Basic studies of human pluripotential stem cells have advanced rapidly and stem cell products are now seeing therapeutic applications. However, questions remain regarding the tumorigenic potential of such cells. Here, we report the tumorigenic potential of induced pluripotent stem cell (iPSC)-derived retinal pigment epithelium (RPE) for the treatment of wet-type, age-related macular degeneration (AMD). First, immunodeficient mouse strains (nude, SCID, NOD-SCID and NOG) were tested for HeLa cells' tumor-forming capacity by transplanting various cell doses subcutaneously with or without Matrigel. The 50% Tumor Producing Dose (TPD50 value) is the minimal dose of transplanted cells that generated tumors in 50% of animals. For HeLa cells, the TPD50 was the lowest when cells were embedded in Matrigel and transplanted into NOG mice (TPD50 = 10(1.1), n = 75). The TPD50 for undifferentiated iPSCs transplanted subcutaneously to NOG mice in Matrigel was 10(2.12); (n = 30). Based on these experiments, 1×10(6) iPSC-derived RPE were transplanted subcutaneously with Matrigel, and no tumor was found during 15 months of monitoring (n = 65). Next, to model clinical application, we assessed the tumor-forming potential of HeLa cells and iPSC 201B7 cells following subretinal transplantation of nude rats. The TPD50 for iPSCs was 10(4.73) (n = 20) and for HeLa cells 10(1.32) (n = 37) respectively. Next, the tumorigenicity of iPSC-derived RPE was tested in the subretinal space of nude rats by transplanting 0.8-1.5×10(4) iPSC-derived RPE in a collagen-lined (1 mm×1 mm) sheet. No tumor was found with iPSC-derived RPE sheets during 6-12 months of monitoring (n = 26). Considering the number of rodents used, the monitoring period, the sensitivity of detecting tumors via subcutaneous and subretinal administration routes and the incidence of tumor formation from the iPSC-derived RPE, we conclude that the tumorigenic potential of the iPSC-derived RPE was negligible.

Stem cells from human exfoliated deciduous teeth (SHED) transplants have been investigated as a possible treatment strategy for spinal cord injuries (SCI) due to their potential for promoting functional recovery. The aim of present study was to investigate the effects of SHED on neuronal death after an experimental model of SCI.

Human pluripotent stem cells (hPSCs) are leading candidate raw materials for cell-based therapeutic products (CTPs). In the development of hPSC-derived CTPs, it is imperative to ensure that they do not form tumors after transplantation for safety reasons. Because cellular immortalization is a landmark of malignant transformation and a common feature of cancer cells, we aimed to develop an in vitro assay for detecting immortalized cells in CTPs. We employed retinal pigment epithelial (RPE) cells as a model of hPSC-derived products and identified a gene encoding slow skeletal muscle troponin T (TNNT1) as a novel marker of immortalized RPE cells by comprehensive microarray analysis. TNNT1 mRNA was commonly upregulated in immortalized RPE cells and human induced pluripotent stem cells (hiPSCs), which have self-renewal ability. Additionally, we demonstrated that TNNT1 mRNA expression is higher in several cancer tissues than in normal tissues. Furthermore, stable expression of TNNT1 in ARPE-19 cells affected actin filament organization and enhanced their migration ability. Finally, we established a simple and rapid qRT-PCR assay targeting TNNT1 transcripts that detected as low as 3% of ARPE-19 cells contained in normal primary RPE cells. Purified hiPSC-derived RPE cells showed TNNT1 expression levels below the detection limit determined with primary RPE cells. Our qRT-PCR method is expected to greatly contribute to process validation and quality control of CTPs.

This article summarizes the recent activity of the International Stem Cell Banking Initiative (ISCBI) held at the California Institute for Regenerative Medicine (CIRM) in California (June 26, 2016) and the Korean National Institutes for Health in Korea (October 19-20, 2016). Through the workshops, ISCBI is endeavoring to support a new paradigm for human medicine using pluripotent stem cells (hPSC) for cell therapies. Priority considerations for ISCBI include ensuring the safety and efficacy of a final cell therapy product and quality assured source materials, such as stem cells and primary donor cells. To these ends, ISCBI aims to promote global harmonization on quality and safety control of stem cells for research and the development of starting materials for cell therapies, with regular workshops involving hPSC banking centers, biologists, and regulatory bodies. Here, we provide a brief overview of two such recent activities, with summaries of key issues raised. Stem Cells Translational Medicine 2017;6:1956-1962.

