A Multi-Omics Approach for Evaluating the Impact of Cytokines and Donor Source on NK Cell Expansion

Introduction As the field of cancer immunotherapy progresses, natural killer (NK) cells have become an ideal candidate for targeting multiple types of cancer. These cells are able to selectively target virally infected cells and tumor cells without damaging the healthy cells of the immune system making them an ideal candidate for treatment in AML, CML, MM, Neuroblastoma, Breast Cancer, and Renal Cell Carcinoma. While NK cells have shown their potential as immunotherapy agents, one of the biggest challenges includes expanding and harvesting enough cells for multiple transfusions when there are limited numbers of starting cells. Our group developed an NK cell expansion platform utilizing membrane bound-IL21 (mbIL-21), which allows for sustained proliferation in order to generate high numbers of cells. Continued research supported that mbIL-21 enables sustained NK cell proliferation compared to mbIL-15 due to mbIL-21 activation through STAT3 signaling. Recent shifts to umbilical cord blood have led to research using cord blood (CB) derived NK cells. However, this presents a challenge as a viable clinical option as others have shown that the expansion of these CB-derived NK cells have been shown to produce less potent NK cells. Here, we will use feeder cells expressing either mbIL15 or mbIL21 to expand NK cells (as is currently done in clinical trials), to deeply compare and contrast the impact of these two cytokines on NK cell biology and simultaneously compare and contrast the use of peripheral-derived NK cells and CB-derived NK cells. Methods NK cells were obtained from healthy donor leukopacks (n =4) and isolated using RosetteSep human NK cell enrichment cocktail. These samples were stimulated with either mbIL-21 or mbIL-15 irradiated feeder cells (IFC) and cultured in AIM-V media supplemented with 100 I/U of IL-2 and immune cell serum replacement (ICSR). Cord Blood NK cells were obtained from OrganaBio (n=4) and stimulated with mbIL-21 IFC . The cells were cultured in AIM-V media supplemented with 100 I/U of IL-2 and ICSR (complete media). All NK cell expansions were stimulated at day 0 and day 7, as previously described, and cell cultures were assessed every 2-3 days keep cells at an optimal concentration by supplementing with complete media. Cells were collected at day 14 to capture a picture before mbIL-15 expanded cells became senescent and were immediately used for cytokine production assays, RNA collected, ATAC-seq collection, and CyTOF fixation. The remaining cells were frozen for cytotoxicity assays, mitochondrial function assays, and degranulation assays. For mitochondrial function and cytotoxicity assays, cells were thawed and cultured in complete media for two days before use. Results Overall, the peripheral blood NK cells expanded with mbIL21 showed an increase in expansion at day 14 when compared to those expanded with mbIL15. NK cells expanded with mbIL21 demonstrated a more consistent cytotoxicity profile against CHLA-136, a neuroblastoma cell line, compared to mbIL21 expanded NK cells when cultured at a 0.5:1 ratio (E:T). While the results were not statistically significant at 14 days, previous data demonstrates that we see increased changes at 21 days. Of note, we had an overall more reproducible serial killing ability in the IL21 expanded samples compared to the variability observed in IL15-expanded samples. We saw a significant difference (p < 0.0001) in peripheral blood NK cells expanded with mbIL21 compared to cb-NK cells expanded with mbIL21 with a 0.5:1 ratio (E:T) against CHLA136. mbIL-21 expanded peripheral blood NK cells had a higher maximum respiration capacity than those expanded with mbIL-15 when measured under mitochondrial stress via seahorse assay (p= 0.016). Conclusions Our data showed that the cells began to show a different phenotypic profile at day 14 depending on the type of NK cell and the type of cytokine used for stimulation. By day 14 data supported that mbIL-21 expanded cells can better survive under low glucose conditions such as tumor microenvironments than mbIL15 expanded cells. The cytotoxicity data demonstrated that peripheral derived NK cells have improved serial killing ability than the cb-NK cells and we will continue to test their ability against additional tumor cell lines that have various HLA-typing. To further elucidate these differences, we will use our RNA-seq, ATAC-seq, and CyTOF data to understand what is causing the different profiles. Figure 1 Figure 1. Disclosures Lee: Kiadis Pharma: Divested equity in a private or publicly-traded company in the past 24 months, Honoraria, Membership on an entity's Board of Directors or advisory committees, Patents & Royalties, Research Funding; Courier Therapeutics: Current holder of individual stocks in a privately-held company.

