that SkinDuoTM may be introduced as an acne-mitigating agent.

Engineering of a Microscale Niche for Pancreatic Tumor Cells Using Bioactive Film Coatings Combined with 3D-Architectured Scaffolds.

Two-photon polymerization has recently emerged as a promising technique to fabricate scaffolds for three-dimensional (3D) cell culture and tissue engineering. Here, we combined 3D-printed microscale scaffolds fabricated using two-photon polymerization with a bioactive layer-by-layer film coating. This bioactive coating consists of hyaluronic acid and poly(l-lysine) of controlled stiffness, loaded with fibronectin and bone morphogenic proteins 2 and 4 (BMP2 and BMP4) as matrix-bound proteins. Planar films were prepared using a liquid handling robot directly in 96-well plates to perform high-content studies of cellular processes, especially cell adhesion, proliferation, and BMP-induced signaling. The behaviors of two human pancreatic cell lines PANC1 (immortalized) and PAN092 (patient-derived cell line) were systematically compared and revealed important context-specific cell responses, notably in response to film stiffness and matrix-bound BMPs (bBMPs). Fibronectin significantly increased cell adhesion, spreading, and proliferation for both cell types on soft and stiff films; BMP2 increased cell adhesion and inhibited proliferation of PANC1 cells and PAN092 on soft films. BMP4 enhanced cell adhesion and proliferation of PANC1 and showed a bipolar effect on PAN092. Importantly, PANC1 exhibited a strong dose-dependent BMP response, notably for bBMP2, while PAN092 was insensitive to BMPs. Finally, we proved that it is possible to combine a microscale 3D Ormocomp scaffold fabricated using the two-photon polymerization technique with the bioactive film coating to form a microscale tumor tissue and mimic the early stages of metastatic cancer.

A Colorectal Cancer 3D Bioprinting Workflow as a Platform for Disease Modeling and Chemotherapeutic Screening.

Colorectal cancer (CRC) is the third most common malignancy and has recently moved up to the second leading cause of death among carcinomas. Prognosis, especially for advanced diseases or certain molecular subtypes of CRC, remains poor, which highlights the urgent need for better therapeutic strategies. However, currently, as little as 0.1% of all drugs make it from bench to bedside because of the inherently high false-positive and false-negative rates of current preclinical and clinical drug testing data. Therefore, the success of developing novel treatment agents lies in the introduction of improved preclinical disease models which resemble in vivo carcinomas closer, possess higher predictive properties, and offer opportunities for individualized therapies. Aiming to address these needs, we have established an affordable, flexible, and highly reproducible 3D bioprinted CRC model. The histological assessment of Caco-2 cells in 3D bioprints revealed the formation of glandular-like structures which show greater pathomorphological resemblance to tumors than monolayer cultures do. RNA expression profiles in 3D bioprinted cells were marked by upregulation of genes involved in cell adhesion, hypoxia, EGFR/KRAS signaling, and downregulation of cell cycle programs. Testing this 3D experimental platform with three of the most commonly used chemotherapeutics in CRC (5-fluoruracil, oxaliplatin, and irinotecan) revealed overall increased resistance compared to 2D cell cultures. Last, we demonstrate that our workflow can be successfully extended to primary CRC samples. Thereby, we describe a novel accessible platform for disease modeling and drug testing, which may present an innovative opportunity for personalized therapeutic screening.

Exploring the Complementarity of Pancreatic Ductal Adenocarcinoma Preclinical Models.

