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Introduction Regenerative solutions of the skin represent a hope for burn victims with extensive skin loss and chronic wound patients. The development of xeno-free workflow is crucial for clinical application in compliance with the directives of the European Medicines Agency. This study aimed at evaluating the outcome of the xeno-free isolation workflow of keratinocytes from human skin biopsy. Methods Skin biopsies were obtained from volunteers. The epidermis was digested with TrypLE™ Select, which was deactivated by dilution or with trypsin, deactivated by media with fetal bovine serum. Freshly isolated cells were compared for total cell number, viability, activity of caspase 3, gene expression and the presence of the keratinocyte surface markers cytokeratin 14. The cells were cultured in xeno-free conditions for one week and characterized regarding the number and viability as well as the metalloproteinase secretion. Results The number of obtained cells was similar in both workflows. The cell viability was less in the TrypLE group, with slight reduction of the cell surface marker cytokeratin 14. Caspase 3 activity was comparable as well as the gene expression of the apoptotic markers BAX, BCL2 and SLUG, as well as the keratinocyte markers cytokeratin 14, stratifin and filaggrin. Upon culture, the number of keratinocytes, their viability and secretion of matrix metalloproteinases 1 and 10 were equal in both groups. Conclusion This study reports the possibility of isolating functioning and viable keratinocytes through a xeno-free workflow for clinical application., Highlights • The xenofree workflows are essential for clinical application of regenerative medicine. • TrypLE based digestion of skin biopsies can be deactivated by dilution in a xenofree workflow to isolate keratinocytes. • Although the xenofree workflow was efficient, keratinocytes viability was better with the classical trypsin-based workflow. • The cell characteristics were equivalent upon culturing the cells.

Bioengineered human skeletal muscle tissues have emerged in the last years as new in vitro systems for disease modeling. These bioartificial muscles are classically fabricated by encapsulating human myogenic precursor cells in a hydrogel scaffold that resembles the extracellular matrix. However, most of these hydrogels are derived from xenogenic sources, and the culture media is supplemented with animal serum, which could interfere in drug testing assays. On the contrary, xeno-free biomaterials and culture conditions in tissue engineering offer increased relevance for developing human disease models. In this work, we used human platelet lysate (PL)-based nanocomposite hydrogels (HUgel) as scaffolds for human skeletal muscle tissue engineering. These hydrogels consist of human PL reinforced with aldehyde-cellulose nanocrystals (a-CNC) that allow tunable mechanical, structural, and biochemical properties for the 3D culture of stem cells. Here, we developed hydrogel casting platforms to encapsulate human muscle satellite stem cells in HUgel. The a-CNC content was modulated to enhance matrix remodeling, uniaxial tension, and self-organization of the cells, resulting in the formation of highly aligned, long myotubes expressing sarcomeric proteins. Moreover, the bioengineered human muscles were subjected to electrical stimulation, and the exerted contractile forces were measured in a non-invasive manner. Overall, our results demonstrated that the bioengineered human skeletal muscles could be built in xeno-free cell culture platforms to assess tissue functionality, which is promising for drug development applications., The authors thank the technical support of MicroFabSpace and Microscopy Characterization Facility, Unit 7 of ICTS 'NANBIOSIS' from CIBER-BBN at IBEC. We would also like to thank the muscle team from the Biosensors for Bioengineering group for their feedback in the review process of this manuscript. Human immortalized muscle satellite stem cells used in this study were kindly provided by Dr Bénédicte Chazaud (Institut NeuroMyoGène (INMG), Lyon, France). This project received financial support from European Research Council program Grant ERC-StG-DAMOC: 714317 (J R-A), European Commission under FET-open program BLOC Project: GA- 863037 (J R-A), Spanish Ministry of Economy and Competitiveness, through the 'Severo Ochoa' Program for Centres of Excellence in R&D: SEV-2016–2019, Spanish Ministry of Economy and Competitiveness: 'Retos de investigación: Proyectos I+D+i': TEC2017-83716-C2-2-R (J R-A), CERCA Programme/Generalitat de Catalunya: 2017-SGR-1079 (J R-A), and Fundación Bancaria 'la Caixa'- Obra Social 'la Caixa': project IBEC-La Caixa Healthy Ageing (J R-A). The authors also acknowledge the European Union's Horizon 2020 research and innovation program under European Research Council Grant Agreement 772817 and Twinning Grant Agreement No. 810850—Achilles. Fundação para a Ciência e a Tecnologia (FCT) for CEECIND/01375/2017 (M G-F) and 2020.03410.CEECIND (R M A D).

As pluripotent stem cell (PSC)‐based reparative cell therapies are reaching the bedside, there is a growing need for the standardization of studies concerning safety of the derived products. Clinical trials using these promising strategies are in development, and treatment for age‐related macular degeneration is one of the first that has reached patients. We have previously established a xeno‐free and defined differentiation protocol to generate functional human embryonic stem cells (hESCs)‐derived retinal pigment epithelial (RPE) cells. In this study, we perform preclinical safety studies including karyotype and whole‐genome sequencing (WGS) to assess genome stability, single‐cell RNA sequencing to ensure cell purity, and biodistribution and tumorigenicity analysis to rule out potential migratory or tumorigenic properties of these cells. WGS analysis illustrates that existing germline variants load is higher than the introduced variants acquired through in vitro culture or differentiation, and enforces the importance to examine the genome integrity at a deeper level than just karyotype. Altogether, we provide a strategy for preclinical evaluation of PSC‐based therapies and the data support safety of the hESC‐RPE cells generated through our in vitro differentiation methodology., Retinal pigment epithelial cells derived from human embryonic stem cells are a promising source for replacement therapy in age‐related macular degeneration. However, preclinical studies concerning safety of the derived products are indispensable. Here, we evaluate safety invitro and invivo, reassuring stability, purity, and lack of tumorigenicity or migratory potential of the derived product for safe clinical translation.

Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation and bone regeneration capacity of mesenchymal stromal cells (MSC). Gingiva-derived progenitor cells (GPC) represent a less invasive alternative to bone marrow MSC (BMSC) for clinical applications. The aim of this study was to test the in vivo bone forming potential of human GPC and BMSC cultured as 3D spheroids or dissociated cells (2D). 2D and 3D cells encapsulated in constructs of human platelet lysate hydrogels (HPLG) and 3D-printed poly (L-lactide-co-trimethylene carbonate) scaffolds (HPLG-PLATMC) were implanted subcutaneously in nude mice; cell-free HPLG-PLATMC constructs served as a control. Mineralization was assessed using micro-computed tomography (µCT), histology, scanning electron microscopy (SEM) and in situ hybridization (ISH). After 4–8 weeks, µCT revealed greater mineralization in 3D-BMSC vs. 2D-BMSC and 3D-GPC (p < 0.05), and a similar trend in 2D-GPC vs. 2D-BMSC (p > 0.05). After 8 weeks, greater mineralization was observed in cell-free constructs vs. all 2D- and 3D-cell groups (p < 0.05). Histology and SEM revealed an irregular but similar mineralization pattern in all groups. ISH revealed similar numbers of 2D and 3D BMSC/GPC within and/or surrounding the mineralized areas. In summary, spheroid culture promoted ectopic mineralization in constructs of BMSC, while constructs of dissociated GPC and BMSC performed similarly. The combination of HPLG and PLATMC represents a promising scaffold for bone tissue engineering applications.

Background Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSC). 3D printing offers the possibility to produce customized scaffolds for complex bone defects. The aim of this study was to compare the potential of human BMSC cultured as 2D monolayers or 3D spheroids encapsulated in constructs of 3D-printed poly-L-lactide-co-trimethylene carbonate scaffolds and modified human platelet lysate hydrogels (PLATMC-HPLG) for bone regeneration. Methods PLATMC-HPLG constructs with 2D or 3D BMSC were assessed for osteogenic differentiation based on gene expression and in vitro mineralization. Subsequently, PLATMC-HPLG constructs with 2D or 3D BMSC were implanted in rat calvarial defects for 12 weeks; cell-free constructs served as controls. Bone regeneration was assessed via in vivo computed tomography (CT), ex vivo micro-CT and histology. Results Osteogenic gene expression was significantly enhanced in 3D versus 2D BMSC prior to, but not after, encapsulation in PLATMC-HPLG constructs. A trend for greater in vitro mineralization was observed in constructs with 3D versus 2D BMSC (p > 0.05). In vivo CT revealed comparable bone formation after 4, 8 and 12 weeks in all groups. After 12 weeks, micro-CT revealed substantial regeneration in 2D BMSC (62.47 ± 19.46%), 3D BMSC (51.01 ± 24.43%) and cell-free PLATMC-HPLG constructs (43.20 ± 30.09%) (p > 0.05). A similar trend was observed in the histological analysis. Conclusion Despite a trend for superior in vitro mineralization, constructs with 3D and 2D BMSC performed similarly in vivo. Regardless of monolayer or spheroid cell culture, PLATMC-HPLG constructs represent promising scaffolds for bone tissue engineering applications.

