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The effect of systemic administration of conditioned media (CM) and extracellular vesicles (EVs) from both human and mouse bone marrow‐derived mesenchymal stromal cells (MSCs) in a murine model of severe experimental asthma was studied. Systemic administration of CM and the isolated EVs significantly ameliorated the challenge‐induced increases in airway hyperreactivity, lung inflammation, and antigen‐specific CD4 T‐cell phenotype. EVs isolated from human MSCs generally were more potent than those from mouse MSCs. An increasing number of studies demonstrate that administration of either conditioned media (CM) or extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) derived from bone marrow and other sources are as effective as the MSCs themselves in mitigating inflammation and injury. The goal of the current study was to determine whether xenogeneic administration of CM or EVs from human bone marrow‐derived MSCs would be effective in a model of mixed Th2/Th17, neutrophilic‐mediated allergic airway inflammation, reflective of severe refractory asthma, induced by repeated mucosal exposure to Aspergillus hyphal extract (AHE) in immunocompetent C57Bl/6 mice. Systemic administration of both CM and EVs isolated from human and murine MSCs, but not human lung fibroblasts, at the onset of antigen challenge in previously sensitized mice significantly ameliorated the AHE‐provoked increases in airway hyperreactivity (AHR), lung inflammation, and the antigen‐specific CD4 T‐cell Th2 and Th17 phenotype. Notably, both CM and EVs from human MSCs (hMSCs) were generally more potent than those from mouse MSCs (mMSCs) in most of the outcome measures. The weak cross‐linking agent 1‐ethyl‐3‐[3‐dimethylaminopropyl]carbodiimide hydrochloride was found to inhibit release of both soluble mediators and EVs, fully negating effects of systemically administered hMSCs but only partly inhibited the ameliorating effects of mMSCs. These results demonstrate potent xenogeneic effects of CM and EVs from hMSCs in an immunocompetent mouse model of allergic airway inflammation and they also show differences in mechanisms of action of hMSCs versus mMSCs to mitigate AHR and lung inflammation in this model. Significance There is a growing experience demonstrating benefit of mesenchymal stromal cell (MSC)‐based cell therapies in preclinical models of asthma. In the current study, conditioned media (CM) and, in particular, the extracellular vesicle fraction obtained from the CM were as potent as the MSCs themselves in mitigating Th2/Th17‐mediated allergic airway inflammation in a mouse model of severe refractory clinical asthma. Moreover, human MSC CM and extracellular vesicles were effective in this immunocompetent mouse model. These data add to a growing scientific basis for initiating clinical trials of MSCs or extracellular vesicles derived from MSCs in severe refractory asthma and provide further insight into the mechanisms by which the MSCs may ameliorate the asthma.

This study used a two‐stage culture system to efficiently produce natural killer (NK) cells from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) in the absence of cell sorting and without need for xenogeneic stromal cells. Although different hESC and iPSC lines had varying efficiencies in hematopoietic development, all cell lines tested could produce functional NK cells. This improved method to develop NK cells from human pluripotent stem cells provides a system for clinical‐scale expansion of antitumor lymphocytes and a genetically amenable platform to study human NK cell development. Adoptive transfer of antitumor lymphocytes has gained intense interest in the field of cancer therapeutics over the past two decades. Human natural killer (NK) cells are a promising source of lymphocytes for anticancer immunotherapy. NK cells are part of the innate immune system and exhibit potent antitumor activity without need for human leukocyte antigen matching and without prior antigen exposure. Moreover, the derivation of NK cells from pluripotent stem cells could provide an unlimited source of lymphocytes for off‐the‐shelf therapy. To date, most studies on hematopoietic cell development from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) have used incompletely defined conditions and been on a limited scale. Here, we have used a two‐stage culture system to efficiently produce NK cells from hESCs and iPSCs in the absence of cell sorting and without need for xenogeneic stromal cells. This novel combination of embryoid body formation using defined conditions and membrane‐bound interleukin 21‐expressing artificial antigen‐presenting cells allows production of mature and functional NK cells from several different hESC and iPSC lines. Although different hESC and iPSC lines had varying efficiencies in hematopoietic development, all cell lines tested could produce functional NK cells. These methods can be used to generate enough cytotoxic NK cells to treat a single patient from fewer than 250,000 input hESCs/iPSCs. Additionally, this strategy provides a genetically amenable platform to study normal NK cell development and education in vitro.

