Feeder Cells Research Articles

Adoptive NK Cell Transfer as a Treatment in Colorectal Cancer Patients: Analyses of Tumour Cell Determinants Correlating With Efficacy In Vitro and In Vivo.

BackgroundColorectal cancer (CRC) is a heterogeneous disease with variable mutational profile and tumour microenvironment composition that influence tumour progression and response to treatment. While chemoresistant and poorly immunogenic CRC remains a challenge, the development of new strategies guided by biomarkers could help stratify and treat patients. Allogeneic NK cell transfer emerges as an alternative against chemoresistant and poorly immunogenic CRC.MethodsNK cell-related immunological markers were analysed by transcriptomics and immunohistochemistry in human CRC samples and correlated with tumour progression and overall survival. The anti-tumour ability of expanded allogeneic NK cells using a protocol combining cytokines and feeder cells was analysed in vitro and in vivo and correlated with CRC mutational status and the expression of ligands for immune checkpoint (IC) receptors regulating NK cell activity.ResultsHLA-I downmodulation and NK cell infiltration correlated with better overall survival in patients with a low-stage (II) microsatellite instability-high (MSI-H) CRC, suggesting a role of HLA-I as a prognosis biomarker and a potential benefit of NK cell immunotherapy. Activated allogeneic NK cells were able to eliminate CRC cultures without PD-1 and TIM-3 restriction but were affected by HLA-I expression. In vivo experiments confirmed the efficacy of the therapy against both HLA+ and HLA− CRC cell lines. Concomitant administration of pembrolizumab failed to improve tumour control.ConclusionsOur results reveal an immunological profile of CRC tumours in which immunogenicity (MSI-H) and immune evasion mechanisms (HLA downmodulation) favour NK cell immunosurveillance at early disease stages. Accordingly, we have shown that allogeneic NK cell therapy can target tumours expressing mutations conferring poor prognosis regardless of the expression of T cell-related inhibitory IC ligands. Overall, this study provides a rationale for a new potential basis for CRC stratification and NK cell-based therapy.

Methanol fixed feeder layers altered the pluripotency and metabolism of bovine pluripotent stem cells

The pluripotency maintenance of pluripotent stem cells (PSCs) requires the suitable microenvironment, which commonly provided by feeder layers. However, the preparation of feeder layers is time consuming and labor exhaustive, and the feeder cells treated with mitomycin C or γ-ray irradiation bring heterologous contamination. In this study, mouse embryonic fibroblasts (MEFs) were treated by methanol to generate chemical fixed feeder cells, and bovine embryonic stem cells F7 (bESC-F7) cultured on this feeder layer. Then the pluripotency and metabolism of bESC-F7 cultured on methanol-fixed MEFs (MT-MEFs) named MT-F7 was compared with mitomycin C treated MEFs (MC-MEFs). The results showed that bESC-F7 formed alkaline phosphatase positive colonies on MT-MEFs, the relative expression of pluripotent markers of these cells was different from the bESCs cultured on the MC-MEFs (MC-F7). The long-term cultured MT-F7 formed embryoid bodies, showed the ability to differentiate into three germ layers similar to MC-F7. The analyses of RNA-seq data showed that MT-MEFs lead bESCs to novel steady expression patterns of genes regulating pluripotency and metabolism. Furthermore, the bovine expanded pluripotent stem cells (bEPSCs) cultured on MT-MEFs formed classical colonies, maintained pluripotency, and elevated metabolism. In conclusion, MT-MEFs were efficient feeder layer that maintain the distinctive pluripotency and metabolism of PSCs.

Efficient expansion of mouse hematopoietic stem cells ex vivo by membrane anchored Angptl2