Unambiguous cell line authentication is essential to avoid loss of association between data and cells. The risk for loss of references increases with the rapidity that new human pluripotent stem cell (hPSC) lines are generated, exchanged, and implemented. Ideally, a single name should be used as a generally applied reference for each cell line to access and unify cell-related information across publications, cell banks, cell registries, and databases and to ensure scientific reproducibility. We discuss the needs and requirements for such a unique identifier and implement a standard nomenclature for hPSCs, which can be automatically generated and registered by the human pluripotent stem cell registry (hPSCreg). To avoid ambiguities in PSC-line referencing, we strongly urge publishers to demand registration and use of the standard name when publishing research based on hPSC lines.

Our group has conducted extensive basic and preclinical studies of the use of human induced pluripotent cell (iPSC)-derived neural stem/progenitor cell (hiPSC-NS/PC) grafts in models of spinal cord injury (SCI). Evidence from animal experiments suggests this approach is safe and effective. We are preparing to initiate a first-in-human clinical study of hiPSC-NS/PC transplantation in subacute SCI.

Decellularization of organs creates an acellular scaffold, ideal for being repopulated by cells. In this work, a low-cost perfusion system was created to be used in the process of liver decellularization and as a bioreactor after recellularization. It consists of a glass chamber to house the organ coupled to a peristaltic pump to promote liquid flow through the organ vascular tree. The rats' liver decellularization was made with a solution of sodium dodecyl sulfate. The recellularization was made with 108 mesenchymal stromal/stem cells and cultivated for seven days. The decellularized matrices showed an absence of DNA while preserving the collagen and glycosaminoglycans quantities, confirming the efficiency of the process. The functional analyses showed a rise in lactate dehydrogenase levels occurring in the first days of the cultivation, suggesting that there is cell death in this period, which stabilized on the seventh day. Histological analysis showed conservation of the collagen web and some groups of cells next to the vessels. It was possible to establish a system for decellularization and a bioreactor to use for the recellularization method. It is easy to assemble, can be ready to use in little time and be easily sterilized.

We introduce a novel approach to determine the critical quality attributes (CQAs) of mesenchymal stem cells (MSCs) expected to exert immunosuppressive effects. MSCs retained homeostatic replication potentials, such as sustainable growth and consistent cell morphology as a population, in early passages, but lost them in late passages. Characteristic surface markers of MSCs (ie, CD73, CD90, and CD105) were no longer expressed at 2 weeks after subcutaneous transplantation into NOG mice when MSCs from late passages were transplanted, but not when MSCs from early passages were transplanted, suggesting that the biological effects of the MSCs differed according to the timing of cell harvesting and highlighting the importance of specifying MSCs that retained homeostatic features to define the CQAs. The homeostatic features of MSCs related to the balance of the redox system, nutrient requirements, and mitochondrial function were also observed until a certain passage. Therefore, we could define the CQAs of MSCs related to manufacturing by selecting process parameters (PPs) underlying the homeostatic features of MSCs and measuring these PPs quantitatively to specify the cell population with homeostatic features by limiting the passage number. The validity of the PPs stipulated in our pilot study was verified using an SKG murine arthritis model, and critical PPs (CPPs) were then selected among the PPs. Thus, CQAs related to manufacturing in the developmental phase could be defined by the CPPs in this manner, and the concept of CQAs could be refined continuously toward commercial manufacturing.

The contamination of human cell-processed therapeutic products (hCTPs) with tumorigenic cells is one of the major concerns in the manufacturing and quality control of hCTPs. However, no quantitative method for detecting the tumorigenic cellular impurities is currently standardized. NOD/Shi-scid IL2Rγnull (NOG) mice have shown high xeno-engraftment potential compared with other well-known immunodeficient strains, e.g. nude mice. Hypothesizing that tumorigenicity test using NOG mice could be a sensitive and quantitative method to detect a small amount of tumorigenic cells in hCTPs, we examined tumor formation after subcutaneous transplantation of HeLa cells, as a model of tumorigenic cells, in NOG mice and nude mice. Sixteen weeks after inoculation, the 50% tumor-producing dose (TPD50) values of HeLa cells were stable at 1.3 × 104 and 4.0 × 105 cells in NOG and nude mice, respectively, indicating a 30-fold higher sensitivity of NOG mice compared to that of nude mice. Transplanting HeLa cells embedded with Matrigel in NOG mice further decreased the TPD50 value to 7.9 × 10 cells, leading to a 5000-fold higher sensitivity, compared with that of nude mice. Additionally, when HeLa cells were mixed with 106 or 107 human mesenchymal stem cells as well as Matrigel, the TPD50 values in NOG mice were comparable to those of HeLa cells alone with Matrigel. These results suggest that the in vivo tumorigenicity test using NOG mice with Matrigel is a highly sensitive and quantitative method to detect a trace amount of tumorigenic cellular impurities in human somatic cells, which can be useful in the quality assessment of hCTPs.