Restoration of keratinocytic phenotypes in autonomous trisomy-rescued cells

Background An extra copy of chromosome 21 in humans can alter cellular phenotypes as well as immune and metabolic systems. Down syndrome is associated with many health-related problems and age-related disorders including dermatological abnormalities. However, few studies have focused on the impact of trisomy 21 (T21) on epidermal stem cells and progenitor cell dysfunction. Here, we investigated the differences in keratinocytic characteristics between Down syndrome and euploid cells by differentiating cells from trisomy 21-induced pluripotent stem cells (T21-iPSCs) and autonomous rescued disomy 21-iPSCs (D21-iPSCs). Methods Our protocol for keratinocytic differentiation of T21-iPSCs and D21-iPSCs was employed. For propagation of T21- and D21-iPSC-derived keratinocytes and cell sheet formation, the culture medium supplemented with Rho kinase inhibitor on mouse feeder cells was introduced as growth rate decreased. Before passaging, selection of a keratinocytic population with differential dispase reactivity was performed. Three-dimensional (3D) air-liquid interface was performed in order to evaluate the ability of iPSC-derived keratinocytes to differentiate and form stratified squamous epithelium. Results Trisomy-rescued disomy 21-iPSCs were capable of epidermal differentiation and expressed keratinocytic markers such as KRT14 and TP63 upon differentiation compared to trisomy 21-iPSCs. The lifespan of iPSC-derived keratinocytes could successfully be extended on mouse feeder cells in media containing Rho kinase inhibitor, to more than 34 population doublings over a period of 160 days. Dispase-based purification of disomy iPSC-derived keratinocytes contributed epidermal sheet formation. The trisomy-rescued disomy 21-iPSC-derived keratinocytes with an expanded lifespan generated 3D skin in combination with a dermal fibroblast component. Conclusions Keratinocytes derived from autonomous trisomy-rescued iPSC have the ability of stratification for manufacturing 3D skin with restoration of keratinocytic functions.

P–797 A novel method for establishing human embryonic stem cells independent of feeder cells

Study question Is there a efficient establishing method of human embryonic stem cells directly from the human blastocysts independent of feeder cells? Summary answer We established a novel method of generating human embryonic stem cells directly from human blastocysts independent of feeder layer cells. What is known already Establishing embryonic stem cells lines mainly needed to coculture ICM clumps with feeder cells (like mouse or human fibroblasts) ,this brought in potential heterogeneous pollution.Although there had be some reports about generating human ESCs independent of feeder cells,but the efficiency was low and conditioned medium were unstable and also had the biological contamination. Study design, size, duration We used ten day5/6 donated human blastocysts from our reproductive center ,most of them were genetically diseased embryos with abnormal PGT diagnosis.After establishing ESCs procedure , all the cell lines were identified with pluripotency and differentiation potential tests.The success rate of system was calculated and compared with the conventional methods. Participants/materials, setting, methods In brief, ICM clumps were separated mechanically by using a micromanipulation system,and then transferred to a 30ul mTESR plus culture media drop pretreated with the geltrex (1:100 dilution) matrix and oxygen concentration was 5%. When cells attached and migrated,we also used laser to destroy the remaining trophoblast cells.About 10 days,the typical ES clone can be mechanically passaged and cells can be cultured in normal oxygen concentrations after passage 2. . Main results and the role of chance Using this method we had successfully established nine embryonic stem cell lines from donated human blastocysts ,the success rate was 90% (9/10). Each cell lines had passed the evaluation test of embryonic stem cell. When compared with the conventional feeder cells dependent method,our novol methods not only eliminated the pollution from heterogeneous cells,but also had higher success rate (90% vs 25%). Limitations, reasons for caution Due to the scarcity of donated human blastocysts, this experiment was a single-center experiment with small samples. Wider implications of the findings: We speculated that the batch differences of culture dishes, matrix and culture medium might affect the establish efficiency , and how to carry out a high level of quality control work might be the key factor to keep the system stable. Trial registration number basic research

P-797 A novel method for establishing human embryonic stem cells independent of feeder cells

Study question Is there a efficient establishing method of human embryonic stem cells directly from the human blastocysts independent of feeder cells? Summary answer We established a novel method of generating human embryonic stem cells directly from human blastocysts independent of feeder layer cells. What is known already Establishing embryonic stem cells lines mainly needed to coculture ICM clumps with feeder cells (like mouse or human fibroblasts), this brought in potential heterogeneous pollution. Although there had be some reports about generating human ESCs independent of feeder cells, but the efficiency was low and conditioned medium were unstable and also had the biological contamination. Study design, size, duration We used ten day5/6 donated human blastocysts from our reproductive center, most of them were genetically diseased embryos with abnormal PGT diagnosis. After establishing ESCs procedure, all the cell lines were identified with pluripotency and differentiation potential tests. The success rate of system was calculated and compared with the conventional methods. Participants/materials, setting, methods In brief, ICM clumps were separated mechanically by using a micromanipulation system,and then transferred to a 30ul mTESR plus culture media drop pretreated with the geltrex (1:100 dilution) matrix and oxygen concentration was 5%. When cells attached and migrated,we also used laser to destroy the remaining trophoblast cells. About 10 days,the typical ES clone can be mechanically passaged and cells can be cultured in normal oxygen concentrations after passage 2.. Main results and the role of chance Using this method we had successfully established nine embryonic stem cell lines from donated human blastocysts, the success rate was 90% (9/10). Each cell lines had passed the evaluation test of embryonic stem cell. When compared with the conventional feeder cells dependent method,our novol methods not only eliminated the pollution from heterogeneous cells,but also had higher success rate (90% vs 25%). Limitations, reasons for caution Due to the scarcity of donated human blastocysts, this experiment was a single-center experiment with small samples. Wider implications of the findings We speculated that the batch differences of culture dishes, matrix and culture medium might affect the establish efficiency, and how to carry out a high level of quality control work might be the key factor to keep the system stable. Trial registration number basic research

