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Gene therapy, cell therapy, and tissue engineering have the potential to revolutionize the treatment of disease and injury. Attaining marketing authorization for such advanced therapy medicinal products (ATMPs) requires a rigorous scientific evaluation by the European Medicines Agency—authorization is only granted if the product can fulfil stringent requirements for quality, safety, and efficacy. However, many ATMPs are being provided to patients under alternative means, such as “hospital exemption” schemes. Holoclar (ex vivo expanded autologous human corneal epithelial cells containing stem cells), a novel treatment for eye burns, is one of the few ATMPs to have been granted marketing authorization and is the first containing stem cells. This review highlights the differences in standards between an authorized and unauthorized medicinal product, and specifically discusses how the manufacture of Holoclar had to be updated to achieve authorization. The result is that patients will have access to a therapy that is manufactured to high commercial standards, and is supported by robust clinical safety and efficacy data. Stem Cells Translational Medicine 2018;7:146–154 The role of clonogenic keratinocytes in generation and renewal of the corneal epithelium. The holoclone differentiation process from highly proliferative self‐renewing holoclones to transiently amplifying cells (meroclones and paraclones). A confocal microscopy image of holoclone stem cells is on the left showing high expression of ΔNp63α, an isoform of the p63 transcription factor.

Mesenchymal stem cells (MSCs) are being exploited in regenerative medicine due to their tri‐lineage differentiation and immunomodulation activity. Currently, there are two major challenges when directing the differentiation of MSCs for therapeutic applications. First, chemical and growth factor strategies to direct osteogenesis in vivo lack specificity for targeted delivery with desired effects. Second, MSC differentiation by gene therapy is difficult as transfection with existing approaches is clinically impractical (viral transfection) or have low efficacy (lipid‐mediated transfection). These challenges can be avoided by directly delivering nonvirally derived recombinant protein transcription factors with the glycosaminoglycan‐binding enhanced transduction (GET) delivery system (P21 and 8R peptides). We used the osteogenic master regulator, RUNX2 as a programming factor due to its stage‐specific role in osteochondral differentiation pathways. Herein, we engineered GET‐fusion proteins and compared sequential osteogenic changes in MSCs, induced by exposure to GET fusion proteins or conventional stimulation methods (dexamethasone and Bone morphogenetic protein 2). By assessing loss of stem cell‐surface markers, upregulation of osteogenic genes and matrix mineralization, we demonstrate that GET‐RUNX2 efficiently transduces MSCs and triggers osteogenesis by enhancing target gene expression directly. The high transduction efficiency of GET system holds great promise for stem cell therapies by allowing reproducible transcriptional control in stem cells, potentially bypassing problems observed with high‐concentration growth‐factor or pleiotropic steroid therapies. Stem Cells Translational Medicine 2017;6:2146–2159 Many regenerative medicine approaches employ the use of mesenchymal stem cells (MSCs) as they can be obtained directly from the patient from a number of tissues, can be expanded in culture, and have been shown to have positive clinical outcomes in a number of trials. These cells are multipotent meaning they have the ability to become different tissue‐type cells (fat, bone, cartilage) with a predisposition to convert into specific tissue types (differentiate) depending on the source tissue from which they were first isolated. Methods to control or change this predisposition will be key to exploiting them to repair tissue in cell therapies. Here, we describe a method to program the gene expression of MSCs to differentiate them efficiently into bone cells. Importantly this technique can overcome the predisposition to become alternatives (such as cartilage) directly at the level of gene expression. Our technology is based upon delivering a recombinant transcription factor protein (RUNX2) which does not genetically modify cells unlike gene therapy. This can now be exploited for programming MSCs when developing strategies for repairing bone trauma and disorders. 1. Schematic representation of directed osteogenesis of human MSCs using P21‐RUNX2‐8R. 2. Fluorescent images of human MSCs showing efficient delivery of P21‐RUNX2‐8R (Scale bar 50 μm). 3. Dual luciferase assay showing transcriptional activity of transduced P21‐RUNX28R. 4. Expression of osteogenic markers post transduction of P21‐RUNX2‐8R (Scale bar 20 μm). 5. Alizarin Red staining showing enhanced matrix mineralization using P21‐RUNX2‐8R (×1.5 magnification).

