Feeder-free Culture Research Articles

A Hydrogel Based 3D Culture System for Hematopoietic Differentiation of Induced Pluripotent Stem Cells

The generation of hematopoietic stem and progenitor cells (HSPCs) from induced pluripotent cells (iPSCs) holds a great potential in development of cell therapies, modeling hematological malignancies, and testing new drugs. Hematopoiesis is regulated by bone marrow (BM) microenvironmental factors such as BM architecture, and cell-cell and cell-matrix interactions. The current methods for iPSC differentiation into HSPCs rely on cell-cell interaction (embryoid body formation and feeder cell co-cultures) or cell-matrix interaction (ECM coated dishes). Generation of a 3-dimensional (3D) culture environment attempts to capture both cell-cell and cell matrix interactions in studying the differentiation of the iPSCs. This study reports development of a 3D culture system for hematopoietic differentiation of iPSCs. iPSC colonies were encapsulated in 3D Matrigel or polyethylene glycol (PEG) based hydrogels containing synthetic integrin binding peptide (GRGDSPC) and enzymatically degradable peptide (GGPQGIWGQGKG) and cultured in feeder-free cell culture and maintenance medium (mTeSR1™, Stem Cell Technology). There were notable morphological differences between the 3D encapsulated and 2D cultured iPSC colonies (Fig 1A). The 3D encapsulated colonies were more compact with a spheroid morphology in PEG compared to Matrigel, whereas colonies in 2D were more diffused. Viability of the encapsulated colonies was evaluated in situ using cytotoxicity kit. The 3D encapsulation did not have an adverse effect on the viability and growth of iPSCs (Fig 1B). The encapsulated iPSCs maintained the pluripotent phenotype in 3D as assessed by TRA-1-60 staining (Fig 1C). To test the efficiency of these 3D model systems to generate HSPCs, the encapsulated iPSCs were subjected to hematopoietic differentiation using STEMdiff™ Hematopoietic Kit (Fig 1D). Following differentiation, the single cells were collected and immunophenotype analysis was performed by flow cytometry. Above 50% population stained positive for HSPC surface marker CD34 both in PEG hydrogel and Matrigel (Fig 1E). To test the robustness of the 3D system in inducing hematopoietic differentiation, we encapsulated the isogenic trisomic 21 (Tri21) iPSCs and subjected to hematopoietic differentiation and flow cytometry analysis as described above. As reported previously in 2D system (Banno et al., 2016, Cell Reports), Tri21 iPSCs showed higher percentage of hematopoietic commitment into erythroid and megakaryocytic lineages compared to disomic 21 (Di21), as characterized by the presence of respective surface markers (Fig 1F). In conclusion, 3D culture system was suitable for inducing hematopoietic differentiation of iPSCs in isogenic disomic and trisomic lines. The 3D model can be used to generate patient-specific HSPCs. Figure 1 Disclosures No relevant conflicts of interest to declare.