A scalable, robust, and integrated differentiation platform for large‐scale production of human pluripotent stem cell‐cardiomyocyte (hPSC‐CM) in a stirred suspension bioreactor as a single‐unit operation was developed. This platform could become a valuable tool for mass production of functional hPSC‐CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications including drug discovery and toxicity assays. Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs), in conjunction with the promising outcomes from preclinical and clinical studies, have raised new hopes for cardiac cell therapy. We report the development of a scalable, robust, and integrated differentiation platform for large‐scale production of hPSC‐CM aggregates in a stirred suspension bioreactor as a single‐unit operation. Precise modulation of the differentiation process by small molecule activation of WNT signaling, followed by inactivation of transforming growth factor‐β and WNT signaling and activation of sonic hedgehog signaling in hPSCs as size‐controlled aggregates led to the generation of approximately 100% beating CM spheroids containing virtually pure (∼90%) CMs in 10 days. Moreover, the developed differentiation strategy was universal, as demonstrated by testing multiple hPSC lines (5 human embryonic stem cell and 4 human inducible PSC lines) without cell sorting or selection. The produced hPSC‐CMs successfully expressed canonical lineage‐specific markers and showed high functionality, as demonstrated by microelectrode array and electrophysiology tests. This robust and universal platform could become a valuable tool for the mass production of functional hPSC‐CMs as a prerequisite for realizing their promising potential for therapeutic and industrial applications, including drug discovery and toxicity assays. Significance Recent advances in the generation of cardiomyocytes (CMs) from human pluripotent stem cells (hPSCs) and the development of novel cell therapy strategies using hPSC‐CMs (e.g., cardiac patches) in conjunction with promising preclinical and clinical studies, have raised new hopes for patients with end‐stage cardiovascular disease, which remains the leading cause of morbidity and mortality globally. In this study, a simplified, scalable, robust, and integrated differentiation platform was developed to generate clinical grade hPSC‐CMs as cell aggregates under chemically defined culture conditions. This approach resulted in approximately 100% beating CM spheroids with virtually pure (∼90%) functional cardiomyocytes in 10 days from multiple hPSC lines. This universal and robust bioprocessing platform can provide sufficient numbers of hPSC‐CMs for companies developing regenerative medicine technologies to rescue, replace, and help repair damaged heart tissues and for pharmaceutical companies developing advanced biologics and drugs for regeneration of lost heart tissue using high‐throughput technologies. It is believed that this technology can expedite clinical progress in these areas to achieve a meaningful impact on improving clinical outcomes, cost of care, and quality of life for those patients disabled and experiencing heart disease.

Although protocols to generate retinal pigmented epithelium (RPE) from human pluripotent stem cells have become more efficient since the first report in 2004, they are still time‐consuming and relatively inefficient. This work describes an experiment wherein the addition of defined factors at specific times led to conversion of approximately 80% of the cells to an RPE phenotype in only 14 days. This protocol should be useful for rapidly generating RPE for transplantation as well as for studying RPE development in vitro. Controlling the differentiation of human pluripotent stem cells is the goal of many laboratories, both to study normal human development and to generate cells for transplantation. One important cell type under investigation is the retinal pigmented epithelium (RPE). Age‐related macular degeneration (AMD), the leading cause of blindness in the Western world, is caused by dysfunction and death of the RPE. Currently, RPE derived from human embryonic stem cells are in clinical trials for the treatment of AMD. Although protocols to generate RPE from human pluripotent stem cells have become more efficient since the first report in 2004, they are still time‐consuming and relatively inefficient. We have found that the addition of defined factors at specific times leads to conversion of approximately 80% of the cells to an RPE phenotype in only 14 days. This protocol should be useful for rapidly generating RPE for transplantation as well as for studying RPE development in vitro.