Joint cartilage damage affects 10-12% of the world's population. Medical treatments improve the short-term quality of life of affected individuals but lack a long-term effect due to injury progression into fibrocartilage. The use of mesenchymal stem cells (MSCs) is one of the most promising strategies for tissue regeneration due to their ability to be isolated, expanded and differentiated into metabolically active chondrocytes to achieve long-term restoration. For this purpose, human adipose-derived MSCs (Ad-MSCs) were isolated from lipectomy and grown in xeno-free conditions. To establish the best differentiation potential towards a stable chondrocyte phenotype, isolated Ad-MSCs were sequentially exposed to five differentiation schemes of growth factors in previously designed three-dimensional biphasic scaffolds with incorporation of a decellularized cartilage matrix as a bioactive ingredient, silk fibroin and bone matrix, to generate a system capable of being loaded with pre-differentiated Ad-MSCs, to be used as a clinical implant in cartilage lesions for tissue regeneration. Chondrogenic and osteogenic markers were analyzed by reverse transcription-quantitative PCR and cartilage matrix generation by histology techniques at different time points over 40 days. All groups had an increased expression of chondrogenic markers; however, the use of fibroblast growth factor 2 (10 ng/ml) followed by a combination of insulin-like growth factor 1 (100 ng/ml)/TGFβ1 (10 ng/ml) and a final step of exposure to TGFβ1 alone (10 ng/ml) resulted in the most optimal chondrogenic signature towards chondrocyte differentiation and the lowest levels of osteogenic expression, while maintaining stable collagen matrix deposition until day 33. This encourages their possible use in osteochondral lesions, with appropriate properties for use in clinical patients.

Das Hochdurchsatz-Screening ist von großer Bedeutung und bildet einen der Eckpfeiler der aktuellen Life-Science-Research wie Biologie, Biotechnologie, Biochemie, Computerwissenschaft, medizinische Chemie und Pharmakologie. Das Interesse an einem Screening mit hohem Durchsatz unter akademischen, mittleren und kleinen Biotech-Unternehmen, staatlichen und gemeinnützigen Screening-Standorten, sowie Auftragsforschungsorganisationen wurde spürbar erhöht, da Hochdurchsatz-Screenings eine effiziente Analyse von einer Vielzahl von Stoffen in kürzester Zeit ermöglicht. Das steigende Interesse an dieser Methode führte zu einer schnellen Entwicklung von Screening-Technologien mit hohem Durchsatz in Kombination mit komplementären Technologien. Die Nachteile herkömmlicher Hochdurchsatz-Screenings beinhalten hohe Kosten und Materialverschwendung, lange Zykluszeiten, geringe Produktivität und die begrenzte Ausbaufähigkeit der existierenden Stoffbibliotheken. Die genannten Nachteile verdeutlichen die Wichtigkeit der Bemühungen zur Entwicklung von verbesserten miniaturisierten und automatisierten Hochdurchsatz-Screening Methoden. Droplet Microarray (DMA) ist eine dieser miniaturisierten Plattformen, welche durch die Vereinigung von Oberflächenchemie, Oberflächenfunktionalisierung und Biologie erschaffen wurde. Die hohe Tröpfchendichte auf DMA senkt die Kosten, Materialverschwendung, Zykluszeit des Screenings und erhöht die Effizienz des Verfahrens, während sie effektiv die Verwendung größerer chemischer und biologischer Bibliotheken unterstützt. Die weitere Entwicklung, Auswertung und Verbesserung der DMAs spielt nicht nur für den Fortschritt der Hochdurchsatz-Screenings eine wichtige Rolle, sondern beherbergt auch unermessliches Potential in pharmalogischen Bereichen und der Stammzellenforschung, in welcher es die Wirksamkeit von Gentransfers und das Kultivieren von undifferenzierten Stammzellen erleichtert. Pharmakologisches Priming (Drug Repurposing) wurde als adjuvante Strategie zur Verbesserung der Effizienz nicht-viraler Genübertragung angesehen. Dennoch ist die facettenreiche Anwendung des pharmakologischen Priming aufgrund der unerschwinglich hohen Kosten für Reagenzien und die Durchführung der Methode unweit verbreitet, was den aufkommenden Drang nach miniaturisierten Plattformen und Drug Repurposing erklärt. Abgesehen von der Dringlichkeit im pharmakologischen Bereich, um klinisches Potenzial zu realisieren, benötigt die Stammzellenforschung auch miniaturisierte Methoden um den Einfluss von Zelloberflächeninteraktionen und Zellkulturmedium auf das Verhalten von Stammzellen und die Effekte von Zusatzstoffen, und Oberflächenbeschichtungen einschließlich oberflächenabsorbierter Proteine auf Stammzellen zu beobachten. Das Ziel für den ersten Teil der Dissertation war es nach kleinen Molekülen (Stoffen) auf DMAs zu suchen, welche transfektionsverstärkende Effekte beinhalten, um eine mögliche Verbesserung in den Bereichen Gentransfer- und Therapie zu bieten. Eine Untersuchung der Einflüsse von 774 Food and Drug Administration (FDA)-zugelassenen Wirkstoffen auf die Transfektionseffizienz mit verschiedenen Zelltypen in miniaturisierter- und hochdurchsatzweise wurde mit Hilfe der DMAs veranlasst. Das Screening der FDA-zugelassenen Arzneimittelbibliothek identifizierte 14 einzelne Verbindungen, die eine zwei- bis fünffache Verbesserung der Transfektion aufwiesen. Diese Treffer wurden verifiziert und in großem Maßstab untersucht. Die Ergebnisse deuten darauf hin, dass der auf DMA basierende Ansatz für das Drug Repurposing beständig ist und zur Untersuchung und Entwicklung wirksamerer nicht-viraler Genübertragungssysteme verwendet werden könnte. Das Ziel des zweiten Abschnitts der Dissertation war es, den Einfluss von Oberflächeneigenschaften und sinkendem Volumen auf die Pluripotenz von human induzierten pluripotenten Stammzellen (hiPSCs) zu bestimmen. Zwei künstliche Oberflächen mit unterschiedlichen chemischen Elementen und deren dazugehörigen DMAs wurden für die Kultivierung und Pluripotenz von hiPSCs in vitro untersucht. Die Oberflächen und DMAs wurden genutzt, um human induzierten pluripotenten Stammzellen in 2 ml und 200 nL-Volumen zu kultivieren. Die Ergebnisse zeigten, dass hiPSCs eine hohe Lebensfähigkeit, sowie die erwartete Morphologie und Pluripotenz in 200 nL Tröpfchen auf Typ A und Typ B DMAs ohne Matrigelbeschichtung nach 24 h Kultivierung aufwiesen. Dies beweist, dass DMAs eine vielseitige und simple Plattform für kurzzeitige und xeno-freie Hochdurchsatz-Screenings von hiPSCs sind. Als Ziel für den dritten Teil der Dissertation wurde das Identifizieren von chemisch definierten Proteinen, welche das Kultivieren und Aufrechterhalten der Plutipotenz von hiPSCs Zellen auf DMAs, sowie Mikrotiterplatten weiter verbessern könnten, angesetzt. Es ist möglich eine Proteinbeschichtung, Zellkultur und Immunfluoreszenzfärbung auf miniaturisierter Ebene parallel mit Hilfe von DMAs durchzuführen, was zu einer Reduzierung von Versuchsfehlern und Verbrauchsmaterialien führt. Auf Grund dessen wurden DMAs als Basis zur Überprüfung von elf verschiedenen Proteinen und deren verwandten binären und ternären Kombinationen (insgesamt 231 verschiedene Gruppen) auf ihre Fähigkeit zur Erhaltung der Pluripotenz von hiPSCs genutzt. Aus diesem Raster wurden zehn Gruppen von ternären Proteinkombinationen identifiziert, welche die Proliferation und das Self-Renewal besser unterstützen könnten als mit Matrigel beschichtete Oberflächen. Die effizientesten Proteinkombinationen des primären Screenings wurden weiterhin in einer Langzeitkultur (fünf Wochen) verifiziert. Zusätzlich wurde die Formation von embryonalen Körperchen der auf den ausgewählten Proteinbeschichtungen kultivierten Zellen erzielt und es folgte die Differenzierung der hiPSCs in drei Keimblätter. Zusammengefasst, wurden DMAs als miniaturisierte Schnellscreening-Plattform verwendet, um mehrere biologische Fragen zu beantworten. Als erstes wurden 776 Wirkstoffe auf Nanoebene untersucht und somit kosten-, zeit- und arbeitssparend verwertet. Vierzehn Stoffe wiesen eine zwei- bis fünffache Verbesserung der Transfektionstechniken auf. Als zweites wurden chemische Komponente von Zellkulturoberflächen und kleinen Volumina (200nl) von Zellkulturmedium gefunden, die zur Erhaltung der Pluripotenz von hiPSCs beitragen. Als letztes wurden zehn Gruppen von ternären Proteinkombinationen identifiziert, welche das Kultivieren von undifferenzierten hiPSCs unterstützen. Zwei von ihnen wurden weiter untersucht, um eine Langzeitkultur von undifferenzierten hiPSCs zu erzielen, gefolgt von ihrer Differenzierung in drei Keimblätter. Eine Zusammenfassung und eine Aussicht auf die Zukunft befinden sich am Ende der Dissertation.

Background Amnion-derived mesenchymal stem cells (AM-MSCs) are an attractive source of stem cell therapy for patients with irreversible liver disease. However, there are obstacles to their use due to low efficiency and xeno-contamination for hepatic differentiation. Methods We established an efficient protocol for differentiating AM-MSCs into hepatic progenitor cells (HPCs) by analyzing transcriptome-sequencing data. Furthermore, to generate the xeno-free conditioned differentiation protocol, we replaced fetal bovine serum (FBS) with polyvinyl alcohol (PVA). We investigated the hepatocyte functions with the expression of mRNA and protein, secretion of albumin, and activity of CYP3A4. Finally, to test the transplantable potential of HPCs, we transferred AM-MSCs along with hepatic progenitors after differentiated days 11, 12, and 13 based on the expression of hepatocyte-related genes and mitochondrial function. Further, we established a mouse model of acute liver failure using a thioacetamide (TAA) and cyclophosphamide monohydrate (CTX) and transplanted AM-HPCs in the mouse model through splenic injection. Results We analyzed gene expression from RNA sequencing data in AM-MSCs and detected downregulation of hepatic development-associated genes including GATA6, KIT, AFP, c-MET, FGF2, EGF, and c-JUN, and upregulation of GSK3. Based on this result, we established an efficient hepatic differentiation protocol using the GSK3 inhibitor, CHIR99021. Replacing FBS with PVA resulted in improved differentiation ability, such as upregulation of hepatic maturation markers. The differentiated hepatocyte-like cells (HLCs) not only synthesized and secreted albumin, but also metabolized drugs by the CYP3A4 enzyme. The best time for translation of AM-HPCs was 12 days from the start of differentiation. When the AM-HPCs were transplanted into the liver failure mouse model, they settled in the damaged livers and differentiated into hepatocytes. Conclusion This study offers an efficient and xeno-free conditioned hepatic differentiation protocol and shows that AM-HPCs could be used as transplantable therapeutic materials. Thus, we suggest that AM-MSC-derived HPCs are promising cells for treating liver disease.

