Scaling up human mesenchymal stem cell manufacturing using bioreactors for clinical uses.

Human mesenchymal stem cells (hMSCs) are multipotent cells and an attractive therapeutic agent in regenerative medicine and intensive clinical research. Despite the great potential, the limitation that needs to be overcome is the necessity of ex vivo expansion because of insufficient number of hMSCs presented within adult organs and the high doses required for a transplantation. As a result, numerous research studies aim to provide novel expansion methods in order to achieve appropriate numbers of cells with preserved therapeutic quality. Bioreactor-based cell expansion provide high-level production of hMSCs in accordance with good manufacturing practice (GMP) and quality standards. This review summarizes current knowledge about the hMSCs manufacturing platforms with a main focus to the application of bioreactors for large-scale production of GMP-grade hMSCs.

A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells.

PURPOSE: Single cell (sc) analyses of key embryonic, fetal and adult stages were performed to generate a comprehensive single cell atlas of all the corneal and adjacent conjunctival cell types from development to adulthood. METHODS: Four human adult and seventeen embryonic and fetal corneas from 10 to 21 post conception week (PCW) specimens were dissociated to single cells and subjected to scRNA- and/or ATAC-Seq using the 10x Genomics platform. These were embedded using Uniform Manifold Approximation and Projection (UMAP) and clustered using Seurat graph-based clustering. Cluster identification was performed based on marker gene expression, bioinformatic data mining and immunofluorescence (IF) analysis. RNA interference, IF, colony forming efficiency and clonal assays were performed on cultured limbal epithelial cells (LECs). RESULTS: scRNA-Seq analysis of 21,343 cells from four adult human corneas and adjacent conjunctivas revealed the presence of 21 cell clusters, representing the progenitor and differentiated cells in all layers of cornea and conjunctiva as well as immune cells, melanocytes, fibroblasts, and blood/lymphatic vessels. A small cell cluster with high expression of limbal progenitor cell (LPC) markers was identified and shown via pseudotime analysis to give rise to five other cell types representing all the subtypes of differentiated limbal and corneal epithelial cells. A novel putative LPCs surface marker, GPHA2, expressed on the surface of 0.41% ± 0.21 of the cultured LECs, was identified, based on predominant expression in the limbal crypts of adult and developing cornea and RNAi validation in cultured LECs. Combining scRNA- and ATAC-Seq analyses, we identified multiple upstream regulators for LPCs and demonstrated a close interaction between the immune cells and limbal progenitor cells. RNA-Seq analysis indicated the loss of GPHA2 expression and acquisition of proliferative limbal basal epithelial cell markers during ex vivo LEC expansion, independently of the culture method used. Extending the single cell analyses to keratoconus, we were able to reveal activation of collagenase in the corneal stroma and a reduced pool of limbal suprabasal cells as two key changes underlying the disease phenotype. Single cell RNA-Seq of 89,897 cells obtained from embryonic and fetal cornea indicated that during development, the conjunctival epithelium is the first to be specified from the ocular surface epithelium, followed by the corneal epithelium and the establishment of LPCs, which predate the formation of limbal niche by a few weeks. CONCLUSIONS: Our scRNA-and ATAC-Seq data of developing and adult cornea in steady state and disease conditions provide a unique resource for defining genes/pathways that can lead to improvement in ex vivo LPCs expansion, stem cell differentiation methods and better understanding and treatment of ocular surface disorders.

Winter is coming: the future of cryopreservation.

The preservative effects of low temperature on biological materials have been long recognised, and cryopreservation is now widely used in biomedicine, including in organ transplantation, regenerative medicine and drug discovery. The lack of organs for transplantation constitutes a major medical challenge, stemming largely from the inability to preserve donated organs until a suitable recipient is found. Here, we review the latest cryopreservation methods and applications. We describe the main challenges-scaling up to large volumes and complex tissues, preventing ice formation and mitigating cryoprotectant toxicity-discuss advantages and disadvantages of current methods and outline prospects for the future of the field.