Purpose: Compare pancreatic ductal adenocarcinoma (PDAC), preclinical models, by their transcriptome and drug response landscapes to evaluate their complementarity. Experimental Design: Three paired PDAC preclinical models-patient-derived xenografts (PDX), xenograft-derived pancreatic organoids (XDPO) and xenograft-derived primary cell cultures (XDPCC)-were derived from 20 patients and analyzed at the transcriptomic and chemosensitivity level. Transcriptomic characterization was performed using the basal-like/classical subtyping and the PDAC molecular gradient (PAMG). Chemosensitivity for gemcitabine, irinotecan, 5-fluorouracil and oxaliplatin was established and the associated biological pathways were determined using independent component analysis (ICA) on the transcriptome of each model. The selection criteria used to identify the different components was the chemosensitivity score (CSS) found for each drug in each model. Results: PDX was the most dispersed model whereas XDPO and XDPCC were mainly classical and basal-like, respectively. Chemosensitivity scoring determines that PDX and XDPO display a positive correlation for three out of four drugs tested, whereas PDX and XDPCC did not correlate. No match was observed for each tumor chemosensitivity in the different models. Finally, pathway analysis shows a significant association between PDX and XDPO for the chemosensitivity-associated pathways and PDX and XDPCC for the chemoresistance-associated pathways. Conclusions: Each PDAC preclinical model possesses a unique basal-like/classical transcriptomic phenotype that strongly influences their global chemosensitivity. Each preclinical model is imperfect but complementary, suggesting that a more representative approach of the clinical reality could be obtained by combining them. Translational Relevance: The identification of molecular signatures that underpin drug sensitivity to chemotherapy in PDAC remains clinically challenging. Importantly, the vast majority of studies using preclinical in vivo and in vitro models fail when transferred to patients in a clinical setting despite initially promising results. This study presents for the first time a comparison between three preclinical models directly derived from the same patients. We show that their applicability to preclinical studies should be considered with a complementary focus, avoiding tumor-based direct extrapolations, which might generate misleading conclusions and consequently the overlook of clinically relevant features.

Characterization of Different Sources of Human MSCs Expanded in Serum-Free Conditions with Quantification of Chondrogenic Induction in 3D.

Mesenchymal stem cells (MSCs) represent alternative candidates to chondrocytes for cartilage engineering. However, it remains difficult to identify the ideal source of MSCs for cartilage repair since conditions supporting chondrogenic induction are diverse among published works. In this study, we characterized and evaluated the chondrogenic potential of MSCs from bone marrow (BM), Wharton's jelly (WJ), dental pulp (DP), and adipose tissue (AT) isolated and cultivated under serum-free conditions. BM-, WJ-, DP-, and AT-MSCs did not differ in terms of viability, clonogenicity, and proliferation. By an extensive polychromatic flow cytometry analysis, we found notable differences in markers of the osteochondrogenic lineage between the 4 MSC sources. We then evaluated their chondrogenic potential in a micromass culture model, and only BM-MSCs showed chondrogenic conversion. This chondrogenic differentiation was specifically ascertained by the production of procollagen IIB, the only type II collagen isoform synthesized by well-differentiated chondrocytes. As a pilot study toward cartilage engineering, we encapsulated BM-MSCs in hydrogel and developed an original method to evaluate their chondrogenic conversion by flow cytometry analysis, after release of the cells from the hydrogel. This allowed the simultaneous quantification of procollagen IIB and α10, a subunit of a type II collagen receptor crucial for proper cartilage development. This work represents the first comparison of detailed immunophenotypic analysis and chondrogenic differentiation potential of human BM-, WJ-, DP-, and AT-MSCs performed under the same serum-free conditions, from their isolation to their induction. Our study, achieved in conditions compliant with clinical applications, highlights that BM-MSCs are good candidates for cartilage engineering.

Development of new 3D human ex vivo models to study sebaceous gland lipid metabolism and modulations.

OBJECTIVES: Sebaceous glands maintain skin homeostasis by producing sebum. Low production can induce hair loss and fragile skin. Overproduction provokes seborrhoea and may lead to acne and inflammatory events. To better study sebaceous gland maintenance, sebocyte maturation, lipid production and ageing or inflammatory processes, we developed innovative 3D ex vivo models for human sebaceous glands. MATERIALS AND METHODS: Culture conditions and analytical methods optimized on sebocyte monolayers were validated on extracted sebaceous glands and allowed the development of two 3D models: (a) "air-liquid" interface and (b) human fibronectin-coated "sandwich" method. Lipid production was assessed with microscopy, fluorometry or flow cytometry analysis after Nile Red staining. Specific lipids (particularly squalene and peroxidized squalene) were measured by Gas or liquid Chromatography and Mass spectrometry. RESULTS: This study allowed us to select appropriate conditions and design Seb4Gln culture medium inducing sebocyte proliferation and neutral lipid production. The "air-liquid" model was appropriate to induce sebocyte isolation. The "sandwich" model enabled sebaceous gland maintenance up to 42 days. A treatment with Insulin Growth Factor-1 allowed validation of the model as we succeeded in mimicking dynamic lipid overproduction. CONCLUSION: Functional sebocyte maturation and physiological maintenance were preserved up to 6 weeks in our models. Associated with functional assays, they provide a powerful platform to mimic physiological skin lipid metabolism and to screen for active ingredients modulating sebum production.