Here, we introduce a new serum-free defined medium (SPM) that supports the cultivation of human pluripotent stem cells (hPSCs) on recombinant human vitronectin-N (rhVNT-N)-coated dishes after seeding with either cell clumps or single cells. With this system, there was no need for an intervening sequential adaptation process after moving hPSCs from feeder layer-dependent conditions. We also introduce a micropatterned dish that was coated with extracellular matrix by photolithographic technology. This procedure allowed the cultivation of hPSCs on 199 individual rhVNT-N-coated small round spots (1 mm in diameter) on each 35-mm polystyrene dish (termed "patterned culture"), permitting the simultaneous formation of 199 uniform high-density small-sized colonies. This culture system supported controlled cell growth and maintenance of undifferentiated hPSCs better than dishes in which the entire surface was coated with rhVNT-N (termed "non-patterned cultures"). Non-patterned cultures produced variable, unrestricted cell proliferation with non-uniform cell growth and uneven densities in which we observed downregulated expression of some self-renewal-related markers. Comparative flow cytometric studies of the expression of pluripotency-related molecules SSEA-3 and TRA-1-60 in hPSCs from non-patterned cultures and patterned cultures supported this concept. Patterned cultures of hPSCs allowed sequential visual inspection of every hPSC colony, giving an address and number in patterned culture dishes. Several spots could be sampled for quality control tests of production batches, thereby permitting the monitoring of hPSCs in a single culture dish. Our new patterned culture system utilizing photolithography provides a robust, reproducible and controllable cell culture system and demonstrates technological advantages for the mass production of hPSCs with process quality control.

It is known that the adhesion molecule ALCAM (CD166) mediates metastasis of malignant cells and organogenesis in embryos. We show here that embryonic day 8.5 (E8.5) murine yolk sac cells express ALCAM protein and that ALCAM expression can be used to define endothelial and cardiac precursors from hematopoietic precursors in E8.5 yolk sacs. ALCAM high+ cells exclusively give rise to endothelial and cardiac cells in matrigel assays but generate no hematopoietic colonies in methylcellulose assays. ALCAM low+ and ALCAM- populations predominantly give rise to hematopoietic cells in methylcellulose, but do not generate any cell clusters in matrigel. The ALCAM high+ population contains both Flk-1+ and Flk-1- cells. The former population exclusively contains endothelial cells whereas the latter give rise to cardiac cells when cultured on OP9 stromal cells. We also show that cardiac lineage marker genes such as Nkx-2.5, and the endothelial marker VE-cadherin are expressed in the ALCAM high+ fraction, whereas the hematopoietic marker GATA1 and Runx1 are expressed in the ALCAM low+/- fraction. However, we did not detect expression of the cardiac structural protein cTn-T in cells from yolk sac cells until these had had been differentiated on OP9 for 5 days. Altogether, these results indicate that cells retaining a potential to differentiate to the cardiac lineage are present in E8.5 yolk sacs and can be isolated as ALCAM high+, Flk-1- cells. Our report provides novel insights into the origin and differentiation process of cardiac cells in the formation of the circulatory system.