Ex vivo Engineering of Red Blood Cells

Culture systems for human in vitro erythropoiesis are now well established. Using our 3-stage feeder-free erythroid culture system we can efficiently differentiate erythroid cells from adult and cord blood (CB) CD34+ cells with >105 fold expansion, enucleation rates of up to 95% and producing packed reticulocyte yields of >12ml post leukofiltration1. The final preparations for a first in man clinical trial of adult cultured reticulocytes produced under good manufacturing practice (RESTORE) are underway. Although we have shown that it is possible to modify the CD34+ derived cells using lentivirus for reengineering or additions to the medium, the finite proliferative capacity of CD34+ cells in culture currently limits yield. We therefore took the alternative approach of immortalising early erythroid cells differentiated from adult bone marrow (BM) CD34+ cells, creating the BEL-A (Bristol Erythroid Line Adult) line2, a sustainable erythroid cell source that recapitulates normal adult erythropoiesis, terminally differentiating to generate enucleated reticulocytes that express normal levels of adult globin. We have created a further 13 lines from BM, adult peripheral blood, CB and iPSC CD34+ cells, demonstrating reproducibility of the approach and its application to create lines from more accessible stem cell sources. Analysis of surface marker and globin profiles demonstrate the lines follow a similar differentiation profile to their respective primary cell source, with comparative proteomics confirming cell source representation of the lines. Lines always established at the pro-erythroblast/early basophilic stage, even when later stage erythroid cells were present in populations. As well as proof of principal as an alternative transfusion product and improved tools for studying erythropoiesis, such lines have far reaching additional applications, a number of which we are now exploring: Diagnostic and 'Universal' transfusion products: Presently serological testing by blood group reference laboratories relies on donated blood, which represent a finite resource and for some blood group phenotypes can be difficult to source. We used CRISPR-Cas9 gene editing to remove blood group antigens in order to generate a sustainable bank of cell lines with useful blood group phenotypes for diagnostic purposes3. Building on this, with the aim of developing a more compatible "universal" transfusion product to meet the needs of chronically transfused patients and those with rare blood group phenotypes, we used combinatorial gene targeting to create sublines deficient in multiple antigens responsible for the most common transfusion incompatibilities (ABO [Bombay], Rh [Rhnull], Kell [K0], Duffy [Fynull], GPB [S-s-U-]). Individual and multiple blood group knockout lines retained the ability to undergo terminal differentiation and enucleation, also illustrating the capacity for coexistence of multiple rare blood group phenotypes within viable reticulocytes3. Cytokine independent lines Cytokines represent a substantial cost contribution to erythroid culture systems. We therefore exploited activating mutations found in patient c-Kit and EPOR that cause hypersensitivity to ligand, to create cytokine independent lines thus increasing economic viability of cultured red cells. Bi-allelic EPOR or c-kit edits were introduced into BEL-A with confirmation and exploration of mechanism on differentiation in the absence, or with substantially reduced levels of cytokines. Model disease systems In addition to potential therapeutic applications we are also creating lines as model cellular disease systems for studying molecular mechanisms and as drug screening platforms, via CRISPR-Cas9 gene editing of BEL-A and by directly immortalising patient stem cells. To date we have made b-thalassemia major, HbE thalassemia and lines with KLF1 mutations. Furthermore, we have shown BEL-A reticulocytes support invasion and growth of Plasmodium falciparum and are utilising the line to study mechanisms of malaria parasite invasion4. Kupzig S, Parsons SF, Curnow E, Anstee DJ, Blair A. Superior survival of ex vivo cultured human reticulocytes following transfusion into mice. 2016;102:476-483Trakarnsanga K, Griffiths RE, Wilson MC, et al. An immortalized adult human erythroid line facilitates sustainable and scalable generation of functional red cells. Nat. Commun. 2017;8:14750Hawksworth J, Satchwell TJ, Meinders M, et al. Enhancement of red blood cell transfusion compatibility using CRISPR-mediated erythroblast gene editing. EMBO Mol. Med. 2018; 10:e8454Satchwell TJ, Wright K, Haydn-Smith K, et al. Genetic manipulation of cell line derived reticulocytes enables dissection of host malaria invasion requirements. Nat. Comm. 2019;10:3806 Disclosures No relevant conflicts of interest to declare.

A Reproducible and Simple Method to Generate Red Blood Cells from Human Pluripotent Stem Cells