Efficient Surface Immobilization of Chemically Modified Hyaluronans for Enhanced Bioactivity and Survival of In Vitro-Cultured Embryonic Salivary Gland Mesenchymal Cells

Embryonic salivary gland mesenchyme (eSGM) secretes various growth factors (bioactives) that support the proper growth and differentiation of salivary gland epithelium. Therefore, eSGM cells can be used as feeder cells for in vitro-cultured artificial salivary gland if their survival and bioactivity are properly maintained. As eSGM is encapsulated in a hyaluronan (HA)-rich developmental milieu, we hypothesized that mimicking this environment in vitro via surface immobilization of HA might enhance survival and bioactivity of eSGM. In this study, various HA derivatives, conjugated with catechol (HA–CA), thiol (HA–SH), or amine (HA–EDA) moieties, respectively, were screened for their efficacy of culturing eSGM-derived feeder cells in vitro. Among these HA derivatives, HA–CA showed the highest surface coating efficiency and growth enhancement effect on the embryonic submandibular gland. In addition, the HA–CA coating enhanced the production of growth factors EGF and FGF7, but not FGF10. These effects were maintained when eSGM cells isolated from the embryonic salivary gland were re-seeded to develop the feeder layer cells. CD44s (a major HA receptor) in eSGM cells were clustered at the cell membrane, and enhanced EGF expression was detected only in CD44 cluster-positive cells, suggesting that membrane clustering of CD44 is the key mechanism for the increased expression of EGF.

Endometrial regeneration with endometrial epithelium: homologous orchestration with endometrial stroma as a feeder

Background Thin endometrium adversely affects reproductive success rates with fertility treatment. Autologous transplantation of exogenously prepared endometrium can be a promising therapeutic option for thin endometrium; however, endometrial epithelial cells have limited expansion potential, which needs to be overcome in order to make regenerative medicine a therapeutic strategy for refractory thin endometrium. Here, we aimed to perform long-term culture of endometrial epithelial cells in vitro. Methods We prepared primary human endometrial epithelial cells and endometrial stromal cells and investigated whether endometrial stromal cells and human embryonic stem cell-derived feeder cells could support proliferation of endometrial epithelial cells. We also investigated whether three-dimensional culture can be achieved using thawed endometrial epithelial cells and endometrial stromal cells. Results Co-cultivation with the feeder cells dramatically increased the proliferation rate of the endometrial epithelial cells. We serially passaged the endometrial epithelial cells on mouse embryonic fibroblasts up to passage 6 for 4 months. Among the human-derived feeder cells, endometrial stromal cells exhibited the best feeder activity for proliferation of the endometrial epithelial cells. We continued to propagate the endometrial epithelial cells on endometrial stromal cells up to passage 5 for 81 days. Furthermore, endometrial epithelium and stroma, after the freeze-thaw procedure and sequential culture, were able to establish an endometrial three-dimensional model. Conclusions We herein established a model of in vitro cultured endometrium as a potential therapeutic option for refractory thin endometrium. The three-dimensional culture model with endometrial epithelial and stromal cell orchestration via cytokines, membrane-bound molecules, extracellular matrices, and gap junction will provide a new framework for exploring the mechanisms underlying the phenomenon of implantation. Additionally, modified embryo culture, so-called “in vitro implantation”, will be possible therapeutic approaches in fertility treatment.

Measurement of the Bio-Mechanical Properties of Two Different Feeder Layer Cells

Introduction: We here present our findings on 2 types of feeder layers, one composed of mouse embryonic fibroblasts (MEF) and the second one of mouse skeletal myoblasts (C2Cl2) feeder cells. Methods: The 2 feeder layers present a dramatic variance of intrinsic stiffness (142.68 ± 17.21 KPa and 45.78 ± 9.81 KPa, respectively). Results and Conclusion: This information could be used for a better understanding of cells and cell microenvironment mechano-physical characteristics that are influencing stem cell commitment, in order to develop a suitable engineered tissue for cardiac and skeletal muscle repair and a bio-actuator.