The ex vivo generation of human red blood cells on a large scale from hematopoietic stem and progenitor cells has been considered as a potential method to overcome blood supply shortages. Here, we report that functional human erythrocytes can be efficiently produced from cord blood (CB) CD34+ cells using a bottle turning device culture system. Safety and efficiency studies were performed in murine and nonhuman primate (NHP) models. With the selected optimized culture conditions, one human CB CD34+ cell could be induced ex vivo to produce up to 200 million erythrocytes with a purity of 90.1% ± 6.2% and 50% ± 5.7% (mean ± SD) for CD235a+ cells and enucleated cells, respectively. The yield of erythrocytes from one CB unit (5 million CD34+ cells) could be, in theory, equivalent to 500 blood transfusion units in clinical application. Moreover, induced human erythrocytes had normal hemoglobin content and could continue to undergo terminal maturation in the murine xenotransplantation model. In NHP model, xenotransplantation of induced human erythrocytes enhanced hematological recovery and ameliorated the hypoxia situation in the primates with hemorrhagic anemia. These findings suggested that the ex vivo‐generated erythrocytes could be an alternative blood source for traditional transfusion products in the clinic. Stem Cells Translational Medicine 2017;6:1698–1709 This study showed an efficient technology for ex vivo generating human erythrocytes. The yield of erythrocytes derived from one cord blood unit (5 million CD34+ cells) could, in theory, be equivalent to 500 blood transfusion units in clinical application. Xenotransfusion studies in mice and nonhuman primates confirmed the safety and efficiency for generated erythrocytes.

The clinical application of the fetal membranes dates back to nearly a century. Their use has ranged from superficial skin dressings to surgical wound closure. The applications of the fetal membranes are constantly evolving, and key to this is the uncovering of multiple populations of stem and stem‐like cells, each with unique properties that can be exploited for regenerative medicine. In addition to pro‐angiogenic and immunomodulatory properties of the stem and stem‐like cells arising from the fetal membranes, the dehydrated and/or decellularized forms of the fetal membranes have been used to support the growth and function of other cells and tissues, including adipose‐derived mesenchymal stem cells. This concise review explores the biological origin of the fetal membranes, a history of their use in medicine, and recent developments in the use of fetal membranes and their derived stem and stem‐like cells in regenerative medicine. Stem Cells Translational Medicine 2017;6:1767–1776 The clinical application of the fetal membranes dates back to nearly a century. The applications of the fetal membranes are constantly evolving. This concise review explores the biological origin of the fetal membranes, a history of their use in medicine, and recent developments in the use of fetal membranes and their derived stem and stem‐like cells in regenerative medicine.

To date, different experimental strategies have been developed for the ex vivo expansion of human hematopoietic stem (HSCs) and progenitor (HPCs) cells. This has resulted in significant advances on the use of such expanded cells in transplantation settings. To this day, however, it is still unclear to what extent those stem and progenitor cells generated in vitro retain the functional and genomic integrity of their freshly isolated counterparts. In trying to contribute to the solving of this issue, in the present study we have selected and purified three different hematopoietic cell populations: HSCs (CD34+ CD38‐ CD45RA‐ CD71‐ Lin‐ cells), myeloid progenitor cells (CD34+ CD38+ CD45RA+ CD71‐ Lin‐ cells), and erythroid progenitor cells (CD34+ CD38+ CD45RA‐ CD71+ Lin‐ cells), obtained directly from fresh human umbilical cord blood (UCB) units or generated in vitro under particular culture conditions. We, then, compared their functional integrity in vitro and their gene expression profiles. Our results indicate that in spite of being immunophenotipically similar, fresh and in vitro generated cells showed significant differences, both in functional and genetic terms. As compared to their fresh counterparts, those HSCs generated in our culture system showed a deficient content of long‐term culture‐initiating cells, and a marked differentiation bias toward the myeloid lineage. In addition, in vitro generated HSCs and HPCs showed a limited expansion potential. Such functional alterations correlated with differences in their gene expression profiles. These observations are relevant in terms of HSC biology and may have implications in UCB expansion and transplantation. Stem Cells Translational Medicine 2018;7:602–614 In vitro biology of hematopoietic stem and progenitor cells. Comparison of cells obtained from human cord blood and those generated in vitro. Significant differences are observed in terms of their cell content and growth capacities.