Erythroid cells generated from human pluripotent stem cells (hPSCs) can potentially offer an unlimited and safe supply of red blood cells (RBCs) for transfusion. Human PSC-derived erythroid cells at various stages of differentiation can also be used to model blood diseases, test new drug candidates, and develop cellular and genetic therapies. Although several protocols for deriving RBCs from hPSCs have been described, these are typically complex, involving multiple culture steps that may include co-culture with feeder cells, and exhibit large variability in erythroid cell yields between hPSC lines and replicate experiments. We have developed a straightforward, serum-free and feeder-free culture method to generate erythroid cells from hPSCs with high yields and high purity. The method has been validated on multiple human embryonic stem (ES) cell lines (H1, H7, H9) and induced pluripotent stem (iPS) cell lines (WLS-1C, STiPS-F016, STiPS-B004). The protocol involves two steps: hematopoietic specification of hPSCs, followed by differentiation of hPSC-derived hematopoietic stem and progenitor cells (HSPCs) into erythroid cells. Lineage specification and differentiation is driven by only three supplements that combine cytokines and other factors to support optimal differentiation efficiency and cell yield across cell lines. In the first step, small hPSC aggregates routinely maintained in feeder-free maintenance medium, are plated onto matrigel-coated microwells, and specification to mesoderm and subsequent hematoendothelial differentiation is induced by addition of successive expansion supplements. This phase promotes extensive hematopoietic progenitor cell generation, with a single hPSC producing on average 142 HSPCs (range: 50 - 360, n = 3 experiments) by day 10 across all six ES and iPS cell lines tested. The average frequency of cells expressing CD43, an embryonic pan-hematopoietic marker, is 92% (range: 85 - 95%), and the frequency of CD34+ cells ranges between 24-55%. In the second, erythroid differentiation step, hPSC-derived HSPCs expand on average 300-fold (range: 80 - 1000) within 10 - 14 days, and the average frequency of GlyA+ cells is 75% (range: 70 - 85%). Cumulatively, this results in the generation of on average 30,000 GlyA+ cells (range: 10,000 - 80,000) per initial hPSC after 20 - 24 days. Further maturation in 7-day cultures containing EPO and human serum resulted in a > 90% pure population of GlyA+ erythroid cells. Notably, no cell loss was observed during the maturation phase, resulting in an average yield of 50,000 GlyA+ cells (range: 10,000 - 220,000) per single initial hPSC on day 31. Differentiated cells were characterized by orthochromatic normoblast morphology and decreased CD71 expression, consistent with erythroid maturation. Erythroid cells generated in this differentiation culture system expressed a mix of 'primitive' and 'definitive' hemoglobin types, but with adult and fetal hemoglobin being expressed at higher levels than embryonic hemoglobin. The observed enucleation rates of hPSC-derived erythroid cells are consistent with current reports and are subject to further optimization. In summary, we have developed a two-step, serum- and feeder-free erythroid differentiation method to generate large numbers of erythroid cells from multiple hPSC lines. This culture system provides a simple, standardized and reproducible platform to generate RBCs from hPSCs with high yields and efficiency for basic and translational research. Disclosures Walasek: STEMCELL Technologies, Inc: Employment. Chau:STEMCELL Technologies, Inc: Employment. Barborini:STEMCELL Technologies, Inc: Employment. Richardson:STEMCELL Technologies, Inc: Employment. Szilvassy:STEMCELL Technologies, Inc: Employment. Louis:STEMCELL Technologies, Inc: Employment. Thomas:STEMCELL Technologies, Inc: Employment. Eaves:STEMCELL Technologies, Inc: Employment. Wognum:STEMCELL Technologies, Inc: Employment.

Maintenance of hPSCs under Xeno-Free and Chemically Defined Culture Conditions.

Previously, the majority of human embryonic stem cells and human induced pluripotent stem cells have been derived on feeder layers and chemically undefined medium. Those media components related to feeder cells, or animal products, often greatly affect the consistency of the cell culture. There are clear advantages of a defined, xeno-free, and feeder-free culture system for human pluripotent stem cells (hPSCs) cultures, since consistency in the formulations prevents lot-to-lot variability. Eliminating all non-human components reduces health risks for downstream applications, and those environments reduce potential immunological reactions from stem cells. Therefore, development of feeder-free hPSCs culture systems has been an important focus of hPSCs research. Recently, researchers have established a variety of culture systems in a defined combination, xeno-free matrix and medium that supports the growth and differentiation of hPSCs. Here we described detailed hPSCs culture methods under feeder-free and chemically defined conditions using vitronetin and TeSR-E8 medium including supplement bioactive lysophospholipid for promoting hPSCs proliferation and maintaining stemness.

Robust protocol for feeder-free adaptation of cryopreserved human pluripotent stem cells.

Human pluripotent stem cells (hPSCs) are conventionally maintained on mouse embryonic fibroblast (MEF) feeder layers. However, downstream applications, such as directed differentiation protocols, are primarily optimized for feeder-free cultures. Therefore, hPSCs must often be adapted to feeder-free conditions. Here we propose a novel feeder-free adaptation protocol using StemFlex medium, which can be directly applied to thawed hPSC lines.The direct feeder-free adaptation protocol using StemFlex culture medium on Geltrex coating led to robust hPSC cultures in approximately 2 weeks. This approach was tested with three human embryonic stem cell (hESC) lines. All lines were confirmed to be pluripotent, expressing POU5F1, SOX2, and NANOG. No chromosomal imbalances were induced by the feeder-free adaptation.StemFlex medium enabled the efficient adaptation of hPSCs to feeder-free conditions directly after thawing. This protocol is easy to implement in laboratories that perform feeder-free cultures, allowing more convenient adaptation and more robust expansion of cryopreserved hPSCs, even in cases when sample quality is low or unknown.