Platform to study intracellular polystyrene nanoplastic pollution and clinical outcomes.

Increased pollution by plastics has become a serious global environmental problem, but the concerns for human health have been raised after reported presence of microplastics (MPs) and nanoplastics (NPs) in food and beverages. Unfortunately, few studies have investigate the potentially harmful effects of MPs/NPs on early human development and human health. Therefore, we used a new platform to study possible effects of polystyrene NPs (PSNPs) on the transcription profile of preimplantation human embryos and human induced pluripotent stem cells (hiPSCs). Two pluripotency genes, LEFTY1 and LEFTY2, which encode secreted ligands of the transforming growth factor-beta, were downregulated, while CA4 and OCLM, which are related to eye development, were upregulated in both samples. The gene set enrichment analysis showed that the development of atrioventricular heart valves and the dysfunction of cellular components, including extracellular matrix, were significantly affected after exposure of hiPSCs to PSNPs. Finally, using the HiPathia method, which uncovers disease mechanisms and predicts clinical outcomes, we determined the APOC3 circuit, which is responsible for increased risk for ischemic cardiovascular disease. These results clearly demonstrate that better understanding of NPs bioactivities and its implications for human health is of extreme importance. Thus, the presented platform opens further aspects to study interactions between different environmental and intracellular pollutions with the aim to decipher the mechanism and origin of human diseases.

The Role of Nerve Growth Factor in Maintaining Proliferative Capacity, Colony-Forming Efficiency, and the Limbal Stem Cell Phenotype.

Nerve growth factor (NGF) has demonstrated great benefit in the treatment of neurotrophic corneal ulcers. There is evidence for multiple modes of action in promoting corneal healing, but only indirect evidence exists for NGF's effects on limbal stem cells (LSCs). Understanding the role of NGF in LSC biology will improve our understanding of paracrine regulation of the limbal niche and the design of stem cell-based therapies for conditions such as LSC deficiency. In this article, we studied the regulation of NGF signaling components during LSC differentiation and the role of NGF in LSC proliferation and maintenance of the stem cell phenotype. LSC differentiation was induced by prolonged (40 day) culture which resulted in a significant increase in cell size, decrease in colony-forming efficiency and expression of putative LSC markers. A protein microarray measuring expression of 248 signaling proteins indicated the low affinity NGF receptor p75NTR to be the most downregulated protein upon differentiation. Further confirmation by Western blotting and real-time quantitative polymerase chain reaction indicated that NGF and p75NTR are expressed in early LSC cultures and downregulated upon differentiation. LSC cultures grown in the presence of anti-NGF antibody showed decreased colony-forming efficiency, DNA replication and expression of putative LSC markers ABCG2 and C/EBPδ. Supplementation of LSC culture medium with NGF extended the life span of LSC cultures in vitro and increased the expression of putative LSC markers ΔNp63α and ABCG2. Taken together, our data indicate that NGF signaling is a key promoter of LSC proliferation, colony-forming efficiency, and a maintainer of the LSC phenotype. Stem Cells 2019;37:139-149.

CD200 Expression Marks a Population of Quiescent Limbal Epithelial Stem Cells with Holoclone Forming Ability.

One of the main challenges in limbal stem cell (LSC) biology and transplantation is the lack of definitive cell surface markers which can be used to identify and enrich viable LSCs. In this study, expression of 361 cell surface proteins was assessed in ex vivo expanded limbal epithelial cells. One marker, CD200 was selected for further characterization based on expression in a small subset of limbal epithelial cells (2.25% ± 0.69%) and reduced expression through consecutive passaging and calcium induced differentiation. CD200 was localized to a small population of cells at the basal layer of the human and mouse limbal epithelium. CD200+ cells were slow cycling and contained the majority of side population (SP) and all the holoclone forming progenitors. CD200+ cells displayed higher expression of LSCs markers including PAX6, WNT7A, CDH3, CK14, CK15, and ABCB5 and lower expression of Ki67 when compared to CD200- . Downregulation of CD200 abrogated the ability of limbal epithelial cells to form holoclones, suggesting an important function for CD200 in the maintenance and/or self-renewal of LSCs. A second marker, CD109, which was expressed in 56.29% ± 13.96% of limbal epithelial cells, was also found to co-localize with ΔNp63 in both human and mouse cornea, albeit more abundantly than CD200. CD109 expression decreased slowly through calcium induced cell differentiation and CD109+ cells were characterized by higher expression of Ki67, when compared to CD109- subpopulation. Together our data suggest that CD200 expression marks a quiescent population of LSCs with holoclone forming potential, while CD109 expression is associated with a proliferative progenitor phenotype. Stem Cells 2018;36:1723-1735.