The ability to generate spiral ganglion neurons (SGNs) from stem cells is a necessary prerequisite for development of cell‐replacement therapies for sensorineural hearing loss. We present a protocol that directs human embryonic stem cells (hESCs) toward a purified population of otic neuronal progenitors (ONPs) and SGN‐like cells. Between 82% and 95% of these cells express SGN molecular markers, they preferentially extend neurites to the cochlear nucleus rather than nonauditory nuclei, and they generate action potentials. The protocol follows an in vitro stepwise recapitulation of developmental events inherent to normal differentiation of hESCs into SGNs, resulting in efficient sequential generation of nonneuronal ectoderm, preplacodal ectoderm, early prosensory ONPs, late ONPs, and cells with cellular and molecular characteristics of human SGNs. We thus describe the sequential signaling pathways that generate the early and later lineage species in the human SGN lineage, thereby better describing key developmental processes. The results indicate that our protocol generates cells that closely replicate the phenotypic characteristics of human SGNs, advancing the process of guiding hESCs to states serving inner‐ear cell‐replacement therapies and possible next‐generation hybrid auditory prostheses. © Stem Cells Translational Medicine 2017;6:923–936

Recent studies have shown evidence for the functional integration of human pluripotent stem cell (hPSC)‐derived ventral midbrain dopamine (vmDA) neurons in animal models of Parkinson’s disease. Although these cells present a sustainable alternative to fetal mesencephalic grafts, a number of hurdles require attention prior to clinical translation. These include the persistent use of xenogeneic reagents and challenges associated with scalability and storage of differentiated cells. In this study, we describe the first fully defined feeder‐ and xenogeneic‐free protocol for the generation of vmDA neurons from hPSCs and utilize two novel reporter knock‐in lines (LMX1A‐eGFP and PITX3‐eGFP) for in‐depth in vitro and in vivo tracking. Across multiple embryonic and induced hPSC lines, this “next generation” protocol consistently increases both the yield and proportion of vmDA neural progenitors (OTX2/FOXA2/LMX1A) and neurons (FOXA2/TH/PITX3) that display classical vmDA metabolic and electrophysiological properties. We identify the mechanism underlying these improvements and demonstrate clinical applicability with the first report of scalability and cryopreservation of bona fide vmDA progenitors at a time amenable to transplantation. Finally, transplantation of xeno‐free vmDA progenitors from LMX1A‐ and PITX3‐eGFP reporter lines into Parkinsonian rodents demonstrates improved engraftment outcomes and restoration of motor deficits. These findings provide important and necessary advancements for the translation of hPSC‐derived neurons into the clinic. Stem Cells Translational Medicine 2017;6:937–948

Despite considerable progress within the cell therapy industry, unmet bioprocessing and logistical challenges associated with the storage and distribution of cells between sites of manufacture and the clinic exist. We examined whether hypothermic (4°C–23°C) preservation of human adipose‐derived stem cells could be improved through their encapsulation in 1.2% calcium alginate. Alginate encapsulation improved the recovery of viable cells after 72 hours of storage. Viable cell recovery was highly temperature‐dependent, with an optimum temperature of 15°C. At this temperature, alginate encapsulation preserved the ability for recovered cells to attach to tissue culture plastic on rewarming, further increasing its effect on total cell recovery. On attachment, the cells were phenotypically normal, displayed normal growth kinetics, and maintained their capacity for trilineage differentiation. The number of cells encapsulated (up to 2 × 106 cells per milliliter) did not affect viable cell recovery nor did storage of encapsulated cells in a xeno‐free, serum‐free,current Good Manufacturing Practice‐grade medium. We present a simple, low‐cost system capable of enhancing the preservation of human adipose‐derived stem cells stored at hypothermic temperatures, while maintaining their normal function. The storage of cells in this manner has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state. Significance Despite considerable advancement in the clinical application of cell‐based therapies, major logistical challenges exist throughout the cell therapy supply chain associated with the storage and distribution of cells between the sites of manufacture and the clinic. A simple, low‐cost system capable of preserving the viability and functionality of human adipose‐derived stem cells (a cell with substantial clinical interest) at hypothermic temperatures (0°C–32°C) is presented. Such a system has considerable potential for extending the shelf life of cell therapy products at multiple stages throughout the cell therapy supply chain. A simple, low‐cost system to enhance the preservation of human adipose‐derived stem cells stored at hypothermic temperatures, while maintaining their normal function, is presented. This system has great potential for extending the time windows for quality assurance and efficacy testing, distribution between the sites of manufacture and the clinic, and reducing the wastage associated with the limited shelf life of cells stored in their liquid state.