Immunophenotyping Reveals the Diversity of Human Dental Pulp Mesenchymal Stromal Cells In vivo and Their Evolution upon In vitro Amplification.

Mesenchymal stromal/stem cells (MSCs) from human dental pulp (DP) can be expanded in vitro for cell-based and regenerative dentistry therapeutic purposes. However, their heterogeneity may be a hurdle to the achievement of reproducible and predictable therapeutic outcomes. To get a better knowledge about this heterogeneity, we designed a flow cytometric strategy to analyze the phenotype of DP cells in vivo and upon in vitro expansion with stem cell markers. We focused on the CD31- cell population to exclude endothelial and leukocytic cells. Results showed that the in vivo CD31- DP cell population contained 1.4% of CD56+, 1.5% of CD146+, 2.4% of CD271+ and 6.3% of MSCA-1+ cells but very few Stro-1+ cells (≤ 1%). CD56+, CD146+, CD271+, and MSCA-1+ cell subpopulations expressed various levels of these markers. CD146+MSCA-1+, CD271+MSCA-1+, and CD146+CD271+ cells were the most abundant DP-MSC populations. Analysis of DP-MSCs expanded in vitro with a medicinal manufacturing approach showed that CD146 was expressed by about 50% of CD56+, CD271+, MSCA-1+, and Stro-1+ cells, and MSCA-1 by 15-30% of CD56+, CD146+, CD271+, and Stro-1+ cells. These ratios remained stable with passages. CD271 and Stro-1 were expressed by <1% of the expanded cell populations. Interestingly, the percentage of CD56+ cells strongly increased from P1 (25%) to P4 (80%) both in all sub-populations studied. CD146+CD56+, MSCA-1+CD56+, and CD146+MSCA-1+ cells were the most abundant DP-MSCs at the end of P4. These results established that DP-MSCs constitute a heterogeneous mixture of cells in pulp tissue in vivo and in culture, and that their phenotype is modified upon in vitro expansion. Further studies are needed to determine whether co-expression of specific MSC markers confers DP cells specific properties that could be used for the regeneration of human tissues, including the dental pulp, with standardized cell-based medicinal products.

Manufacturing of dental pulp cell-based products from human third molars: current strategies and future investigations.

In recent years, mesenchymal cell-based products have been developed to improve surgical therapies aimed at repairing human tissues. In this context, the tooth has recently emerged as a valuable source of stem/progenitor cells for regenerating orofacial tissues, with easy access to pulp tissue and high differentiation potential of dental pulp mesenchymal cells. International guidelines now recommend the use of standardized procedures for cell isolation, storage and expansion in culture to ensure optimal reproducibility, efficacy and safety when cells are used for clinical application. However, most dental pulp cell-based medicinal products manufacturing procedures may not be fully satisfactory since they could alter the cells biological properties and the quality of derived products. Cell isolation, enrichment and cryopreservation procedures combined to long-term expansion in culture media containing xeno- and allogeneic components are known to affect cell phenotype, viability, proliferation and differentiation capacities. This article focuses on current manufacturing strategies of dental pulp cell-based medicinal products and proposes a new protocol to improve efficiency, reproducibility and safety of these strategies.

Production of Human Dental Pulp Cells with a Medicinal Manufacturing Approach.