Human-Induced Pluripotent Stem Cells Generate Light Responsive Retinal Organoids with Variable and Nutrient-Dependent Efficiency.

The availability of in vitro models of the human retina in which to perform pharmacological and toxicological studies is an urgent and unmet need. An essential step for developing in vitro models of human retina is the ability to generate laminated, physiologically functional, and light-responsive retinal organoids from renewable and patient specific sources. We investigated five different human-induced pluripotent stem cell (iPSC) lines and showed a significant variability in their efficiency to generate retinal organoids. Despite this variability, by month 5 of differentiation, all iPSC-derived retinal organoids were able to generate light responses, albeit immature, comparable to the earliest light responses recorded from the neonatal mouse retina, close to the period of eye opening. All iPSC-derived retinal organoids exhibited at this time a well-formed outer nuclear like layer containing photoreceptors with inner segments, connecting cilium, and outer like segments. The differentiation process was highly dependent on seeding cell density and nutrient availability determined by factorial experimental design. We adopted the differentiation protocol to a multiwell plate format, which enhanced generation of retinal organoids with retinal-pigmented epithelium (RPE) and improved ganglion cell development and the response to physiological stimuli. We tested the response of iPSC-derived retinal organoids to Moxifloxacin and showed that similarly to in vivo adult mouse retina, the primary affected cell types were photoreceptors. Together our data indicate that light responsive retinal organoids derived from carefully selected and differentiation efficient iPSC lines can be generated at the scale needed for pharmacology and drug screening purposes. Stem Cells 2018;36:1535-1551.

Differences in the Activity of Endogenous Bone Morphogenetic Protein Signaling Impact on the Ability of Induced Pluripotent Stem Cells to Differentiate to Corneal Epithelial-Like Cells.

Cornea is a clear outermost layer of the eye which enables transmission of light onto the retina. The transparent corneal epithelium is regenerated by limbal stem cells (LSCs), whose loss/dysfunction results in LSCs deficiency (LSCD). Ex vivo expansion of autologous LSCs obtained from patient's healthy eye followed by transplantation onto the LSCs damaged/deficient eye, has provided a successful treatment for unilateral LSCD. However, this is not applicable to patient with total bilateral LSCD, where LSCs are lost/damaged from both eyes. We investigated the potential of human induced pluripotent stem cell (hiPSC) to differentiate into corneal epithelial-like cells as a source of autologous stem cell treatment for patients with total bilateral LSCD. Our study showed that combined addition of bone morphogenetic protein 4 (BMP4), all trans-retinoic acid and epidermal growth factor for the first 9 days of differentiation followed by cell-replating on collagen-IV-coated surfaces with a corneal-specific-epithelial cell media for an additional 11 days, resulted in step wise differentiation of human embryonic stem cells (hESC) to corneal epithelial progenitors and mature corneal epithelial-like cells. We observed differences in the ability of hiPSC lines to undergo differentiation to corneal epithelial-like cells which were dependent on the level of endogenous BMP signaling and could be restored via the activation of this signaling pathway by a specific transforming growth factor ß inhibitor (SB431542). Together our data reveal a differential ability of hiPSC lines to generate corneal epithelial cells which is underlined by the activity of endogenous BMP signaling pathway. Stem Cells 2018;36:337-348.