Funder: Tampereen Yliopisto (University of Tampere); doi: https://doi.org/10.13039/501100004371, Funder: Tekes (Finnish Funding Agency for Innovation); doi: https://doi.org/10.13039/501100003406, Human pluripotent stem cell (hPSC)-derived neurons provide exciting opportunities for in vitro modeling of neurological diseases and for advancing drug development and neurotoxicological studies. However, generating electrophysiologically mature neuronal networks from hPSCs has been challenging. Here, we report the differentiation of functionally active hPSC-derived cortical networks on defined laminin-521 substrate. We apply microelectrode array (MEA) measurements to assess network events and compare the activity development of hPSC-derived networks to that of widely used rat embryonic cortical cultures. In both of these networks, activity developed through a similar sequence of stages and time frames; however, the hPSC-derived networks showed unique patterns of bursting activity. The hPSC-derived networks developed synchronous activity, which involved glutamatergic and GABAergic inputs, recapitulating the classical cortical activity also observed in rodent counterparts. Principal component analysis (PCA) based on spike rates, network synchronization and burst features revealed the segregation of hPSC-derived and rat network recordings into different clusters, reflecting the species-specific and maturation state differences between the two networks. Overall, hPSC-derived neural cultures produced with a defined protocol generate cortical type network activity, which validates their applicability as a human-specific model for pharmacological studies and modeling network dysfunctions.

Osteoarthritis (OA) in articular joints is a prevalent disease. With increasing life expectancy, the need for therapies other than knee replacement arises. The intrinsic repair capacity of cartilage is limited, therefore alternative strategies for cartilage regeneration are being explored. The purpose of this study is first to investigate the potential of platelet lysate (PL) as a xeno-free alternative in expansion of human OA chondrocytes for cell therapy, and second to assess the effects of PL on redifferentiation of expanded chondrocytes in 3D pellet cultures. Chondrocytes were isolated from human OA cartilage and subjected to PL in monolayer culture. Cell proliferation, morphology, and expression of chondrogenic genes were assessed. Next, PL-expanded chondrocytes were cultured in 3D cell pellets and cartilage matrix production was assessed after 28 days. In addition, the supplementation of PL to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by quantitative biochemical analyses, as well as by (immuno)histochemistry. A dose-dependent effect of PL on chondrocyte proliferation was found, but expression of chondrogenic markers was decreased when compared to FBS-expanded cells. After 28 days of subsequent 3D pellet culture, GAG production was significantly higher in pellets consisting of chondrocytes expanded with PL compared to controls. However, when used to supplement redifferentiation medium for chondrocyte pellets, PL significantly decreased the production of GAGs and collagen. In conclusion, chondrocyte proliferation is stimulated by PL and cartilage production in subsequent 3D culture is maintained. Furthermore, the presences of PL during redifferentiation of 3D chondrocyte strongly inhibits GAG and collagen content. The data presented in the current study indicate that while the use of PL for expansion in cartilage cell therapies is possibly beneficial, intra-articular injection of the product in the treatment of OA might be questioned.

Three-dimensional (3D) bioprinting holds the promise to fabricate tissue and organ substitutes for regenerative medicine. However, the lack of bioactive inks to fabricate and support functional living constructs is one of the main limitations hindering the progress of this technology. In this study, a biofunctional human-based nanocomposite bioink (HUink) composed of platelet lysate hydrogels reinforced by cellulose nanocrystals is reported. When combined with suspended bioprinting technologies, HUink allows the biofabrication of 3D freeform constructs with high resolution and integrity, mimicking the hierarchical nano-to-macro fibrillary composition of native tissues. Remarkably, HUink supports bioprinting of stem cells with high viability immediately after extrusion and over long-term cell culture without the need for additional biochemical or animal-derived media supplementation. As opposed to typical polymer-based bioinks, the pool of growth factors, cytokines and adhesion proteins in HUink boosts cell spreading and proliferation, stimulating the fast production of cell-secreted extracellular matrix. This innovative bioprinting platform with unpaired biofunctionality allows the fabrication of complex freeform cell-laden constructs that can ultimately be applied in the development of xeno-free 3D tissue models for in vitro research or to develop tissue and organ surrogates for clinical applications., The authors thank Hospital da Prelada (Porto, Portugal) for providing adipose tissue samples and Hospital São João (Porto, Portugal) for providing platelet concentrates. Authors acknowledge the financial support from project NORTE-010145-FEDER-000021 supported by Norte Portugal Regional Operational Programme (NORTE 2020), under the PORTUGAL 2020 Partnership Agreement, through the European Regional Development Fund (ERDF); the European Union Framework Programme for Research and Innovation HORIZON 2020, under the TEAMING Grant agreement No 739572 - The Discoveries CTR EU, Marie Skłodowska-Curie grant agreement No 706996 and European Research Council grant agreement No 726178; FCT/MCTES (Fundação para a Ciência e a Tecnologia/ Ministério da Ciência, Tecnologia, e Ensino Superior) and the Fundo Social Europeu através do Programa Operacional do Capital Humano (FSE/POCH) in the framework of PhD grant PD/59/2013 - PD/BD/113807/2015 for BBM, Post-Doc grant SFRH/BPD/112459/2015 for RMD. The authors would like to thank Tram Le, Carlos Guimarães and Luca Gasperini for their support to acquire pictures and design the drawings. The authors would also like to thank Syeda Mahwish Bakht for the gelatin rhelogical data acquisiton.

Three-dimensional retinal organoids derived from human induced pluripotent stem cells (hiPSCs) are gaining much attention as a possible source for cell transplantation to treat retinal degenerative conditions. However, the protocol for producing retinal organoids is time and labor intensive, involving a sequence of precise steps, and thus has yet to be successfully translated into a Good Manufacturing Practice (GMP)-compliant procedure. This review seeks to define the progress that has already been made in the pursuit of designing a GMP-compliant, streamlined, and automated protocol for retinal organoid production for optimal clinical success. The reviewed literature compares various approaches for cell culture automation, appropriate xeno-free conditions, and cell sources for iPSC line generation; yet, there are still important gaps for these three key considerations that remain to be addressed. Thus, the authors also discuss further potential strategies to successfully achieve GMP-compliant production of retinal organoids for eventual safe and efficient use in clinical trials. Translational Relevance Designing a GMP-compliant protocol for three-dimensional retinal organoid production is of urgent need in order to bring transplantation of hiPSC-derived retinal tissue and derived cells to clinical trials – and ultimately patient treatment – for retinal degenerative diseases.

Summary Human embryonic stem cells (hESCs) are promising in regenerative medicine. Although several hESC-based clinical trials are under way, a widely accepted standard of clinical-grade cells remains obscure. To attain a completely xeno-free clinical-grade cell line, the system must be free of xenogenic components, the cells must have a comprehensive set of functions, and good manufacturing practice conditions must be used. In this study, following these criteria, we successfully derived two hESC lines, which were thereby considered “clinical-grade embryonic stem cells”. In addition to the primary capacity for pluripotency, these two cell lines were efficiently differentiated into various types of clinical-grade progeny. Importantly, the cells were recognized by the National Institutes for Food and Drug Control of China for further eligible accreditation. These data indicate that we have established completely xeno-free clinical-grade hESC lines and their derivatives, which will be valuable for the foundation of an international standard for clinical-grade cells for therapy., Graphical Abstract, Highlights • Two clinical-grade hESC lines were generated • The hESCs were demonstrated to have sustained pluripotency • The hESCs could be differentiated into functional cells • The hESCs passed biosafety tests and were recognized by the NIFDC, In this article, Zhou, Hao, and colleagues generate two completely xeno-free clinical-grade human embryonic stem cell lines which have demonstrated pluripotency and biosafety under the investigation from an accredited organization.

Summary A defined protocol for efficiently deriving endothelial cells from human pluripotent stem cells was established and vascular morphogenesis was used as a model system to understand how synthetic hydrogels influence global biological function compared with common 2D and 3D culture platforms. RNA sequencing demonstrated that gene expression profiles were similar for endothelial cells and pericytes cocultured in polyethylene glycol (PEG) hydrogels or Matrigel, while monoculture comparisons identified distinct vascular signatures for each cell type. Endothelial cells cultured on tissue-culture polystyrene adopted a proliferative phenotype compared with cells cultured on or encapsulated in PEG hydrogels. The proliferative phenotype correlated to increased FAK-ERK activity, and knockdown or inhibition of ERK signaling reduced proliferation and expression for cell-cycle genes while increasing expression for “3D-like” vasculature development genes. Our results provide insight into the influence of 2D and 3D culture formats on global biological processes that regulate cell function., Graphical Abstract, Highlights • Defined, high-efficiency differentiation of human PSCs to endothelial cell • Comprehensive genome-wide comparisons of 2D and 3D cell-culture formats • Gene expression profiles for endothelial cells and pericytes in 3D cell culture • Highly proliferative phenotypes on tissue-culture polystyrene surfaces, Murphy, Schwartz and colleagues report a genome-wide analysis of biological function for endothelial cells and pericytes cultured using 2D or 3D formats. Global gene expression patterns are comparable for vascular cells cultured in synthetic hydrogels or Matrigel during vascular network formation, while cells cultured on tissue-culture polystyrene surfaces adopt a proliferative phenotype that correlates to increased FAK-ERK activity.