Reprogramming of spermatogonial stem cells into pluripotent stem cells in the spheroidal state

ABSTRACT Spermatogonial stem cells (SSCs) are unipotent adult stem cells, capable of differentiating into sperm cells. SSCs can be cultured in vitro for a long time. SSCs expressing Oct4, a pluripotency marker, and are the only adult cells which pluripotency can be induced under defined culture conditions. However, because 2D culture imposes limitations in cell junction formation, cell shape, metabolism, response to stimuli, and cell interface with medium, mechanistic studies on reprogramming of SSCs using feeder cells still have many challenges. Recent studies have shown that a culture system using a bio-matrix can be used in long-term feeder-free SSCs culture and for induction of pluripotency in SSCs. However, the bio-matrix cannot be the optimal micro-environment in mechanistic studies because it creates a physical barrier to growth factors and other signaling molecules. To overcome this effect of the matrix, we reprogrammed SSCs into pluripotent ESC-like cells, so-called germline-derived pluripotent stem cells (gPSCs) by using a 3D scaffold, in which cells are less responsive to external stimuli than in 2D cultures. Thus, we confirm the possibility of SSC reprogramming in the spheroidal state and suggest the utility of 3D scaffolds as a tool for studying the mechanism of SSC reprogramming into gPSCs without a bio-matrix.

Robust induction of retinal pigment epithelium cells from human induced pluripotent stem cells by inhibiting FGF/MAPK signaling

Functional decline and loss of the retinal pigment epithelium (RPE) cause retinal diseases. Clinical studies using human embryonic stem cell (hESC)- or induced pluripotent stem cell (hiPSC)-derived RPE cells have shown the safety and potential efficacy of hESC/iPSC-RPE cell transplantation. However, the production of RPE cells remains somewhat problematic. hESCs/iPSCs co-cultured with mouse feeder cells carry the risk of xeno-transmitted infections and immune reactions. Moreover, increasing the rate of cell division to ensure the quantity and purity of cells with low differentiation efficiency elevates the risk of gene mutations and chromosomal abnormalities. Here, we show that the transient inhibition of the FGF/MAPK signaling pathway during the hiPSC maintenance period markedly promotes RPE differentiation efficiency under feeder-free culture conditions. Blockage of FGF/MAPK signal induces neural differentiation and generates RPE cells without subsequent inhibition of Wnt and Nodal signals, which is known to be effective for retinal specification. We also found that additional inhibition of the PKC or BMP signaling pathway together with FGF/MAPK signal inhibition further elevates RPE differentiation efficiency. Our study will be helpful for producing clinical-grade RPE cells and will facilitate the development of therapies using hESC/hiPSC-RPE cells.

Synergistic effect of basic fibroblast growth factor (bFGF) and epidermal growth factor on derivation of camel (Camelus dromedarius) trophoblast stem cells.

This study aimed to optimize the derivation of trophectoderm from in vitro-produced camel embryos under feeder-free culture conditions using the basement membrane matrix Matrigel. Trophoblastic vesicles were obtained through mechanical microdissection of in vitro-produced camel (Camelus dromedarius) embryos. Supplementing the culture medium with 10 ng/ml of epidermal growth factor and 10 ng/ml fibroblast growth factor improved the attachment and subsequent outgrowths of cultured trophoblastic vesicles when compared with the control group and the groups supplemented individually with each growth factor. The expression levels of pluripotency genes octamer-binding transcription factor 4 (Oct4), sex determining region Y-box 2 (Sox2), myelocytomatosis proto-oncogene (c-Myc) and anti-apoptotic gene B-cell lymphoma 2 (Bcl2) were increased in trophoblastic vesicles supplemented with both growth factors when compared with the control group. Conversely, both growth factors decreased the expression of apoptotic genes tumour protein p53 (p53) and Bcl-2-associated X protein (Bax). To the best of our knowledge, this may be the first report describing the derivation of trophoblast stem cells from in vitro-produced camel embryos.

Transposon mediated reprogramming of buffalo fetal fibroblasts to induced pluripotent stem cells in feeder free culture conditions

Commonly, induced pluripotent stem (iPS) cells are generated by viral transduction of four core reprogramming genes, but recent evidences suggest that slightly different combination of transcription factors improve the efficiency and quality of generated iPS cells. However, vectors like retro- and lentiviral may cause insertional mutagenesis due to its integrating ability. Hence, alternate methods with safety concerns are needed to be investigated. Therefore, the present study was undertaken to reprogram buffalo fibroblasts using non-viral piggyBac (PB) transposon mediated transfer of six transcription factors. To generate buffalo iPS cells, fibroblasts were isolated from buffalo fetus at passage 2. The cells were co-electroporated with a PB transposon having CAGGS promoter driven cassette of Oct4, Sox2, Klf4, cMyc, Nanog, and Lin28 transcription factors separated by self-cleaving 2A peptide and a helper plasmid pCMV-PB transposase. After 12–14 days post electroporation, fibroblast cells morphology was observed to change to round structures which formed loose aggregates of cells on day 18. Putative iPS cell colonies were propagated in feeder free system and characterized through expression of pluripotency markers such as alkaline phosphatase, SSEA-1, SSEA-4, SSEA-5, TRA-1-81, Oct4, Nanog and Sox2 and endogenous genes supported the stemness property of the generated cells. These cells differentiated in vitro to form embryoid bodies and were found to express three germ layers markers. In conclusion, generation of buffalo iPS cells using transposon system provides insights into viral-free iPS technology which will facilitate genetic modification of the buffalo genome and help in the production of transgenic animals using genetically modified iPS cells.