Mindfulness-Based Treatment for Bipolar Disorder: A Systematic Review of the Literature.

Despite the increasing number of studies examining the effects of mindfulness interventions on symptoms associated with Bipolar Disorder (BD), the effectiveness of this type of interventions remains unclear. The aim of the present systematic review was to (i) critically review all available evidence on Mindfulness Based Cognitive Therapy (MBCT) as a form of intervention for BD; (ii) discuss clinical implications of MBCT in treating patients with BD; and (iii) provide a direction for future research. The review presents findings from 13 studies (N = 429) that fulfilled the following selection criteria: (i) included BD patients; (ii) presented results separately for BD patients and control groups (where a control group was available); (iii) implemented MBCT intervention; (iv) were published in English; (v) were published in a peer reviewed journal; and (vi) reported results for adult participants. Although derived from a relatively small number of studies, results from the present review suggest that MBCT is a promising treatment in BD in conjunction with pharmacotherapy. MBCT in BD is associated with improvements in cognitive functioning and emotional regulation, reduction in symptoms of anxiety depression and mania symptoms (when participants had residual manic symptoms prior to MBCT). These, treatment gains were maintained at 12 month follow up when mindfulness was practiced for at least 3 days per week or booster sessions were included. Additionally, the present review outlined some limitations of the current literature on MBCT interventions in BD, including small study sample sizes, lack of active control groups and idiosyncratic modifications to the MBCT intervention across studies. Suggestions for future research included focusing on factors underlying treatment adherence and understanding possible adverse effects of MBCT, which could be of crucial clinical importance.

An Induced Pluripotent Stem Cell Patient Specific Model of Complement Factor H (Y402H) Polymorphism Displays Characteristic Features of Age-Related Macular Degeneration and Indicates a Beneficial Role for UV Light Exposure.

Age-related macular degeneration (AMD) is the most common cause of blindness, accounting for 8.7% of all blindness globally. Vision loss is caused ultimately by apoptosis of the retinal pigment epithelium (RPE) and overlying photoreceptors. Treatments are evolving for the wet form of the disease; however, these do not exist for the dry form. Complement factor H polymorphism in exon 9 (Y402H) has shown a strong association with susceptibility to AMD resulting in complement activation, recruitment of phagocytes, RPE damage, and visual decline. We have derived and characterized induced pluripotent stem cell (iPSC) lines from two subjects without AMD and low-risk genotype and two patients with advanced AMD and high-risk genotype and generated RPE cells that show local secretion of several proteins involved in the complement pathway including factor H, factor I, and factor H-like protein 1. The iPSC RPE cells derived from high-risk patients mimic several key features of AMD including increased inflammation and cellular stress, accumulation of lipid droplets, impaired autophagy, and deposition of "drüsen"-like deposits. The low- and high-risk RPE cells respond differently to intermittent exposure to UV light, which leads to an improvement in cellular and functional phenotype only in the high-risk AMD-RPE cells. Taken together, our data indicate that the patient specific iPSC model provides a robust platform for understanding the role of complement activation in AMD, evaluating new therapies based on complement modulation and drug testing. Stem Cells 2017;35:2305-2320.

An important role for adenine, cholera toxin, hydrocortisone and triiodothyronine in the proliferation, self-renewal and differentiation of limbal stem cells in vitro.