The use of non-optimal preparations of mesenchymal stem cells (MSCs), such as extensively expanded cells, might be necessary to obtain the large numbers of cells needed for many clinical applications. We previously demonstrated that minimally expanded (early passage) MSCs can be pre-activated as spheroids to produce potentially therapeutic factors in 3D cultures. Here, we used extensively expanded (late passage) MSCs and studied their 3D-culture activation potential. MSCs were culture-expanded as 2D monolayers, and cells from various passages were activated by 3D culture in hanging drops with either fetal bovine serum (FBS)-containing media or a more clinically-applicable animal product-free (xeno-free) media. Gene expression analyses demonstrated that MSC spheroids prepared from passage 3, 5, and 7 cells were similar to each other but different from 2D MSCs. Furthermore, the expression of notable anti-inflammatory/immune-modulatory factors cyclooxygenase-2 (PTGS2), TNF alpha induced protein 6 (TNFAIP6), and stanniocalcin 1 (STC-1) were up-regulated in all spheroid preparations. This was confirmed by the detection of secreted prostaglandin E2 (PGE-2), tumor necrosis factor-stimulated gene 6 (TSG-6, and STC-1. This study demonstrated that extensively expanded MSCs can be activated in 3D culture through spheroid formation in both FBS-containing and xeno-free media. This work highlights the possibility of activating otherwise less useable MSC preparations through 3D culture generating large numbers of potentially therapeutic MSCs.

Three-dimensional clumps of mesenchymal stem cell (MSC)/extracellular matrix (ECM) complexes (C-MSCs) consist of cells and self-produced ECM. We demonstrated previously that C-MSCs can be transplanted into bone defect regions with no artificial scaffold to induce bone regeneration. To apply C-MSCs in a clinical setting as a reliable bone regenerative therapy, the present study aimed to generate C-MSCs in xeno-free/serum-free conditions that can exert successful bone regenerative properties and to monitor interactions between grafted cells and host cells during bone healing processes. Human bone marrow-derived MSCs were cultured in xeno-free/serum-free medium. To obtain C-MSCs, confluent cells that had formed on the cellular sheet were scratched using a micropipette tip and then torn off. The sheet was rolled to make a round clump of cells. Then, C-MSCs were transplanted into an immunodeficient mouse calvarial defect model. Transplantation of C-MSCs induced bone regeneration in a time-dependent manner. Immunofluorescence staining showed that both donor human cells and host mice cells contributed to bone reconstruction. Decellularized C-MSCs implantation failed to induce bone regeneration, even though the host mice cells can infiltrate into the defect area. These findings suggested that C-MSCs generated in xeno-free/serum-free conditions can induce bone regeneration via direct and indirect osteogenesis.

STUDY QUESTION Does the X chromosome inactivation (XCI) of Klinefelter syndrome (KS)-derived human induced pluripotent stem cells (hiPSCs) correspond to female human pluripotent stem cells (hPSCs) and reflect the KS genotype? SUMMARY ANSWER Our results demonstrate for the first time that KS-derived hiPSCs show similar XCI behavior to female hPSCs in culture and show biological relevance to KS genotype-related clinical features. WHAT IS KNOWN ALREADY So far, assessment of XCI of KS-derived hiPSCs was based on H3K27me3 staining and X-inactive specific transcript gene expression disregarding the at least three XCI states (XaXi with XIST coating, XaXi lacking XIST coating, and XaXe (partially eroded XCI)) that female hPSCs display in culture. STUDY DESIGN, SIZE, DURATION The study used hiPSC lines generated from two azoospermic patients with KS and included two healthy male (HM) and one healthy female donor. PARTICIPANTS/MATERIALS, SETTING, METHODS In this study, we derived hiPSCs by reprograming fibroblasts with episomal plasmids and applying laminin 521 as culture substrate. hiPSCs were characterized by karyotyping, immunocytochemistry, immunohistochemistry, quantitative PCR, teratoma formation, and embryoid body differentiation. XCI and KS hiPSC relevance were assessed by whole genome transcriptomics analysis and immunocytochemistry plus FISH of KS, HM and female fibroblast, and their hiPSC derivatives. MAIN RESULTS AND THE ROLE OF CHANCE Applying whole genome transcriptomics analysis, we could identify differentially expressed genes (DEGs) between KS and HM donors with enrichment in gene ontology terms associated with fertility, cardiovascular development, ossification, and brain development, all associated with KS genotype-related clinical features. Furthermore, XCI analysis based on transcriptomics data, RNA FISH, and H3K27me3 staining revealed variable XCI states of KS hiPSCs similar to female hiPSCs, showing either normal (XaXi) or eroded (XaXe) XCI. KS hiPSCs with normal XCI showed nevertheless upregulated X-linked genes involved in nervous system development as well as synaptic transmission, supporting the potential use of KS-derived hiPSCs as an in vitro model for KS. LIMITATIONS, REASONS FOR CAUTION Detailed clinical information for patients included in this study was not available. Although a correlation between DEGs and the KS genotype could be observed, the biological relevance of these cells has to be confirmed with further experiments. In addition, karyotype analysis for two hiPSC lines was performed at passage 12 but not repeated at a later passage. Nevertheless, since all XCI experiments for those lines were performed between passage 11 and 15 the authors expect no karyotypic changes for those experiments. WIDER IMPLICATIONS OF THE FINDINGS As KS patients have variable clinical phenotypes that are influenced by the grade of aneuploidy, mosaicism, origin of the X chromosome, and XCI ‘escapee’ genes, which vary not only among individuals but also among different tissues within the same individual, differentiated KS hiPSCs could be used for a better understanding of KS pathogenesis. STUDY FUNDING/COMPETING INTEREST(S) This study was supported by grants from the Knut and Alice Wallenberg Foundation (2016.0121 and 2015.0096), Ming Wai Lau Centre for Reparative Medicine (2-343/2016), Ragnar Söderberg Foundation (M67/13), Swedish Research Council (2013-32485-100360-69), the Centre for Innovative Medicine (2–388/2016–40), Kronprinsessan Lovisas Förening För Barnasjukvård/Stiftelsen Axel Tielmans Minnesfond, Samariten Foundation, Jonasson Center at the Royal Institute of Technology, Sweden, and Initial Training Network Marie Curie Program ‘Growsperm’ (EU-FP7-PEOPLE-2013-ITN 603568). The authors declare no conflicts of interest.

Human pluripotent stem cells (hPSC) are self-renewing cells having the potential of differentiation into the three lineages of somatic cells and thus can be medically used in diverse cellular therapies. One of the requirements for achieving these clinical applications is development of completely defined xeno-free systems for large-scale cell expansion and differentiation. Previously, we demonstrated that microcarriers (MCs) coated with mouse laminin-111 (LN111) and positively charged poly-l-lysine (PLL) critically enable the formation and evolution of cells/MC aggregates with high cell yields obtained under agitated conditions. In this article, we further improved the MC system into a defined xeno-free MC one in which the MCs are coated with recombinant human laminin-521 (LN521) alone without additional positive charge. The high binding affinity of the LN521 to cell integrins enables efficient initial HES-3 cell attachment (87%) and spreading (85%), which leads to generation of cells/MC aggregates (400 μm in size) and high cell yields (2.4–3.5×106 cells/mL) within 7 days in agitated plate and scalable spinner cultures. The universality of the system was demonstrated by propagation of an induced pluripotent cells line in this defined MC system. Long-term pluripotent (>90% expression Tra-1-60) cell expansion and maintenance of normal karyotype was demonstrated after 10 cell passages. Moreover, tri-lineage differentiation as well as directed differentiation into cardiomyocytes was achieved. The new LN521-based MC system offers a defined, xeno-free, GMP-compatible, and scalable bioprocessing platform for the production of hPSC with the quantity and quality compliant for clinical applications. Use of LN521 on MCs enabled a 34% savings in matrix and media costs over monolayer cultures to produce 108 cells.

Background Human mesenchymal stem cells are a strong candidate for cell therapies owing to their regenerative potential, paracrine regulatory effects, and immunomodulatory activity. Yet, their scarcity, limited expansion potential, and age-associated functional decline restrict the ability to consistently manufacture large numbers of safe and therapeutically effective mesenchymal stem cells for routine clinical applications. To overcome these limitations and advance stem cell treatments using mesenchymal stem cells, researchers have recently derived mesenchymal progenitors from human-induced pluripotent stem cells. Human-induced pluripotent stem cell-derived progenitors resemble adult mesenchymal stem cells in morphology, global gene expression, surface antigen profile, and multi-differentiation potential, but unlike adult mesenchymal stem cells, it can be produced in large numbers for every patient. For therapeutic applications, however, human-induced pluripotent stem cell-derived progenitors must be produced without animal-derived components (xeno-free) and in accordance with Good Manufacturing Practice guidelines. Methods In the present study we investigate the effects of expanding mesodermal progenitor cells derived from two human-induced pluripotent stem cell lines in xeno-free medium supplemented with human platelet lysates and in a commercial high-performance Good Manufacturing Practice-compatible medium (Unison Medium). Results The results show that long-term culture in xeno-free and Good Manufacturing Practice-compatible media somewhat affects the morphology, expansion potential, gene expression, and cytokine profile of human-induced pluripotent stem cell-derived progenitors but supports cell viability and maintenance of a mesenchymal phenotype equally well as medium supplemented with fetal bovine serum. Conclusions The findings support the potential to manufacture large numbers of clinical-grade human-induced pluripotent stem cell-derived mesenchymal progenitors for applications in personalized regenerative medicine. Graphical abstract Electronic supplementary material The online version of this article (10.1186/s13287-018-1119-3) contains supplementary material, which is available to authorized users.