INTRODUCTION: Human dental pulp cells (HDPCs) are generally isolated and cultured with xenogeneic products and in stress conditions that may alter their biological features. However, guidelines from the American Food and Drug Administration and the European Medicines Agency currently recommend the use of protocols compliant with medicinal manufacturing. Our aim was to design an ex vivo procedure to produce large amounts of HDPCs for dentin/pulp and bone engineering according to these international recommendations. METHODS: HDPC isolation was performed from pulp explant cultures. After appropriate serum-free medium selection, cultured HDPCs were immunophenotyped with flow cytometry. Samples were then cryopreserved for 510 days. The post-thaw cell doubling time was determined up to passage 4 (P4). Karyotyping was performed by G-band analysis. Osteo/odontoblastic differentiation capability was determined after culture in a differentiation medium by gene expression analysis of osteo/odontoblast markers and mineralization quantification. RESULTS: Immunophenotyping of cultured HDPCs revealed a mesenchymal profile of the cells, some of which also expressed the stem/progenitor cell markers CD271, Stro-1, CD146, or MSCA-1. The post-thaw cell doubling times were stable and similar to fresh HDPCs. Cells displayed no karyotype abnormality. Alkaline phosphatase, osteocalcin, and dentin sialophosphoprotein gene expression and culture mineralization were increased in post-thaw HDPC cultures performed in differentiation medium compared with cultures in control medium. CONCLUSIONS: We successfully isolated, cryopreserved, and amplified human dental pulp cells with a medicinal manufacturing approach. These findings may constitute a basis on which to investigate how HDPC production can be optimized for human pulp/dentin and bone tissue engineering.

Osteoblastic differentiation of Wharton jelly biopsy specimens and their mesenchymal stromal cells after serum-free culture.

BACKGROUND: Cleft lip and cleft palate are increasingly being detected by prenatal ultrasound, which raises the opportunity of using the patient's own osteogenicity from umbilical cord mesenchymal cells for bony repair. The authors address the growth of the cells under a fully defined and regulated protocol. METHODS: Wharton jelly-derived mesenchymal stromal cells were isolated and expanded as a monolayer with defined serum-free medium. Osteoblastic differentiation was tested in the cells and in the entire Wharton jelly biopsy specimens. The serum-free-cultured cells were included in hydroxyapatite granule-fibrin constructs and, without predifferentiation, subcutaneously implanted into immunoincompetent mice. RESULTS: Isolation and expansion of Wharton jelly-derived mesenchymal stromal cells were consistently successful under serum-free conditions, and the cells expressed standard mesenchymal stromal cell markers. The serum-free-cultivated cells produced a mineralized extracellular matrix under osteogenic differentiation, with a significant increase of osteoblastic lineage gene expression (Hox-A10 and Runx2) and an up-regulation of downstream osteogenic genes (OSX, OCN, ALPL, and BSP2). In vivo, they formed a dense matrix adjacent to the granules after 8 weeks, but no lamellar bone. serum-free-cultivated entire Wharton jelly biopsy specimens produced a mineralized extracellular matrix within the collagen matrix of the Wharton jelly. CONCLUSIONS: The osteogenic differentiation potential of Wharton jelly-derived mesenchymal stromal cells was maintained under serum-free isolation and expansion techniques. The cells without predifferentiation form a dense collagen matrix but not bone in vivo. Moreover, entire Wharton jelly biopsy specimens showed periosteal-like mineralization under osteogenic differentiation, which offers new options for autologous bone tissue engineering, including cleft palate surgery.

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.

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.

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.

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.

The performance of laminin-containing cryogel scaffolds in neural tissue regeneration.

Currently, there are no effective therapies to restore lost brain neurons, although rapid progress in stem cell biology and biomaterials development provides new tools for regeneration of central nervous system. Here we describe neurogenic properties of bioactive scaffolds generated by cryogelation of dextran or gelatin linked to laminin - the main component of brain extracellular matrix. We showed that such scaffolds promoted differentiation of human cord blood-derived stem cells into artificial neural tissue in vitro. Our experiments revealed that optimal range of scaffolds' pore size for neural tissue engineering was 80-100 microns. We found that scaffold seeded with undifferentiated, but not neutrally committed stem cells, gave optimal cell adhesion and proliferation in "niche"-like structures. Subsequent differentiation resulted in generation of mature 3D networks of neurons (MAP2+) and glia (S100beta+) cells. We showed that cryogel scaffolds could be transplanted into the brain tissue or organotypic hippocampal slices in a rat models. The scaffolds did not induced inflammation mediated by microglial cells (ED1-, Ox43-, Iba1-) and prevented formation of glial scar (GFAP-). Contrary, laminin-rich scaffolds attracted infiltration of host's neuroblasts (NF200+, Nestin+) indicating high neuroregeneration properties.