Xeno-free and feeder-free culture and differentiation of human embryonic stem cells on recombinant vitronectin-grafted hydrogels.

Recombinant vitronectin-grafted hydrogels were developed by adjusting surface charge of the hydrogels with grafting of poly-l-lysine for optimal culture of human embryonic stem cells (hESCs) under xeno- and feeder-free culture conditions, with elasticity regulated by crosslinking time (10-30 kPa), in contrast to conventional recombinant vitronectin coating dishes, which have a fixed stiff surface (3 GPa). hESCs proliferated on the hydrogels for over 10 passages and differentiated into the cells derived from three germ layers indicating the maintenance of pluripotency. hESCs on the hydrogels differentiated into cardiomyocytes under xeno-free culture conditions with much higher efficiency (80% of cTnT+ cells) than those on conventional recombinant vitronectin or Matrigel-coating dishes just only after 12 days of induction. It is important to have an optimal design of cell culture biomaterials where biological cues (recombinant vitronectin) and physical cues (optimal elasticity) are combined for high differentiation of hESCs into specific cell lineages, such as cardiomyocytes, under xeno-free and feeder-free culture conditions.

In Vitro Differentiation of T Cell: From Human iPS Cells in Feeder-Free Condition.

In order to differentiate T cells in vitro, co-culture systems with Notch ligand-expressing feeder cells have been in use for a long time. Here we describe a feeder-free culture condition for differentiating T cells from hematopoietic cells that are cultured on Fc-DLL4-coated plate with T-lineage cytokines. This condition is capable of efficiently differentiating hematopoietic progenitor cells (HPCs) to immature T cells expressing both CD4 and CD8. To mature those cells into functional T cells, further stimulation and culture is necessary.

An Improved Method to Produce Clinical-Scale Natural Killer Cells from Human Pluripotent Stem Cells.

Human natural killer (NK) cell-based adoptive anticancer immunotherapy has gained intense interest with many clinical trials actively recruiting patients to treat a variety of both hematological malignancies and solid tumors. Most of these trials use primary NK cells isolated either from peripheral blood (PB-NK cells) or umbilical cord blood (UCB-NK cells), though these sources require NK cell collection for each patient leading to donor variability and heterogeneity in the NK cell populations. In contrast, NK cells derived human embryonic stem cells (hESC-NK cells) or induced pluripotent stem cells (hiPSC-NK cells) provide more homogeneous cell populations that can be grown at clinical scale, and genetically engineered if needed. These characteristics make hESC-/iPSC-derived NK cells an ideal cell population for developing standardized, "off-the-shelf" immunotherapy products. Additionally, production of NK cells from undifferentiated human pluripotent stem cells enables studies to better define pathways that regulate human NK cell development and function. Our group previously has established a stromal-free, two-stage culture system to derive NK cells from hESC/hiPSC in vitro followed by clinical-scale expansion of these cells using interleukin (IL)-21 expressing artificial antigen-presenting cells. However, prior to differentiation, this method requires single-cell adaptation of hESCs/hiPSCs which takes months. Recently we optimized this method by adapting the mouse embryonic fibroblast-dependent hESC/hiPSC to feeder-free culture conditions. These feeder-free hESCs/hiPSCs are directly used to form embryoid body (EB) to generate hemato-endothelial precursor cells. This new method produces mature, functional NK cells with higher efficiency to enable rapid production of an essentially unlimited number of homogenous NK cells that can be used for standardized, targeted immunotherapy for the treatment of refractory cancers and infectious diseases.