Human induced pluripotent stem cells (iPSCs) can be differentiated into vascular endothelial (iEC) and smooth muscle (iSMC) cells. However, because iECs and iSMCs are not derived from an intact blood vessel, they represent an immature phenotype. Hemodynamics and heterotypic cell:cell communication play important roles in vascular cell phenotypic modulation. Here we tested the hypothesis that hemodynamic exposure of iECs in coculture with iSMCs induces an in vivo‐like phenotype. iECs and iSMCs were cocultured under vascular region‐specific blood flow hemodynamics, and compared to hemodynamic cocultures of blood vessel‐derived endothelial (pEC) and smooth muscle (pSMC) cells. Hemodynamic flow‐induced gene expression positively correlated between pECs and iECs as well as pSMCs and iSMCs. While endothelial nitric oxide synthase 3 protein was lower in iECs than pECs, iECs were functionally mature as seen by acetylated‐low‐density lipoprotein (LDL) uptake. SMC contractile protein markers were also positively correlated between pSMCs and iSMCs. Exposure of iECs and pECs to atheroprone hemodynamics with oxidized‐LDL induced an inflammatory response in both. Dysfunction of the transforming growth factor β (TGFβ) pathway is seen in several vascular diseases, and iECs and iSMCs exhibited a transcriptomic prolife similar to pECs and pSMCs, respectively, in their responses to LY2109761‐mediated transforming growth factor β receptor I/II (TGFβRI/II) inhibition. Although there are differences between ECs and SMCs derived from iPSCs versus blood vessels, hemodynamic coculture restores a high degree of similarity in their responses to pathological stimuli associated with vascular diseases. Thus, iPSC‐derived vascular cells exposed to hemodynamics may provide a viable system for modeling rare vascular diseases and testing new therapeutic approaches. Stem Cells Translational Medicine 2017;6:1673–1683 Human induced pluripotent stem cells differentiated into vascular endothelial (iECs) and smooth muscle (iSMCs) cells were cocultured to preserve heterotypic cell:cell communication and exposed to relevant physiological hemodynamics. This application restores a more in vivo‐like phenotype of these cell types.

Transplantation of small cord blood (CB) units, or of autologous ex vivo‐genetically modified adult hematopoietic stem cells (HSC), face the common challenge of suboptimal HSC doses for infusion and impaired engraftment of the transplanted cells. Ex vivo expansion of HSCs, using either cell‐based coculture approaches or especially small molecules have been successfully tested mainly in CB and in prolonged cultures. Here, we explored whether innovative combinations of small molecules can sufficiently, after short culture, expand adult HSCs while retaining their functionality in vivo. We found that 5‐day cultured cells, in the presence of the small molecule combinations tested, achieved higher engraftment levels in NSG mice than both their uncultured and their cytokine only‐cultured counterparts. Surprisingly, the engraftment levels were neither concordant to the numbers of phenotypically similar HSCs expanded under different small molecule combinations, nor explained by their distinct companion cells present. Transcriptomic comparative analysis of sorted, phenotypically similar, ex vivo generated HSCs transplanted in equal numbers, suggested that HSCs generated under expansion conditions that maintain low expression of the Rap1/Ras/PI3K‐AKT pathway exhibit a superior functional profile in vivo. Stem Cells Translational Medicine 2017;6:1852–1858 Adult hematopoietic stem cells (HSCs) (CD34+/CD38–/CD90+) expanded ex vivo in the presence of SR1+Ly, have enhanced engraftment in vivo compared with phenotypically similar HSCs expanded under different protocols. Reduction in Ras/Rap 1/PI3k‐AKT signaling observed under SR1+Ly seems to preserve their self‐renewal in vivo.