The cornea is a self-renewing tissue located at the front of the eye. Its transparency is essential for allowing light to focus onto the retina for visual perception. The continuous renewal of corneal epithelium is supported by limbal stem cells (LSCs) which are located in the border region between conjunctiva and cornea known as the limbus. Ex vivo expansion of LSCs has been successfully applied in the last two decades to treat patients with limbal stem cell deficiency (LSCD). Various methods have been used for their expansion, yet the most widely used culture media contains a number of ingredients derived from animal sources which may compromise the safety profile of human LSC transplantation. In this study we sought to understand the role of these components namely adenine, cholera toxin, hydrocortisone and triiodothyronine with the aim of re-defining a safe and GMP compatible minimal media for the ex vivo expansion of LSCs on human amniotic membrane. Our data suggest that all four components play a critical role in maintaining LSC proliferation and promoting LSC self-renewal. However removal of adenine and triiodothyronine had a more profound impact and led to LSC differentiation and loss of viability respectively, suggesting their essential role for ex vivo expansion of LSCs. Replacement of each of the components with GMP-grade reagents resulted in equal growth to non-GMP grade media, however an enhanced differentiation of LSCs was observed, suggesting that additional combinations of GMP grade reagents need to be tested to achieve similar or better level of LSC maintenance in the same manner as the traditional LSC media.

Galectin-3 Plays an Important Pro-inflammatory Role in the Induction Phase of Acute Colitis by Promoting Activation of NLRP3 Inflammasome and Production of IL-1ß in Macrophages.

BACKGROUND AND AIMS: Galectin-3 [Gal-3] is an endogenous lectin with a broad spectrum of immunoregulatory effects: it plays an important role in autoimmune/inflammatory and malignant diseases, but the precise role of Gal-3 in pathogenesis of ulcerative colitis is still unknown. METHODS: We used a model of dextran sulphate sodium [DSS]-induced acute colitis. The role of Gal-3 in pathogenesis of this disease was tested by evaluating disease development in Gal-3 deficient mice and administration of Gal-3 inhibitor. Disease was monitored by clinical, histological, histochemical, and immunophenotypic investigations. Adoptive transfer was used to detect cellular events in pathogenesis. RESULTS: Genetic deletion or pharmacological inhibition of Gal-3 significantly attenuate DSS-induced colitis. Gal-3 deletion suppresses production of pro-inflammatory cytokines in colonic macrophages and favours their alternative activation, as well as significantly reducing activation of NOD-like receptor family, pyrin domain containing 3 [NLRP3] inflammasome in macrophages. Peritoneal macrophages isolated from untreated Gal-3(-/-) mice and treated in vitro with bacterial lipopolysaccharide or DSS produce lower amounts of tumour necrosis factor alpha [TNF-α] and interleukin beta [IL-1ß] when compared with wild type [WT] cells. Genetic deletion of Gal-3 did not directly affect total neutrophils, inflammatory dendritic cells [DCs] or natural killer [NK] T cells. However, the total number of CD11c+ CD80+ DCs which produce pro-inflammatory cytokines, as well as TNF-α and IL-1ß producing CD45+ CD11c- Ly6G+ neutrophils were significantly lower in colons of Gal-3(-/-) DSS-treated mice. Adoptive transfer of WT macrophages significantly enhanced the severity of disease in Gal-3(-/-) mice. CONCLUSIONS: Gal-3 expression promotes acute DSS-induced colitis and plays an important pro-inflammatory role in the induction phase of colitis by promoting the activation of NLRP3 inflammasome and production of IL-1ß in macrophages.

Gal-3 regulates the capacity of dendritic cells to promote NKT-cell-induced liver injury.

Galectin-3 (Gal-3), an endogenous lectin, exhibits pro- and anti-inflammatory effects in various disease conditions. In order to explore the role of Gal-3 in NKT-cell-dependent pathology, we induced hepatitis in C57BL/6 WT and Gal-3-deficient mice by using specific ligand for NKT cells: α-galactosylceramide, glycolipid Ag presented by CD1d. The injection of α-galactosylceramide significantly enhanced expression of Gal-3 in liver NKT and dendritic cells (DCs). Genetic deletion or selective inhibition of Gal-3 (induced by Gal-3-inhibitor TD139) abrogated the susceptibility to NKT-cell-dependent hepatitis. Blood levels of pro-inflammatory cytokines (TNF-α, IFN-γ, IL-12) and their production by liver DCs and NKT cells were also downregulated. Genetic deletion or selective inhibition of Gal-3 alleviated influx of inflammatory CD11c(+) CD11b(+) DCs in the liver and favored tolerogenic phenotype and IL-10 production of liver NKT and DCs. Deletion of Gal-3 attenuated the capacity of DCs to support liver damage in the passive transfer experiments and to produce pro-inflammatory cytokines in vitro. Gal-3-deficient DCs failed to optimally stimulate production of pro-inflammatory cytokines in NKT cells, in vitro and in vivo. In conclusion, Gal-3 regulates the capacity of DCs to support NKT-cell-mediated liver injury, playing an important pro-inflammatory role in acute liver injury.