CD19 Redirected CAR NK Cells Are Equally Effective but Less Toxic Than CAR T Cells

Based on the clinical success observed in acute lymphoblastic leukemia (ALL) with chimeric antigen receptor engineered T (CAR T), we hypothesized that combining the specificity of a CAR with the innate allo-reactivity of KIR-mismatched NK cells might provide a powerful tool for adoptive cell therapy. The use of a third-party bank of CAR-NK cells offers the advantage of an immediate availability to be exploited in the allogenic setting and could be associated with a lower toxicity profile than CAR-T cells.In order to overcome regulatory and manufacturing hurdles associated with generation of CAR-NK cells, we developed a feeder-free culture resulting in a 3.2-log expansion after 20 days of culture. Specifically, natural cytotoxicity receptors (NCR) expressed on NK cells are stimulated in the presence of pleiotropic cytokines and expanded in GMP grade bioreactors. Expanded NK cells from healthy donors preserve a high percentage of CD56+ CD57- cells (85±13%), associated with high proliferative capability, and maintain the surface expression and the responsiveness of NCR and CD16.We proved that NK cells generated from 10 different healthy donors have high ability to recognize and eliminate different tumor types, including acute myeloid leukemia (AML) and ALL.After genetic modification with a retroviral vector encoding a CAR specific for CD19 antigen, transduction of activated NK cells averaged 38%±15% and the CAR.CD19 expression was stable over extended in vitro culture (60 days). Detailed phenotypic characterization of CAR-NK cells showed that CAR expression was not limited to the more mature NKG2A-/KIR+ cells, but rather was distributed across different NK subsets.We also demonstrated that NK and CAR-NK cells display significant anti-leukemia activity towards CD19+ leukemia and lymphoma cell lines (LCL 721.221, DAUDI and BV173) and primary blasts obtained from patients with B-cell precursor ALL (Bcp-ALL). Co-culture experiments using a 1:5 E/T ratio, showed that, while the anti-tumor activity was already remarkable with non-modified effector NK cells (60±30%, 71±33% and 54±23% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p<0.05 vs T cells), it reached the highest level when CAR-NK cells were used as effectors (7±9%, 16±30% and 22±16% of residual LCL 721.221, DAUDI and BV173 cells, respectively; p<0.05 vs non-transduced NK cells). Importantly, INF-g production was significantly lower upon CAR-NK activation compared to CAR-T cells (DAUDI 384±194 ng/ml vs 1860±678 ng/ml, p=0.002). Functional analysis on primary Bcp-ALL blasts, demonstrate that CAR-NK cells exert high degree of leukemia control (on average 2.1±2% vs 5.4±1.6% with non-modified NK cells as effectors; p=0.04).An in vivo model of leukemia xenograft immunodeficient mice was used to evaluate whether CAR-NK cells are associated with a lower toxicity profile compared to CAR-T cells. While the in vivo antileukemia activity was superimposable between CAR-T and CAR-NK cells (mouse bioluminenscence at 20 days, 4.9x105 vs 6.6x105 photons/second, respectively; p=n.s. Figure A), mice treated with two i.v. infusions (day 0 and day 15) of 10x106 CAR.CD19 NK cells had a 100% overall survival (OS of 5 out of 5 mice) at 50 days compared to 20% of mice (1 out of 5) receiving 10x106 CAR.CD19 T cells (Figure B; p=0.01). Cytokine plasma level monitoring, performed on day +7 and +30 after effector cell infusion in the absence of leukemia persistence (as evidenced by a lack of bioluminescence signal), showed that mice engrafted with CD19+ leukemia and treated with CAR.CD19-NK cells have lower levels of circulating hIFN-g cytokine compared to mice treated with CAR.CD19-T cells at both day 7 (42±82 vs 330±346 ng/ml; p=0.05) and day 30 (0.9±0.7 vs 4148±667 ng/ml; p=0.05).These in vitro and in vivo data demonstrate the feasibility of clinical scale feeder-free expansion of non-modified NK cells and stably transduced CAR-NK cells. Both non-modified and gene-modified cells were capable of significant tumor killing, suggesting a multi-modal adoptive cell approach to treatment of leukemia. Since NK cells have been shown to be safely used in third-party setting (St. Jude Children's Research Hospital, USA; NCT00640796), we suggest that ex-vivo expanded, feeder-free NK cells can be universally applied for ‘off-the-shelf’ immuno-gene-therapy, and that their innate allo-reactivity can be safely harnessed to potentiate allogeneic cell therapy. [Display omitted] DisclosuresLocatelli:Amgen: Honoraria, Membership on an entity's Board of Directors or advisory committees; bluebird bio: Consultancy; Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Miltenyi: Honoraria; Bellicum: Consultancy, Membership on an entity's Board of Directors or advisory committees.