This study compares maintenance of human embryonic stem cells (hESCs) and their differentiation into retinal pigmented epithelial cells (RPE) using Matrigel, Synthemax‐R, and Synthemax II‐SC. Expression of RPE‐specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium‐derived factor. Results suggest that Synthemax II‐SC is a suitable substrate for hESC culture and the xeno‐free derivation of RPE for cellular therapies. Age‐related macular degeneration (AMD), a leading cause of blindness, is characterized by the death of the retinal pigmented epithelium (RPE), which is a monolayer posterior to the retina that supports the photoreceptors. Human embryonic stem cells (hESCs) can generate an unlimited source of RPE for cellular therapies, and clinical trials have been initiated. However, protocols for RPE derivation using defined conditions free of nonhuman derivatives (xeno‐free) are preferred for clinical translation. This avoids exposing AMD patients to animal‐derived products, which could incite an immune response. In this study, we investigated the maintenance of hESCs and their differentiation into RPE using Synthemax II‐SC, which is a novel, synthetic animal‐derived component‐free, RGD peptide‐containing copolymer compliant with good manufacturing practices designed for xeno‐free stem cell culture. Cells on Synthemax II‐SC were compared with cultures grown with xenogeneic and xeno‐free control substrates. This report demonstrates that Synthemax II‐SC supports long‐term culture of H9 and H14 hESC lines and permits efficient differentiation of hESCs into functional RPE. Expression of RPE‐specific markers was assessed by flow cytometry, quantitative polymerase chain reaction, and immunocytochemistry, and RPE function was determined by phagocytosis of rod outer segments and secretion of pigment epithelium‐derived factor. Both hESCs and hESC‐RPE maintained normal karyotypes after long‐term culture on Synthemax II‐SC. Furthermore, RPE generated on Synthemax II‐SC are functional when seeded onto parylene‐C scaffolds designed for clinical use. These experiments suggest that Synthemax II‐SC is a suitable, defined substrate for hESC culture and the xeno‐free derivation of RPE for cellular therapies.

Schwann cells (SCs) play pivotal roles in the maintenance and regeneration of the peripheral nervous system. Although transplantation of SCs enhances repair of experimentally damaged peripheral and central nerve tissues, it is difficult to prepare a sufficient number of functional SCs for transplantation therapy without causing adverse events for the donor. Here, we generated functional SCs by somatic cell reprogramming procedures and demonstrated their capability to promote peripheral nerve regeneration. Normal human fibroblasts were phenotypically converted into SCs by transducing SOX10 and Krox20 genes followed by culturing for 10 days resulting in approximately 43% directly converted Schwann cells (dSCs). The dSCs expressed SC‐specific proteins, secreted neurotrophic factors, and induced neuronal cells to extend neurites. The dSCs also displayed myelin‐forming capability both in vitro and in vivo. Moreover, transplantation of the dSCs into the transected sciatic nerve in mice resulted in significantly accelerated regeneration of the nerve and in improved motor function at a level comparable to that with transplantation of the SCs obtained from a peripheral nerve. The dSCs induced by our procedure may be applicable for novel regeneration therapy for not only peripheral nerve injury but also for central nerve damage and for neurodegenerative disorders related to SC dysfunction. Stem Cells Translational Medicine 2017;6:1207–1216

Neural differentiation of human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) can produce a valuable and robust source of human neural cell subtypes, holding great promise for the study of neurogenesis and development, and for treating neurological diseases. However, current hESCs and hiPSCs neural differentiation protocols require either animal factors or embryoid body formation, which decreases efficiency and yield, and strongly limits medical applications. Here we develop a simple, animal‐free protocol for neural conversion of both hESCs and hiPSCs in adherent culture conditions. A simple medium formula including insulin induces the direct conversion of >98% of hESCs and hiPSCs into expandable, transplantable, and functional neural progenitors with neural rosette characteristics. Further differentiation of neural progenitors into dopaminergic and spinal motoneurons as well as astrocytes and oligodendrocytes indicates that these neural progenitors retain responsiveness to instructive cues revealing the robust applicability of the protocol in the treatment of different neurodegenerative diseases. The fact that this protocol includes animal‐free medium and human extracellular matrix components avoiding embryoid bodies makes this protocol suitable for the use in clinic. Stem Cells Translational Medicine 2017;6:1217–1226