Large graphene quantum dots alleviate immune-mediated liver damage.

We investigated the effect of large (40 nm) graphene quantum dots (GQDs) in concanavalin A (Con A; 12 mg/kg i.v.)-induced mouse hepatitis, a T cell-mediated liver injury resembling fulminant hepatitis in humans. Intravenously injected GQDs (50 mg/kg) accumulated in liver and reduced Con A-mediated liver damage, as demonstrated by histopathological analysis and a decrease in liver lipid peroxidation and serum levels of liver transaminases. The cleavage of apoptotic markers caspase-3/PARP and mRNA levels of proapoptotic mediators Puma, Noxa, Bax, Bak1, Bim, Apaf1, and p21, as well as LC3-I conversion to autophagosome-associated LC3-II and expression of autophagy-related (Atg) genes Atg4b, Atg7, Atg12, and beclin-1, were attenuated by GQDs, indicating a decrease in both apoptosis and autophagy in the liver tissue. This was associated with the reduced liver infiltration of immune cells, particularly the T cells producing proinflammatory cytokine IFN-γ, and a decrease in IFN-γ serum levels. In the spleen of GQD-exposed mice, mRNA expression of IFN-γ and its transcription factor T-bet was reduced, while that of the IL-33 ligand ST2 was increased. The hepatoprotective effect of GQDs was less pronounced in ST2-deficient mice, indicating that it might depend on ST2 upregulation. In vitro, GQDs inhibited splenocyte IFN-γ production, reduced the activation of extracellular signal-regulated kinase in macrophage and T cell lines, inhibited macrophage production of the free radical nitric oxide, and reduced its cytotoxicity toward hepatocyte cell line HepG2. Therefore, GQDs alleviate immune-mediated fulminant hepatitis by interfering with T cell and macrophage activation and possibly by exerting a direct hepatoprotective effect.

Stem cells as new agents for the treatment of infertility: current and future perspectives and challenges.

Stem cells are undifferentiated cells that are present in the embryonic, fetal, and adult stages of life and give rise to differentiated cells that make up the building blocks of tissue and organs. Due to their unlimited source and high differentiation potential, stem cells are considered as potentially new therapeutic agents for the treatment of infertility. Stem cells could be stimulated in vitro to develop various numbers of specialized cells including male and female gametes suggesting their potential use in reproductive medicine. During past few years a considerable progress in the derivation of male germ cells from pluripotent stem cells has been made. In addition, stem cell-based strategies for ovarian regeneration and oocyte production have been proposed as future clinical therapies for treating infertility in women. In this review, we summarized current knowledge and present future perspectives and challenges regarding the use of stem cells in reproductive medicine.

Dental stem cells--characteristics and potential.

Soft dental tissues have been identified as easily accessible sources of multipotent postnatal stem cells. Dental stem cells are mesenchymal stem cells (MSC) capable of differentiating into at least three distinct cell lineages: osteo/odontogenic, adipogenic and neurogenic. They express various markers including those specific for MSC, embryonic stem cells and neural cells. Five different types of dental stem cells have been isolated from mature and immature teeth: dental pulp stem cells, stem cells from exfoliated deciduous teeth, periodontal ligament stem cells, stem cells from apical papilla and dental follicle progenitor cells. Dental stem cells may be used in dental tissue engineering including dental, enamel and periodontal tissue regeneration. They could also be used as a promising tool in potential treatment of neurodegenerative, ischemic and immune diseases.