Generation of a human iPSC line, INMi002-A, carrying the most prevalent USH2A variant associated with Usher syndrome type 2

We generated an induced pluripotent stem cell (iPSC) line using dermal fibroblasts from a patient with Usher syndrome type 2 (USH2). This individual was homozygous for the most prevalent variant reported in the USH2A gene, c.2299delG localized in exon 13. Reprogramming was performed using the non-integrative Sendai virus reprogramming method and the human OSKM transcription factor cocktail under feeder-free culture conditions. This iPSC line will be an invaluable tool for studying the pathophysiology of USH2 and for testing the efficacy of novel treatments.

Testicular endothelial cells are a critical population in the germline stem cell niche

Maintenance of adult tissues depends on stem cell self-renewal in local niches. Spermatogonial stem cells (SSC) are germline adult stem cells necessary for spermatogenesis and fertility. We show that testicular endothelial cells (TECs) are part of the SSC niche producing glial cell line-derived neurotrophic factor (GDNF) and other factors to support human and mouse SSCs in long-term culture. We demonstrate that FGF-2 binding to FGFR1 on TECs activates the calcineurin pathway to produce GDNF. Comparison of the TEC secretome to lung and liver endothelial cells identified 5 factors sufficient for long-term maintenance of human and mouse SSC colonies in feeder-free cultures. Male cancer survivors after chemotherapy are often infertile since SSCs are highly susceptible to cytotoxic injury. Transplantation of TECs alone restores spermatogenesis in mice after chemotherapy-induced depletion of SSCs. Identifying TECs as a niche population necessary for SSC self-renewal may facilitate fertility preservation for prepubertal boys diagnosed with cancer.

Transcriptional landscape changes during human embryonic stem cell derivation.

What are the transcriptional changes occurring during the human embryonic stem cell (hESC) derivation process, from the inner cell mass (ICM) to post-ICM intermediate stage (PICMI) to hESC stage, that have downstream effects on pluripotency states and differentiation? We reveal that although the PICMI is transcriptionally similar to the hESC profile and distinct from ICM, it exhibits upregulation of primordial germ cell (PGC) markers, dependence on leukemia inhibitory factor (LIF) signaling, upregulation of naïve pluripotency-specific signaling networks and appears to be an intermediate switching point from naïve to primed pluripotency. It is currently known that the PICMI exhibits markers of early and late-epiblast stage. It is suggested that hESCs acquire primed pluripotency features due to the upregulation of post-implantation genes in the PICMI which renders them predisposed towards differentiation cues. Despite this current knowledge, the transcriptional landscape changes during hESC derivation from ICM to hESC and the effect of PICMI on pluripotent state is still not well defined. To gain insight into the signaling mechanisms that may govern the ICM to PICMI to hESC transition, comparative RNA sequencing (RNA-seq) analysis was performed on preimplantation ICMs, PICMIs and hESCs in biological and technical triplicates (n = 3). Primed hESCs (XX) were maintained in feeder-free culture conditions on Matrigel for two passages and approximately 50 cells were collected in biological and technical triplicates (n = 3). For ICM sample collection, Day 3, frozen-thawed human embryos were cultured up to day five blastocyst stage and only good quality blastocysts were subjected to laser-assisted micromanipulation for ICM collection (n = 3). Next, day six expanded blastocysts were cultured on mouse embryonic fibroblasts and manual dissection was performed on the PICMI outgrowths between post-plating Day 6 and Day 10 (n = 3). Sequencing of these samples was performed on NextSeq500 and statistical analysis was performed using edgeR (false discovery rate (FDR) < 0.05). Comparative RNA-seq data analysis revealed that 634 and 560 protein-coding genes were significantly up and downregulated in hESCs compared to ICM (FDR < 0.05), respectively. Upon ICM to PICMI transition, 471 genes were expressed significantly higher in the PICMI compared to ICM, while 296 genes were elevated in the ICM alone (FDR < 0.05). Principle component analysis showed that the ICM was completely distinct from the PICMI and hESCs while the latter two clustered in close proximity to each other. Increased expression of E-CADHERIN1 (CDH1) in ICM and intermediate levels in the PICMI was observed, while CDH2 was higher in hESCs, suggesting a role of extracellular matrix components in facilitating pluripotency transition during hESC derivation. The PICMI also showed regulation of naïve-specific LIF and bone morphogenetic protein signaling, differential regulation of primed pluripotency-specific fibroblast growth factor and NODAL signaling pathway components, upregulation of phosphatidylinositol 3-kinase (PI3K)/AKT/mammalian target of rapamycin (mTOR) pathway (PI3K/AKT/mTORC), as well as predisposition towards the germ cell lineage, further confirmed by gene ontology analysis. Hence, the data suggest that the PICMI may serve as an intermediate pluripotency stage which, when subjected to an appropriate culture niche, could aid in enhancing naïve hESC derivation and germ cell differentiation efficiency. Gene Expression Omnibus (GEO) Accession number GSE119378. Owing to the limitation in sample availability, the sex of ICM and PICMI have not been taken into consideration. Obtaining cells from the ICM and maintaining them in culture is not feasible as it will hamper the formation of PICMI and hESC derivation. Single-cell quantitative real-time PCR on low ICM and PICMI cell numbers, although challenging due to limited availability of human embryos, will be advantageous to further corroborate the RNA-seq data on transcriptional changes during hESC derivation process. We elucidate the dynamics of transcriptional network changes from the naïve ICM to the intermediate PICMI stage and finally the primed hESC lines. We provide an in-depth understanding of the PICMI and its role in conferring the type of pluripotent state which may have important downstream effects on differentiation, specifically towards the PGC lineage. This knowledge contributes to our limited understanding of the true nature of the human pluripotent state in vitro. This research is supported by the Concerted Research Actions funding from Bijzonder Onderzoeksfonds University Ghent (BOF GOA 01G01112).The authors declare no conflict of interest.