Novel experimental conditions of cancer cell line culture have evolved throughout the recent years, with significantly growing interest in xeno-free, serum-free and three-dimensional culture variants. The choice of proper culture media may enable to mimic tumor microenvironment and promotion of cancer stem cells proliferation. To assess whether stem-like phenotype inducing media may be applied in renal cancer stem cell research, we performed a widespread screening of 13 cell culture media dedicated for mesenchymal cells, stem cells as well as mesenchymal stem cells. We have also screened extracellular matrix compounds and selected optimal RCC 3D—ECM supported culture model. Our results revealed that 786-O as well as HKCSCs cell line cultures in xeno-free media (NutriStem/StemXvivo) and laminin coated plates provide a useful tool in RCC cancer biology research and at the same time enable effective drug toxicity screening. We propose bio-mimic 3D RCC cell culture model with specific low-serum and xeno-free media that promote RCC cell viability and stem-like phenotype according to the tested genes encoding stemness factors including E-cadherin, N-cadherin, HIF1, HIF2, VEGF, SOX2, PAX2 and NESTIN. Electronic supplementary material The online version of this article (10.1007/s10616-018-0273-x) contains supplementary material, which is available to authorized users.

Vascularized adipose tissue models are highly demanded as alternative to existing animal models to elucidate the mechanisms of widespread diseases, screen for new drugs or asses corresponding safety levels. Standardly used animal-derived sera therein, are associated to ethical concerns, the risk of contaminations and many uncertainties in their composition and impact on cells. Therefore their use should be completely omitted. In this study we developed a serum-free, defined co-culture medium and implemented it to set up an adipocyte-endothelial cell (EC) co-culture model. Human adipose-derived stem cells were differentiated under defined conditions (diffASCs) and, like human microvascular ECs (mvECs), cultured in a developed defined co-culture medium in mono-, indirect or direct co-culture for 14 days. The developed defined co-culture medium was superior to compared mono-culture media and facilitated the functional maintenance and maturation of diffASCs including perilipin A expression, lipid accumulation and glycerol and leptin release. The medium equally allowed mvEC maintenance, confirmed by the expression of CD31 and vWF and acLDL uptake. Thereby mvECs showed a strong dependency on EC-specific factors. Additionally the development of vascular structures by mvECs was facilitated when directly co-cultured with diffASCs. The completely defined co-culture system allows for the serum-free setup of adipocyte/EC co-cultures and thereby represents a valuable and ethically acceptable tool for the setup of vascularized adipose tissue models.

Although the biological properties of mesenchymal stem cells (MSC) are well-characterized in vitro, MSC clinical application is still far away to be achieved, mainly due to the need of xenogeneic substances for cell expansion, such as fetal bovine serum (FBS). FBS presents risks regarding pathogens transmissions and internalization of animal’s proteins, which can unleash antigenic responses in patients after MSC implantation. A wide range of venous blood derivatives (VBD) has been reported as FBS substitutes showing promising results. Thus, the aim of this study was to conduct a systematic scoping review to analyze whether VBD are effective FBS substitutes for MSC ex vivo expansion. The search was performed in SciVerse ScopusTM, PubMed, Web of ScienceTM, BIREME, Cochrane library up to January 2016. The keywords were selected using MeSH and entry terms. Two independent reviewers scrutinized the records obtained considering specific inclusion criteria. The included studies were evaluated in accordance with a modified Arksey and O’ Malley’s framework. From 184 found studies, 90 were included. Bone marrow mesenchymal stem cells (BMMSC) were presented in most of these studies. Overall, VBD allowed for either, maintenance of MCS’s fibroblast-like morphology, high proliferation, high colony-formation ability and maintenance of multipotency. Besides. MSC expanded in VBD supplements presented higher mitogen activity than FBS. VBD seems to be excellent xeno-free serum for ex vivo expansion of mesenchymal stem cells. However, an accentuated heterogeneity was observed between the carried out protocols for VBD isolation did not allowing for direct comparisons between the included studies. Resumo Embora as propriedades biológicas das células-tronco mesenquimais (MSC) sejam bem caracterizadas in vitro, a aplicação clínica das MSC ainda está longe de ser alcançada, principalmente devido à necessidade de substâncias xenogênicas para expansão celular, como o soro fetal bovino (FBS). O FBS apresenta riscos quanto às transmissões de patógenos e à internalização de proteínas animais, o que pode desencadear respostas antigênicas em pacientes após a implantação das MSC. Uma vasta gama de derivados do sangue venoso (VBD) têm sido relatada como substitutos do FBS mostrando resultados promissores. Assim, o objetivo deste estudo foi conduzir uma revisão de escopo sistemática para analisar se VBD poderiam ser substitutos do FBS eficazes para expansão das MSC em condições ex vivo. A pesquisa foi realizada no SciVerse Scopus, PubMed, Web of Science, BIREME e biblioteca Cochrane até janeiro de 2016. As palavras-chave foram selecionadas usando MeSH e entre termos. Dois revisores independentes examinaram os registros obtidos considerando critérios de inclusão específicos. Os estudos incluídos foram avaliados de acordo com uma estrutura modificada de Arksey e O ‘Malley. Dos 184 estudos encontrados, 90 foram incluídos. As células-tronco da medula óssea (BMMSC) foram utilizadas na maior parte destes estudos. Em geral, o VBD permitiu tanto a manutenção da morfologia semelhante a fibroblastos das MCS, alta proliferação, alta capacidade de formação de colônias e manutenção de multipotêncialidade. Além disso, as MSC expandidas em suplementos derivados do sangue venoso apresentaram uma maior atividade mitogênica do que as expandidas em FBS. Os VBD parecem ser excelentes soro livres de agentes xenogênicos para expansão ex vivo de MSC. Entretanto, observou-se uma heterogeneidade acentuada entre os protocolos realizados para o isolamento VBD, não permitindo assim comparações diretas entre os estudos incluídos.

Background While a shift towards non-viral and animal component-free methods of generating induced pluripotent stem (iPS) cells is preferred for safer clinical applications, there is still a shortage of reliable cell sources and protocols for efficient reprogramming. Methods Here, we show a robust episomal and xeno-free reprogramming strategy for human iPS generation from dental pulp stem cells (DPSCs) which renders good efficiency (0.19%) over a short time frame (13–18 days). Results The robustness of DPSCs as starting cells for iPS induction is found due to their exceptional inherent stemness properties, developmental origin from neural crest cells, specification for tissue commitment, and differentiation capability. To investigate the epigenetic basis for the high reprogramming efficiency of DPSCs, we performed genome-wide DNA methylation analysis and found that the epigenetic signature of DPSCs associated with pluripotent, developmental, and ecto-mesenchymal genes is relatively close to that of iPS and embryonic stem (ES) cells. Among these genes, it is found that overexpression of PAX9 and knockdown of HERV-FRD improved the efficiencies of iPS generation. Conclusion In conclusion, our study provides underlying epigenetic mechanisms that establish a robust platform for efficient generation of iPS cells from DPSCs, facilitating industrial and clinical use of iPS technology for therapeutic needs. Electronic supplementary material The online version of this article (10.1186/s13287-018-0796-2) contains supplementary material, which is available to authorized users.

Safety concerns over cell-derived pharmaceutical products being manufactured in supplements of fetal bovine serum (FBS) have ignited pleas to replace FBS. Herein, four newly marketed alternatives to FBS were compared: a xeno-free product called Cell-Ess®, a human platelet lysate marketed as GroPro®, and two mixtures of adult bovine serum varying in their proportions of neonatal growth factors, called Liporo® and FetalGro®. An endothelial cell line (C2BBe1) and a neuronal cell line (SHSY5Y) near confluency in media with 10% FBS were selectively scraped and taken through a 25-day step-wise algorithm to replace FBS, and another human endothelial cell line (HRA-19) was studied to replicate C2BBe1. Cells were stained, counted, and compared for viability, migration, and spheroids. The C2BBe1 and HRA-19 cell lines failed to proliferate in 10% Cell-Ess® but grew in 10% GroPro® or 10% FetalGro® reasonably well compared to reference 10% FBS. With SH-SY5Y, only FetalGro® approached FBS's efficacy. These were all inferior to 11 different branded lots of FBS (positive controls), but five days into switching just amongst the FBS brands, 4 of 11 supported less proliferation than reference FBS in endothelial HRA-19 ( p < 0.004). Moreover, neurospheres were enriched in two branded lots of FBS and FetalGro® (each p < 0.004), neurospheres being an unwanted phenotype for any neuronal cell application. Because platelet-derived GroPro® stood out amongst the non-FBS growth supplements to allow proliferation without inducing spheroids, it seems the best (mindful that the cells still grew slower in it compared to FBS). While no perfect replacement was found amongst the alternatives to FBS, the algorithm for switching should be useful in future testing of new alternatives to FBS as the need arises to switch from FBS and expand pharmaceutical products with safety for human use.