Mesenchymal stem cells (MSCs) are considered a potential autologous therapy for tissue engineering. The available procedures for MSC retrieval from patients are invasive, and their limited in vitro proliferation restricts their use in the treatment of damaged tissues. Therefore, it is important to establish an alternative and safe source of MSCs. The objective of this study was to demonstrate induced pluripotent stem cell (iPSC) generation from a combination of an accessible source tissue and an integration-free method; we also attempted the differentiation of iPSCs into MSC-like cells (MSLCs) for future autologous tissue engineering. iPSCs were derived from human gingival tissues, which are easily accessible in the field of dentistry, via the use of non-integrating episomal plasmids. Established iPSCs expressed embryonic stem (ES) cell-specific markers, as assessed by gene analysis and immunocytochemistry. Embryoid bodies and teratoma formation were formed from iPSCs, showing their capacity to differentiate into three germ layers. Furthermore, we were successful in differentiating iPSCs into MSLCs. They tested positively for their capacity of trilineage differentiation. Our results demonstrate that human gingival integration-free iPSCs, readily accessible stem cells generated using episomal plasmid vectors, are a promising source of MSLCs, which can be used in tissue regeneration.

The generation of human embryonic stem cell banking networks has ensured that well-characterized and quality controlled stem cell lines are broadly accessible to researchers worldwide. Here, we provide recommendations for engaging these established networks in efforts to build similar resources for the distribution and collection of induced pluripotent stem cells.

We show that pigment epithelium-derived factor (PEDF), which is secreted from primary or iPSC-derived retinal pigment epithelium (RPE), dramatically inhibits the growth of iPSCs. PEDF is detected abundantly in culture supernatants of primary or iPSC-derived RPE. Apoptotic cell death is induced in iPSC when co-cultured with RPE, a process that is significantly blocked by addition of antibody against PEDF. Indeed, addition of recombinant PEDF to the iPSC cell culture induces apoptotic cell death in iPSCs, but the expression of pluripotency related-genes is maintained, suggesting that PEDF causes cell death, not differentiation, of iPSCs. To recapitulate this event in vivo, we examined tumor formation in NOG mice after subcutaneous injection of iPSCs with or without an iPSC-derived RPE sheet (2.5 × 10(5) RPE cells). We observed that the tumor forming potential of iPSCs was significantly suppressed by simultaneous transplantation with an iPSC-derived RPE sheet.

Mesenchymal stem cells (MSCs) are multipotent cells capable of differentiating into cells from the mesenchymal lineage. The hypoimmunogenic characteristic of MSCs has encouraged studies using allogeneic MSCs for the treatment of autoimmune diseases and inflammatory conditions. Promising preclinical results and the safety of allogeneic MSC transplantation have created the possibility of "off-the-shelf" clinical application of allogeneic cells. This study has aimed to evaluate the survival of untreated and IFN-γ- and TNF-α-treated (preactivated) allogeneic MSCs transplanted under the kidney capsule of immunocompetent mice together with the role of preactivated MSCs after cotransplantation with allogeneic islets. The preactivation of MSCs upregulated the gene expression of anti-inflammatory molecules and also enhanced their immunomodulatory capacity in vitro. In vivo, allogeneic MSCs provoked an immunogenic response, with the infiltration of inflammatory cells at the transplant site and full graft rejection in both the untreated and preactivated groups. Allogeneic islets cotransplanted with preactivated MSCs prolonged graft survival for about 6 days, compared with islet alone. The present results corroborate the hypothesis that allogeneic MSCs are not immune-privileged and that after playing their therapeutic role they are rejected. Strategies that reduce allogeneic MSC immunogenicity can potentially prolong their in vivo persistence and improve the therapeutic effects.

(1) Background: Nanotechnology is an emerging field that can be applied in the biomedical area. In this study, Eudragit nanocapsules (NCs) containing nicotine were produced. Nicotine is the main alkaloid found in tobacco and has anti-inflammatory properties. NCs containing nicotine may be used as an adjuvant therapy in the treatment of inflammation in the central nervous system. (2) Methods: Nanocapsules were prepared by the interfacial deposition of the pre-formed polymer method and characterized in terms of zeta potential, diameter, polydispersity index, pH, encapsulation efficiency (EE), stability and sustained release profile. In vitro tests with the PC12 cell line were performed, such as MTT, LIVE/DEAD and ELISA assays, to verify their cytotoxic and anti-inflammatory effects. (3) Results: The nanocapsules presented satisfactory values of the characterization parameters; however, poor encapsulation was obtained for nicotine (8.17% ± 0.47). The in vitro tests showed that the treatment with nanocapsules reduced cell viability, which suggests that the Eudragit or the amount of polymer on top of the cells may be detrimental to them, as the cells were able to survive when treated with bulk nicotine. ELISA showed an increment in the expression of IL-6 and IL-1β, corroborating the hypothesis that NCs were toxic to the cells because of the increase in the levels of these pro-inflammatory cytokines. (4) Conclusions: This study demonstrates that NCs of Eudragit present toxicity. It is therefore necessary to improve NC formulation to obtain better values for the encapsulation efficiency and reduce toxicity of these nanodevices.