Ocular Surface Reconstruction with the Autologous Conjunctival Epithelium and Establishment of a Feeder-Free and Serum-Free Culture System

Transplantation of the autologous cultured corneal limbal epithelium and oral mucosal epithelium is a standard technique for ocular surface reconstruction under corneal limbal stem cell deficiency. As an option for bilateral cases, we recommend utilization of autologous conjunctivae for ocular surface reconstruction. Autologous conjunctival epithelium sheet transplantation was effective for bilateral corneal limbal stem cell deficiency without symblepharon or severe keratinization. Moreover, we established a feeder-free and serum-free culture system of the limbal epithelium. This system can be applied for culturing conjunctival epithelia. Autologous cultured conjunctival epithelium transplantation is a practical option for treating bilateral corneal limbal stem cell deficiency.

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models

Defined Xeno-free and Feeder-free Culture Conditions for the Generation of Human iPSC-derived Retinal Cell Models.

The production of specialized cells from pluripotent stem cells provides a powerful tool to develop new approaches for regenerative medicine. The use of human-induced pluripotent stem cells (iPSCs) is particularly attractive for neurodegenerative disease studies, including retinal dystrophies, where iPSC-derived retinal cell models mark a major step forward to understand and fight blindness. In this paper, we describe a simple and scalable protocol to generate, mature, and cryopreserve retinal organoids. Based on medium changing, the main advantage of this method is to avoid multiple and time-consuming steps commonly required in a guided differentiation of iPSCs. Mimicking the early phases of retinal development by successive changes of defined media on adherent human iPSC cultures, this protocol allows the simultaneous generation of self-forming neuroretinal structures and retinal pigmented epithelial (RPE) cells in a reproducible and efficient manner in 4 weeks. These structures containing retinal progenitor cells (RPCs) can be easily isolated for further maturation in a floating culture condition enabling the differentiation of RPCs into the seven retinal cell types present in the adult human retina. Additionally, we describe quick methods for the cryopreservation of retinal organoids and RPE cells for long-term storage. Combined together, the methods described here will be useful to produce and bank human iPSC-derived retinal cells or tissues for both basic and clinical research.

Derivation of induced pluripotent stem cells in Japanese macaque (Macaca fuscata)

Non-human primates are our closest relatives and are of special interest for ecological, evolutionary and biomedical research. The Japanese macaque (Macaca fuscata) has contributed to the progress of primatology and neurosciences over 60 years. Despite this importance, the molecular and cellular basis of the Japanese macaque remains unexplored since useful cellular tools are lacking. Here we generated induced pluripotent stem cells (iPSCs) from skin fibroblasts of the Japanese macaque with Sendai virus or plasmid vectors. The Japanese macaque iPSCs (jm-iPSCs) were established under feeder-free culture conditions, but feeder cells turned out to be essential for their maintenance. The jm-iPSCs formed human iPSC-like flat colonies which were positive for pluripotent antigens including alkaline phosphatase, SSEA4, and TRA-1-81. They also expressed endogenous OCT3/4, SOX2, L-MYC, and KLF4 and other pluripotent marker genes. The potential to differentiate into all three germ layers and neural stem cells was confirmed by embryoid body and neurosphere formation, respectively. The jm-iPSCs will provide a robust in vitro tool for investigating the underlying mechanisms of development and physiology studies with the Japanese macaque.