Background Mesenchymal stem cells (MSCs) show promising characteristics for their use in advanced therapy medicinal products. However, there are some unresolved concerns, such as the use of animal components for their expansion. In this study we assessed the suitability of a xeno-free supplement for cell culture (SCC) derived from human plasma, to culture and expand human MSCs (hMSCs) from different origins. Characteristics of viable cultured hMSCs such as genetic stability, phenotype and multipotentiality were qualitatively evaluated. Methods hMSCs from adipose tissue (AT), bone marrow (BM) and umbilical cord (UC) and supplier sources (commercial/non-commercial) were used. After hMSCs expansion in a xeno-free medium, classical hMSCs markers were studied by immunocytochemistry, and genetic stability was tested by classic karyotyping. The capacity of hMSCs to differentiate into adipogenic, osteogenic, and chondrogenic cells in differentiation media was assessed using different staining. Different lots of SCC were used to assure consistency between batches. Results All hMSCs tested maintained their morphology and adherence to plastic during their expansion, and preserved their genetic stability, phenotype and differentiation potential. No differences were observed when using different lots of SCC. Moreover, the proliferation rate, evaluated as population doubling time (PDT) of commercial BM and AT hMSCs, was higher in the xeno-free medium than in the control media provided by the suppliers of the cells (PDT of 4.6 for BM-hMSC and 6.4 for AT-hMSC in xeno-free medium, and 7.0 and 14.7 respectively in the commercial media). UC-hMSCs PDT was similar in all the media tested. When using non-commercial BM-hMSCs, PDT was lower in the xeno-free medium, but reverted to the control level with the addition of growth factors. Conclusions SCC-containing medium can be a feasible xeno-free alternative to expand hMSCs for advanced therapies.

Growing evidence indicates that intracellular signaling mediated by extracellular vesicles (EVs) released by stem cells plays a considerable role in triggering the regenerative program upon transplantation. EVs from umbilical cord mesenchymal stem cells (UC-MSC-EVs) have been shown to enhance tissue repair in animal models. However, translating such results into clinical practice requires optimized EV collection procedures devoid of animal-originating agents. Thus, in this study, we analyzed the influence of xeno-free expansion media on biological properties of UC-MSCs and UC-MSC-EVs for future applications in cardiac repair in humans. Our results show that proliferation, differentiation, phenotype stability, and cytokine secretion by UC-MSCs vary depending on the type of xeno-free media. Importantly, we found distinct molecular and functional properties of xeno-free UC-MSC-EVs including enhanced cardiomyogenic and angiogenic potential impacting on target cells, which may be explained by elevated concentration of several pro-cardiogenic and pro-angiogenic microRNA (miRNAs) present in the EVs. Our data also suggest predominantly low immunogenic capacity of certain xeno-free UC-MSC-EVs reflected by their inhibitory effect on proliferation of immune cells in vitro. Summarizing, conscious selection of cell culture conditions is required to harvest UC-MSC-EVs with the optimal desired properties including enhanced cardiac and angiogenic capacity, suitable for tissue regeneration. Key message Type of xeno-free media influences biological properties of UC-MSCs in vitro.Certain xeno-free media promote proliferation and differentiation ability of UC-MSCs.EVs collected from xeno-free cultures of UC-MSCs are biologically active.Xeno-free UC-MSC-EVs enhance cardiac and angiogenic potential of target cells.Type of xeno-free media determines immunomodulatory effects mediated by UC-MSC-EVs. Electronic supplementary material The online version of this article (doi:10.1007/s00109-016-1471-7) contains supplementary material, which is available to authorized users.

The challenge arises among researchers when hair follicle stem cells (HFSCs) derived from a human hair follicle remain poorly expanded in defined culture medium. In this study, we isolated the HFSCs and they became confluent after 10 days of cultivation. Comparing the viability of HFSCs cultured in defined keratinocytes serum free medium (KSFM) in a coated plate and CnT07 medium in an uncoated plate, the number of HFSCs cultured in CnT07 was significantly higher at days 2, 4, 6 and 8 (P=0.004). The population doubling time of HFSCs was 21.48±0.44 hours in non-coated plates with CnT07 and 30.73±0.75 hours in coated plates with KSFM. Our primary HFSC cultures were positive for CD200 and K15 with brownish color. Flow cytometry analysis showed that the percentage of HFSCs expressing CD200 and K15 were 65.20±3.16 and 72.07±6.62 respectively. After reaching 100% confluence, the HFSCs were differentiated into an epidermal layer in vitro using CnT02-3D defined media. HFSCs were differentiated into an epidermal layer after 2 weeks of induction. Involucrin- and K6-positive cells were detected in the differentiated epidermal layer. This method is a simple technique for HFSC isolation and has a lower cost of processing and labor, and it represents a promising tool for skin tissue engineering.

Background Amnion-derived stem cells have been proposed for cell replacement therapy and tissue regeneration. An easily accessible cell source, the placenta, allows us to potentially establish a bio-bank of cells for immunotype matched clinical applications. Several xeno-free (XF) cryopreservation media are currently available for pluripotent stem cells, however, these media have not yet been evaluated for the cryopreservation of amnion-derived stem cells. Methods Human amniotic epithelial cells were collected using standard protocols, and stored at −160 °C in one of five commercially available media. Cells frozen in standard media containing fetal bovine serum served as controls. Cells were then thawed, and evaluated for viability, mitochondrial membrane stability, and senescence status. Quantitative real time PCR was utilized to assess for expression of stem cell genes, and flow cytometry was used to identify the stem cell surface markers. Results Cell recovery and repopulation assays indicated no significant difference between XF media versus standard cryopreservation medium. In addition, no impact was observed on the senescence status, the cytostructural or mitochondrial morphology between the tested cryopreservation media. Differences were observed on the expression of stem cell marker genes (OCT4, SOX2, and NANOG) and a cell surface marker (TRA1-60) following cryopreservation in different chemically defined XF media, however, these were not statistically significant. Conclusions Xeno-free cryopreservation of human amnion-derived stem cells is feasible and can be standardized to establish a bio-bank with human amnion-derived stem cells for future clinical application. Optimization of this media may allow for improved preservation of stem cell-like characteristics.

Summary Human embryonic stem cell (hESC)-derived retinal pigment epithelial (RPE) cells could replace lost tissue in geographic atrophy (GA) but efficacy has yet to be demonstrated in a large-eyed model. Also, production of hESC-RPE has not yet been achieved in a xeno-free and defined manner, which is critical for clinical compliance and reduced immunogenicity. Here we describe an effective differentiation methodology using human laminin-521 matrix with xeno-free and defined medium. Differentiated cells exhibited characteristics of native RPE including morphology, pigmentation, marker expression, monolayer integrity, and polarization together with phagocytic activity. Furthermore, we established a large-eyed GA model that allowed in vivo imaging of hESC-RPE and host retina. Cells transplanted in suspension showed long-term integration and formed polarized monolayers exhibiting phagocytic and photoreceptor rescue capacity. We have developed a xeno-free and defined hESC-RPE differentiation method and present evidence of functional integration of clinically compliant hESC-RPE in a large-eyed disease model., Highlights • Xeno-free and defined differentiation of hES-RPE cells using recombinant laminin-521 • Functional monolayer integration of hES-RPE cells in a novel large-eyed disease model • Rescue of photoreceptors from induced degeneration by transplanted hES-RPE cells, Lanner and colleagues present an effective xeno-free and defined hES-RPE cell differentiation methodology using a recombinant human laminin-521 matrix. They further present evidence of long-term functional integration and photoreceptor rescue by transplanted hES-RPE cells in a large-eyed disease model.

Traumatic brain injury (TBI) in humans can result in permanent tissue damage and has been linked to cognitive impairment that lasts years beyond the initial insult. Clinically effective treatment strategies have yet to be developed. Transplantation of human neural stem cells (hNSCs) has the potential to restore cognition lost due to injury, however, common methods for the generation of human embryonic stem cell (hESC) derived neural stem cells result in cells with potentially compromised safety profiles due to maintenance of cells in conditions containing non-human proteins (e.g. in bovine serum or on mouse fibroblast feeders) as well an increased risk of teratoma formation. Additionally, the vast majority of rodent TBI/hNSC studies to date have evaluated cognition only at early time points, typically less than 1 month post-injury and cell transplantation, and human cell engraftment and long-term survival in rodent models of TBI has been difficult to achieve due to host immunorejection of the transplanted human cells. To overcome these shortfalls, we have generated hNSCs in completely “Xeno-Free” (human only) culture conditions. Furthermore, we have enriched the hNSCs for the cell surface marker CD133 via magnetic sorting, which has led to an increase in the expansion rate and neuronal fate specification of the hNSCs in vitro, and we have confirmed neural lineage specificity and a lack of teratoma formation upon sorted hNSC transplantation into the immunodeficient NOD-scid mouse brain. We then developed a novel TBI xenotransplantation model that utilizes immunodeficient athymic nude (ATN) rats as the host recipient for the post-TBI transplantation of human embryonic stem cell (hESC) derived NSCs and have furthermore evaluated cognition in these animals at long-term (≥2 months) time points. We report that immunodeficient ATN rats demonstrate hippocampal-dependent spatial memory deficits at 2-3 months post-TBI, confirming that ATN rats recapitulate some of the cognitive deficits found in immunosufficient animal strains. hNSCs survived for at least 5 months post-transplantation and differentiated into cells from all three neural cell lineages. Furthermore, hNSC transplantation facilitated cognitive recovery after TBI, even in the absence of gross histological modulation of lesion or total spared hippocampal tissue volume. Importantly, we have found an overall increase in host hippocampal neuron survival in hNSC transplanted animals and demonstrate that a correlation exists between hippocampal neuron survival and cognitive performance. Together, these findings support the use of immunodeficient rodents in models of TBI that involve the transplantation of human cells, and suggest that hNSC transplantation may be a viable, long-term therapy to restore cognition after TBI.