Umbilical cord blood (UCB) transplants in adults have slower hematopoietic recovery compared to bone marrow (BM) or peripheral blood (PB) stem cells mainly due to low number of total nucleated cells and hematopoietic stem and progenitor cells (HSPC). As such in this study, we aimed to perform ex vivo expansion of UCB HSPC from non‐enriched mononucleated cells (MNC) using novel azole‐based small molecules. Freshly‐thawed UCB–MNC were cultured in expansion medium supplemented with small molecules and basal cytokine cocktail. The effects of the expansion protocol were measured based on in vitro and in vivo assays. The proprietary library of >50 small molecules were developed using structure‐activity‐relationship studies of SB203580, a known p38‐MAPK inhibitor. A particular analog, C7, resulted in 1,554.1 ± 27.8‐fold increase of absolute viable CD45+CD34+CD38–CD45RA– progenitors which was at least 3.7‐fold higher than control cultures (p < .001). In depth phenotypic analysis revealed >600‐fold expansion of CD34+/CD90+/CD49f+ rare HSPCs coupled with significant (p < .01) increase of functional colonies from C7 treated cells. Transplantation of C7 expanded UCB grafts to immunodeficient mice resulted in significantly (p < .001) higher engraftment of human CD45+ and CD45+CD34+ cells in the PB and BM by day 21 compared to non‐expanded and cytokine expanded grafts. The C7 expanded grafts maintained long‐term human multilineage chimerism in the BM of primary recipients with sustained human CD45 cell engraftment in secondary recipients. In conclusion, a small molecule, C7, could allow for clinical development of expanded UCB grafts without pre‐culture stem cell enrichment that maintains in vitro and in vivo functionality. Stem Cells Translational Medicine 2018;7:376–393 This study identified a novel structural analog of SB203580 (p38‐MAPK inhibitor) that expands hematopoietic stem and progenitor cells (HSPC) from non‐enriched, frozen‐thawed umbilical cord blood (UCB)–mononucleated cells. The UCB graft expanded with the lead small molecule C7 consists of primitive HSPC phenotype (CD34+CD90+CD49f+), maintains enhanced in vitro colony formation capacity and engrafts both primary and secondary immunodeficient mice. If CD34 selection is further used, this molecule is also able to attain superior HSPC expansion compared to current technologies.

The stability of in vitro cell cultures is an important issue for any clinical, bio‐industrial, or pharmacological use. Embryonic stem cells are pluripotent; consequently, they possess the ability to differentiate into all three germ layers and are inherently prone to respond to differentiation stimuli. However, long‐term culture inevitably yields clones that are best adapted to the culture conditions, passaging regimes, or differentiation sensitivity. This cellular plasticity is a major obstacle in the development of bio‐industrial or clinical‐grade cultures. At present, the quality control of cell cultures is limited by the lack of reliable (epi)genetic or molecular markers or by the focus on a particular type of instability such as karyotype abnormalities or adverse phenotypic traits. Therefore, there is an ongoing need for robust, feasible, and sensitive methods of determining or confirming cell status and for revealing potential divergences from the optimal state. We modeled both intrinsic and extrinsic changes in human embryonic stem cell (hESC) states using different experimental strategies and addressed the changes in cell status by intact cell mass spectrometry fingerprinting. The analysis of spectral fingerprints by methods routinely used in analytical chemistry clearly distinguished the morphologically and biochemically similar populations of hESCs and provided a biomarker‐independent tool for the quality control of cell culture. Stem Cells Translational Medicine 2018;7:109–114 Unraveling of hidden alterations to cell phenotype by intact cell mass spectrometry and artificial neural networks as a quality control tool with biomedical applicability – workflow 1 to 4.

The application of conditional reprogramming culture (CRC) methods to nasal airway epithelial cells would allow more wide-spread incorporation of primary airway epithelial culture models into complex lung disease research. In this study, we adapted the CRC method to nasal airway epithelial cells, investigated the growth advantages afforded by this technique over standard culture methods, and determined the cellular and molecular basis of CRC cell culture effects. We found that the CRC method allowed the production of 7.1 × 1010 cells after 4 passages, approximately 379 times more cells than were generated by the standard bronchial epithelial growth media (BEGM) method. These nasal airway epithelial cells expressed normal basal cell markers and could be induced to form a mucociliary epithelium. Progenitor cell frequency was significantly higher using the CRC method in comparison to the standard culture method, and progenitor cell maintenance was dependent on addition of the Rho-kinase inhibitor Y-27632. Whole-transcriptome sequencing analysis demonstrated widespread gene expression changes in Y-27632–treated basal cells. We found that Y-27632 treatment altered expression of genes fundamental to the formation of the basal cell cytoskeleton, cell–cell junctions, and cell–extracellular matrix (ECM) interactions. Importantly, we found that Y-27632 treatment up-regulated expression of unique basal cell intermediate filament and desmosomal genes. Conversely, Y-27632 down-regulated multiple families of protease/antiprotease genes involved in ECM remodeling. We conclude that Y-27632 fundamentally alters cell–cell and cell–ECM interactions, which preserves basal progenitor cells and allows greater cell amplification.