Three-dimensional retinal organoids (3D-retinas) are a promising graft source for transplantation therapy. We previously developed self-organizing culture for 3D-retina generation from human pluripotent stem cells (hPSCs). Here we present a quality control method and preclinical studies for tissue-sheet transplantation. Self-organizing hPSCs differentiated into both retinal and off-target tissues. Gene expression analyses identified the major off-target tissues as eye-related, cortex-like, and spinal cord-like tissues. For quality control, we developed a qPCR-based test in which each hPSC-derived neuroepithelium was dissected into two tissue-sheets: inner-central sheet for transplantation and outer-peripheral sheet for qPCR to ensure retinal tissue selection. During qPCR, tissue-sheets were stored for 3-4 days using a newly developed preservation method. In a rat tumorigenicity study, no transplant-related adverse events were observed. In retinal degeneration model rats, retinal transplants differentiated into mature photoreceptors and exhibited light responses in electrophysiology assays. These results demonstrate our rationale toward self-organizing retinal sheet transplantation therapy.

Contamination with tumorigenic cellular impurities is one of the most pressing concerns for human cell-processed therapeutic products (hCTPs). The soft agar colony formation (SACF) assay, which is a well-known in vitro assay for the detection of malignant transformed cells, is applicable for the quality assessment of hCTPs. Here we established an image-based screening system for the SACF assay using a high-content cell analyzer termed the digital SACF assay. Dual fluorescence staining of formed colonies and the dissolution of soft agar led to accurate detection of transformed cells with the imaging cytometer. Partitioning a cell sample into multiple wells of culture plates enabled digital readout of the presence of colonies and elevated the sensitivity for their detection. In practice, the digital SACF assay detected impurity levels as low as 0.00001% of the hCTPs, i.e. only one HeLa cell contained in 10,000,000 human mesenchymal stem cells, within 30 days. The digital SACF assay saves time, is more sensitive than in vivo tumorigenicity tests, and would be useful for the quality control of hCTPs in the manufacturing process.

Peripheral nerve injury is an important cause of incapability and has limited available treatment. Autologous donor nerve implant is the golden standard treatment, however, may cause secondary deficits. Stem cells show positive results in preclinical settings, preserving tissue and function. We tested the efficacy of stem cells derived from human exfoliated deciduous teeth seeded in poly (lactide-co-glycolide) scaffolds in sciatic nerve transection model. Seventy-two adult male Wistar rats had 7-mm nerve gap bridge using scaffolds with (or without) stem cells. Animals were randomly divided into: sham-operated; sham-operated without scaffold; sham-operated + scaffold + stem cells; sciatic transection + no treatment; sciatic transection + acellular scaffolds; sciatic transection + scaffold + stem cells. Sciatic Functional Index and Ladder Rung Walking tests were performed before (-1), 14 and 28 days after surgery. Morphometric nerve measurement and muscle weights were assessed. Scaffolds with stem cells improved function in Sciatic Functional Index. Acellular scaffold was effective, promoting functional recovery and nerve regeneration following nerve injury. Scaffolds provide better nerve regeneration and functional recovery after sciatic transection. Despite cell therapy promoting faster recovery after sciatic transection in the Sciatic Index Score, stem cells did not improve functional and morphological recovery after nerve injury. This is the first study testing the potential use of scaffolds combined with stem cells in the early stages after injury. Scaffolds with stem cells could accelerate nerve recovery and favor adjuvant therapies, evidencing the need for further studies to increase the knowledge about stem cells' mechanisms.

Seven human induced pluripotent stem cell (iPSC) lines were generated from fibroblasts from three neonatal individuals using non-integrative reprogramming. Most control iPSCs are derived from adults, so these iPSCs meet the need for control iPSCs from young individuals. Donors were from different ethnicities and these lines provide unique genetic profiles. All iPSCs have normal karyotypes, express stem cell markers, and exhibit pluripotency, as assessed by capacity to differentiate into three germ layers. These lines are valuable to study human development, as age-matched controls for disorder-specific iPSCs, and as platforms for gene editing to control for age and ethnicity.