Purpose Retinal pigment epithelium (RPE) dysfunction underlies the retinal degenerative process in age-related macular degeneration (AMD), and thus RPE cell replacement provides an optimal treatment target. We characterized longitudinally the efficacy of RPE cells derived under xeno-free conditions from clinical and xeno-free grade human embryonic stem cells (OpRegen) following transplantation into the subretinal space of Royal College of Surgeons (RCS) rats. Methods Postnatal (P) day 20 to 25 RCS rats (n = 242) received a single subretinal injection of 25,000 (low)-, 100,000 (mid)-, or 200,000 (high)-dose xeno-free RPE cells. BSS+ (balanced salt solution) (vehicle) and unoperated eyes served as controls. Optomotor tracking (OKT) behavior was used to quantify functional efficacy. Histology and immunohistochemistry were used to evaluate photoreceptor rescue and transplanted cell survival at 60, 100, 150, and 200 days of age. Results OKT was rescued in a dose-dependent manner. Outer nuclear layer (ONL) was significantly thicker in cell-treated eyes than controls up to P150. Transplanted RPE cells were identified in both the subretinal space and integrated into the host RPE monolayer in animals of all age groups, and often contained internalized photoreceptor outer segments. No pathology was observed. Conclusions OpRegen RPE cells survived, rescued visual function, preserved rod and cone photoreceptors long-term in the RCS rat. Thus, these data support the use of OpRegen RPE cells for the treatment of human RPE cell disorders including AMD. Translational relevance Our novel xeno-free RPE cells minimize concerns of animal derived contaminants while providing a promising prospective therapy to the diseased retina.

© 2014 Wiley Periodicals, Inc. Human embryonic stem cells (hESC) and induced pluripotent stem cells (hiPSC) can differentiate into many cell types and are important for regenerative medicine; however, further work is needed to reliably differentiate hESC and hiPSC into neural-restricted multipotent derivatives or specialized cell types under conditions that are free from animal products. Toward this goal, we tested the transition of hESC and hiPSC lines onto xeno-free (XF) / feeder-free conditions and evaluated XF substrate preference, pluripotency, and karyotype. Critically, XF transitioned H9 hESC, Shef4 hESC, and iPS6-9 retained pluripotency (Oct-4 and NANOG), proliferation (MKI67 and PCNA), and normal karyotype. Subsequently, XF transitioned hESC and hiPSC were induced with epidermal growth factor (EGF) and basic fibroblast growth factor (bFGF) to generate neuralized spheres containing primitive neural precursors, which could differentiate into astrocytes and neurons, but not oligoprogenitors. Further neuralization of spheres via LIF supplementation and attachment selection on CELLstart substrate generated adherent human neural stem cells (hNSC) with normal karyotype and high proliferation potential under XF conditions. Interestingly, adherent hNSC derived from H9, Shef4, and iPS6-9 differentiated into significant numbers of O4+ oligoprogenitors (~20-30%) with robust proliferation; however, very few GalC+ cells were observed (~2-4%), indicative of early oligodendrocytic lineage commitment. Overall, these data demonstrate the transition of multiple hESC and hiPSC lines onto XF substrate and media conditions, and a reproducible neuralization method that generated neural derivatives with multipotent cell fate potential and normal karyotype.

Age-related macular degeneration (AMD) is the leading cause of blindness in the elderly and is characterized by the death of the retinal pigmented epithelium (RPE), the cell layer located behind the retina. The RPE maintains the health of the primary cells responsible for vision, the photoreceptors. As AMD progresses, the RPE degrades, which causes the death of the photoreceptors and a debilitating loss of sight. Human embryonic stem cells (hESCs) can generate a limitless source of RPE for cellular therapies, therefore efforts to derive RPE from human embryonic stem cells (hESCs) to graft into AMD patients are under development. This thesis addresses two of the obstacles that hinder the production of these therapeutical cells. First, to manufacture cells for clinical use, it is desirable for procedures to be performed under defined conditions free of non-human reagents (xeno-free). Therefore, a novel, biomimetic, xeno-free RGD-containing copolymer designed for cell culture was investigated and found to support healthy hESC cultures and permit their differentiation into functional RPE. These stem cells and hESC-RPE demonstrate similar gene expression, protein localization and phagocytic function as cultures grown on a xenogeneic substrate. The second obstacle pertains to the intense time requirement needed to differentiate hESCs into RPE. Therefore, we recapitulated the signaling events during early embryonic development to expedite the production of hESC-RPE from 1-3 months to 14 days. However, this protocol employs full length recombinant growth factors and animal derivatives. These components represent a challenge in employing these cells as therapies due to the risk of exposure to non-human immunogens. Preliminary work to replace the xenogeneic substrate and recombinant growth factors with small molecules is presented. These pilot studies demonstrate that a xeno-free substrate and dual-Smad inhibition via small molecules can rapidly and efficiently differentiate hESCs into a population of cells expressing a pigmentation marker. These studies contribute to the development of defined, xeno-free methods for cellular manufacture which can further the translation of stem cell therapies to the clinic.

Introduction Adipose tissue is an attractive and abundant source of multipotent stem cells. Human adipose stem cells (ASCs) have shown to have therapeutic relevancy in diverse clinical applications. Nevertheless, expansion of ASCs is often necessary before performing clinical studies. Standard in vitro cell-culture techniques use animal-derived reagents that should be avoided in clinical use because of safety issues. Therefore, xeno- and serum-free (XF/SF) reagents are highly desirable for enhancing the safety and quality of the transplanted ASCs. Methods In the current study, animal component-free isolation and cell-expansion protocols were developed for ASCs. StemPro MSC SFM XF medium with either CELLstart™ CTS™ coating or Coating Matrix Kit were tested for their ability to support XF/SF growth. Basic stem-cell characteristics such as immunophenotype (CD3, CD11a, CD14, CD19, CD34, CD45RO, CD54, CD73, CD80, CD86, CD90, CD105, HLA-DR), proliferation, and differentiation potential were assessed in XF/SF conditions and compared with human serum (HS) or traditionally used fetal bovine serum (FBS) cultures. Results ASCs cultured in XF/SF conditions had significantly higher proliferation rates compared with HS/FBS cultures. Characteristic immunophenotypes of ASCs were maintained in every condition; however, cells expanded in XF/SF conditions showed significantly lower expression of CD54 (intercellular adhesion molecule 1, ICAM-1) at low passage number. Further, multilineage differentiation potential of ASCs was maintained in every culture condition. Conclusions Our findings demonstrated that the novel XF/SF conditions maintained the basic stem cell features of ASCs and the animal-free workflow followed in this study has great potential in clinical cell therapies. BioMed Central open access

Ihmisen alkion kantasoluista (hESC) ja geneettisesti pluripotenttiin tilaan uudelleen ohjelmoiduista indusoiduista kantasoluista (hiPSC) voidaan tuottaa rajattomasti erilaisia solutyyppejä kliinisiin kantasoluhoitoihin. Yksi lupaavimmista käyttökohteista on sokeutumiseen johtavien verkkokalvon rappeumasairauksien hoito. HESC soluista erilaistettuja verkkokalvon pigmenttiepiteelisoluja (RPE) testataankin parhaillaan silmänpohjan ikärappeuman hoitoon ensimmäisissä kliinisissä kokeissa. Tällä hetkellä yleisesti käytössä olevat kantasolujen laboratorioviljelyolosuhteet sisältävät tuntemattomia ja eläinperäisiä ainesosia, jotka altistavat kantasolut eläinpatogeeneille ja immunogeenisille eläinperäisille molekyyleille. Kliiniseen käyttöön tarkoitetut kantasolut tulisi eristää, viljellä ja erilaistaa Good Manufacturing Practise (GMP)-laatustandardiston mukaisesti, ilman eläinperäisiä materiaaleja. Myös kantasolujen erilaistumista verkkokalvon soluiksi ohjaavat tekijät tunnetaan heikosti ja tehokkaampien erilaistusmenetelmien kehittäminen on tärkeää. Tässä väitöskirjatyössä testattiin ja optimoitiin eläinperäisiä ainesosia sisältämättömiä ja GMP-soveltuvia erilaistumattomien kantasolujen viljelyliuoksia, tukisoluja ja tukisoluttomia viljelymenetelmiä. Lisäksi tutkittiin tukisolujen merkitystä erilaistumattomien kantasolujen viljelyssä ja erilaistamisessa RPE-soluiksi. Tutkimuksen tuloksena perustettiin eläinkomponentittomia tukisolulinjoja ja tukisoluille optimoitiin kliiniseen käyttöön soveltuvat viljelymenetelmät. Näitä prosesseja voidaan tulevaisuudessa käyttää myös kliiniseen käyttöön tarkoitettujen hiPSC-solujen tuottamiseen ihon sidekudossoluista. Kantasoluja tukevien ja tukemattomien tukisolujen todettiin eroavan soluväliaineen proteiinien, etenkin laminiinien, ja niiden integriinireseptorien tuotossa. Laminiini-511 tunnistettiin kantasolujen kasvua ylläpitäväksi tukisolujen erittämäksi laminiiniksi. Tutkimuksen tuloksena identifioitiin myös uusia mahdollisia kantasolujen kasvua ylläpitäviä tukisolujen tuottamia tekijöitä. Tukisolujen erittämien tekijöiden todettiin myös edistävän kantasolujen erilaistumista RPE-soluiksi, minkä todettiin osittain johtuvan aktiviini A -kasvutekijän erityksestä. Lisäämällä aktiviini A -kasvutekijää viljelyliuokseen, saatiin erilaistumista merkittävästi tehostettua. Tämän tutkimuksen tulokset ovat osaltaan myötävaikuttaneet kantasolujen kliiniseen käyttöön soveltuvien viljelymenetelmien kehittämiseen ja laajentaneet näkemystä kantasolujen erilaistumattomuuden säätelystä ja erilaistumisesta RPE-soluiksi. The establishment of human pluripotent stem cells (hPSCs) has raised great excitement for their prospective use in the field of regenerative medicine. Human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) can self-renew indefinitely and generate virtually any differentiated cell type, thus serving as an unlimited cell source for cell replacement therapies. One promising clinical application of hPSC-based cell therapies is the treatment of retinal degenerative diseases such as age-related macular degeneration (AMD), which is the leading cause of blindness among the elderly in the western world. Phase one clinical trial on the treatment of AMD with retinal pigment epithelial (RPE) cells differentiated from hESCs was recently commenced. The standard in vitro culture conditions for undifferentiated hPSCs contain undefined and xenogeneic compounds, exposing the cells to infectious and immunogenic molecules. Although hESCs cultured under such conditions have recently been approved for clinical trials, the use of xenogeneic cell culture material is highly undesirable. Ideally, clinical grade hPSC lines are established using xeno-free culture protocols and according to Good Manufacturing Practice (GMP)-quality requirements. Also, the molecular mechanisms controlling stem cell differentiation to the eye field and further to the RPE fate are poorly understood. Consequently more efficient and defined differentiation protocols are required. This thesis aimed at testing and optimising xeno-free and GMP-compatible culture protocols for hESCs regarding hESC culture media, feeder-independent culture methods, and feeder cells. Further, the feeder cell contribution to maintenance of pluripotent hESC status and the induction of RPE differentiation were studied. As a result, human serum (HS) supplemented hESC culture medium was found to suboptimally support xeno-free hESC culture on human feeder cells. Commercial feeder-independent culture methods were found to support long-term hESC culture while feeder-independent and xeno-free methods published by academic research groups were found to be unsupportive. Xeno-free, defined media as well as matrices based on human extracellular matrix (ECM) proteins are today on the market for both feeder-dependent and feeder-independent hPSC culture. Xeno-free human feeder cells were established and clinical grade culture protocols for the feeder cells were optimized. These processes could in the future also serve in the establishment of clinical grade hiPSC lines from dermal tissue. Human dermal fibroblast (hDF) feeder cells were found unsupportive of hESCs while commercial human foreskin fibroblasts (hFF) supported long-term hESC propagation in xeno-free culture system. The feeder cells were found to differ significantly in their ECM protein, especially laminin, and corresponding integrin receptor expression. Laminin-511 was found to contribute to hESC maintenance in feeder-dependent culture as laminin-511 was secreted only by hESC supportive feeder cells. Other factors with a possible role in the maintenance of pluripotency were identified. Feeder cell conditioned media were found to enhance RPE differentiation of hESCs and hiPSCs. Higher activin A secretion by mouse embryonic fibroblast (mEF) feeder cells was hypothesized to be an inductive agent and a corresponding level of activin A significantly augmented hESC-RPE differentiation. The results of this thesis have contributed to the translation of hPSC research to the clinic by developing suitable culture conditions and provided insights into the mechanisms of maintenance of hESC pluripotency and induction of hPSC-RPE differentiation.

Human adipose stromal/stem cells (ASCs) are an abundant, readily available population of adult stem cells that reside in adipose tissue and that have a great potential utility for tissue engineering and regenerative medicine therapeutic applications. Several preclinical studies have shown that ASCs have therapeutic applicability, but a standardized isolation and expansion methodology for clinical cell therapy has yet to be established. ASC are typically isolated and expanded using reagents with xenogenic components and this may pose certain risks and safety issues, such as exposure to infectious agents and immune reactions, creating further obstacles to the translation of ASC based therapies to clinical scenarios. The objective of this study was to determine the suitability and efficacy of various alternative enzymatic products, CLS1 (Worthington), CLSAFA (Worthington), NB4 (SERVA), and Liberase (Roche), for the digestion of adipose tissue and subsequent isolation of ASCs, assessing cells functionality concerning their proliferation and differentiation ability. Results show that there are no statistically significant differences on yield and proliferation of cells isolated after enzymatic digestion with any of the studied products. The differentiation potential of the cells was not affected and cell surface markers expression was similar amongst all products. We concluded that clinical grade products can replace current research grade products effectively in our cell isolation protocols without any negative effect in the yield or function of human ASCs., P.P.C. acknowledges the Portuguese Foundation for Science and Technology (FCT) for a Ph.D. scholarship (SFRH/BD/44128/2008) and financial support of the project MIT/ECE/0047/2009. The authors thank the following individuals for their assistance: Ana Frias for her help with the flow cytometry; Alessandra Zonari for her help preparing histological samples. The authors would also like to thank Worthington Biochemical Corp, USA; SERVA Electrophoresis GmbH, Germany; and Roche Applied Sciences, Portugal, for kindly providing samples of their products for this study.

Analysis of cryopreserved peripheral mononuclear cells (PBMC) is important for evaluating new vaccines in immune based therapies and in pathogenesis studies. To ensure comparable assay results from different laboratories and points of time, collaborative research in multicenter trials needs reliable and reproducible cryopreservation protocols that maintain cell viability and functionality. Current cryomedia consist largely of fetal bovine serum (FBS), a natural mix of growth factors, cytokines, and undefined compounds. Standardized procedures are not possible, as FBS can affect the antigen-specific T-cell response, the most important parameter in functionality assays. Also, worldwide sample exchange is complicated by the strict import restrictions on FBS, because of transfection risk.After establishing a serum-free cryopreservation protocol that maintains cell viability, recovery and antigen-specific T-cell response of PBMC comparably to FBS-based cryomedia (Germann et al., 2011), the aim of this study was the complete avoidance of animal proteins and products in combination with efficient cryopreservation.As long-term stability of the cryopreservation process is crucial for retrospective evaluation of samples at different points of time, PBMC were analyzed after storage for maximal four weeks and again after approximately six months.The cryopreservation efficiency of the protein-free and fully chemically defined cryomedium was comparable to FBS-medium after storage for few weeks and several months. Directly after thawing, this medium yielded viabilities over 97% and recovery values over 84%. Also, the specific T-cell functionality was preserved. Additionally, short-term and six month cryopreservation gave comparable results. The fully chemically defined medium presented here will increase standardization and reproducibility of analysis in multicenter-studies or in retrospective evaluation.

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Introduction In the last decades, adipose tissue (AT) has attracted attention of the scientific and industrial communities for its high therapeutic potential. When focusing on the clinical translation of such type of cells, aspects such as Good Manufacturing Practices (GMP) compliance, scalability, reproducibility, and protocol validation must be ensured. Manufacturing of cells must warrant high quality concerning cell viability, cell yield immunophenotype, and absence of any microbial or pathological contamination. This project focused on the development of a complete manufacturing and cryopreservation process of therapeutic cells for human clinical delivery, ensuring all parameters and characteristics listed above. Materials and Methods The new protocol developed is particularly compact and ensures economies of scale when increasing the volume of processed AT, which reduces manufacturing cost. In this project 100-200g AT/human donor, obtained from liposuction or lipectomy procedures (n=15) were processed under GMP conditions. Stromal vascular fraction (SVF) isolation and cryopreservation was performed using xeno-free and GMP grade reagents. Cell number and viability, before and after cryopreservation, was determined by trypan blue exclusion dye by three operators. Immunophenotype of SVF subpopulations was evaluated by flow cytometry (FACSCanto and FACSDiva software) for the expression of mesenchymal, endothelial, hematopoietic and pericytic markers: CD31, CD34, CD45, CD73, CD90, CD105, CD146 and HLA-DR. Mesenchymal trilineage differentiation was performed to demonstrate potential of the isolated cells. Results The developed xeno-free cGMP procedure for isolation of clinical grade SVF cells resulted in a cell yield of 2.4x105 ±0.9 cells/g AT, and cell viability of 85.4%±7.1, which demonstrated differentiation into mesodermal lineages. After xeno-free cryopreservation cell viability was 80.0%±10.1. Regarding immunophenotype of SVF subpopulations (Fig.1), when considering mesenchymal markers co-expressed with CD341, 12%±5 of the cells presented the CD90+CD34+CD73+CD105+ phenotype, while 1.3%±0.8 of cells expressed concomitant mesenchymal markers in the absence of CD34. Regarding endothelial markers, 15%±6 of the cells are CD45-CD34+CD31+. Preliminary analysis of samples demonstrated no significant difference of phenotypic expression before and after xeno-free cryopreservation. Discussion and Conclusions To the best of our knowledge this project is pioneer in providing clinical grade cryopreserved hSVF, ensuring extremely high viability post-thaw, and maintenance of immunophenotypic characteristics of its sub-populations. This achievement is of great relevance for therapeutic use of adipose tissue regenerative cells. References 1 Scherberich A. et al. World J Stem Cells 5, 1, 2013; 2 Bourin P. et al. Cytotherapy. 0, 1, 2013.

Human embryonic stem cell-derived retinal pigment epithelial cells (hESC-RPE) are a promising cell source to treat age-related macular degeneration (AMD). Despite several ongoing clinical studies, a detailed mapping of transient cellular states during invitro differentiation has not been performed. Here, we conduct single-cell transcriptomic profiling of an hESC-RPE differentiation protocol that has been developed for clinical use. Differentiation progressed through a culture diversification recapitulating early embryonic development, whereby cells rapidly acquired a rostral embryo patterning signature before converging toward the RPE lineage. At intermediate steps, we identified and examined the potency of an NCAM1+ retinal progenitor population and showed the ability of the protocol to suppress non-RPE fates. We demonstrated that the method produces a pure RPE pool capable of maturing further after subretinal transplantation in a large-eyed animal model. Our evaluation of hESC-RPE differentiation supports the development of safe and efficient pluripotent stem cell-based therapies for AMD.