Hematopoietic stem cell (HSC) transplantation represents an important curative therapy for numerous hematological and immune diseases. Many efforts have been applied to achieve attainable ex vivo HSC expansion. We previously showed that angiopoietin-like proteins 2 (Angptl2) binds and activates the immune inhibitory receptor human leukocyte immunoglobulin (Ig)-like receptor B2 (LILRB2) to support the expansion of HSC. However, soluble Angptl2 is unstable and the downstream signaling would be attenuated by ligand-binding triggered receptor endocytosis, compromising the potential of Angptl2 to expand HSCs. We proposed that membrane anchored Angptl2 will overcome these limitations. In this study, we constructed the C-terminal and N-terminal anchored membrane Angptl2 (Cm-Angptl2 and Nm-Angptl2) by adding a transmembrane domain at the C-terminal or an anchor sequence at the N-terminal respectively. Both forms of Angptl2 showed efficient expression on the surface of feeder cells. Nm-Angptl2, but not Cm-Angptl2, induces a potent activation of LILRB2 reporter, indicating the fibronectin (FBN) domain at the C-terminus of Angptl2 is essential to stimulate LILRB2 signaling. Compared to soluble Angptl2, Nm-Angptl2 displays higher activities to activate LILRB2 reporter, and to promote the expansion of mouse HSCs as determined by transplantation and limiting dilution assay. Our study revealed the importance of FBN domain for Angptl2 to activate LILRB2 and demonstrated that Nm-Angptl2 have enhanced activities than the soluble protein in LILRB2 activation and HSC expansion, providing a strategy to explore the mode of ligand induced receptor signaling, and an optimized approach to expand HSCs ex vivo.

NKG2C+ NK Cells for Immunotherapy of Glioblastoma Multiforme.

In glioblastoma, non-classical human leucocyte antigen E (HLA-E) and HLA-G are frequently overexpressed. HLA-E loaded with peptides derived from HLA class I and from HLA-G contributes to inhibition of natural killer (NK) cells with expression of the inhibitory receptor CD94/NKG2A. We investigated whether NK cells expressing the activating CD94/NKG2C receptor counterpart were able to exert anti-glioma effects. NKG2C+ subsets were preferentially expanded by a feeder cell line engineered to express an artificial disulfide-stabilized trimeric HLA-E ligand (HLA-E*spG). NK cells expanded by a feeder cell line, which facilitates outgrowth of conventional NKG2A+, and fresh NK cells, were included for comparison. Expansion via the HLA-E*spG feeder cells selectively increased the fraction of NKG2C+ NK cells, which displayed a higher frequency of KIR2DL2/L3/S2 and CD16 when compared to expanded NKG2A+ NK cells. NKG2C+ NK cells exhibited increased cytotoxicity against K562 and KIR:HLA-matched and -mismatched primary glioblastoma multiforme (GBM) cells when compared to NKG2A+ NK cells and corresponding fresh NK cells. Cytotoxic responses of NKG2C+ NK cells were even more pronounced when utilizing target cells engineered with HLA-E*spG. These findings support the notion that NKG2C+ NK cells have potential therapeutic value for treating gliomas.

Evaluation of allogeneic and autologous membrane-bound IL-21–expanded NK cells for chronic lymphocytic leukemia therapy

AbstractSuccesses with anti-CD20 antibodies in chronic lymphocytic leukemia (CLL) and enhanced activity of Fc-engineered vs unmodified antibody therapy suggest a potentially impactful role for natural killer (NK) cells and other innate immune cells in controlling this disease. Stimulated NK cells have shown promise as a cellular therapy, but their application has been constrained by limited expansion capacity and low cytotoxic activity against CLL cells. Here, we demonstrate that both healthy donor-derived and CLL patient-derived NK cells expand rapidly when stimulated with feeder cells expressing membrane-bound interleukin-21 (mbIL-21) and have potent cytotoxic activity against allogeneic or autologous CLL cells. Combination with anti-CD20 antibodies significantly enhances NK recognition and killing of CLL targets. As any CLL immune therapy would likely be given in combination, we assess commonly used treatments and demonstrate that ibrutinib has mixed suppressive and protective effects on expanded NK cells, whereas expanded NKs are highly resistant to venetoclax. We demonstrate efficacy in vivo in 2 xenograft mouse models of human CLL that support building upon a regimen of venetoclax and obinutuzumab with mbIL-21–expanded NK cells. Collectively, these data support development of mbIL-21–expanded NKs combined with the CD20 antibody obinutuzumab and venetoclax in the treatment of CLL.

Robust Serum- and Feeder-Free Expansion of Mouse B Cells In Vitro

Abstract Mouse B cells are used in workflows to discover novel therapeutic antibodies or develop preclinical applications using genetically engineered B cells. In vitro expansion of B cells offers the opportunity to improve the recovery of antibody sequences and aids in the potential increase in the diversity of the B cell repertoire. Achieving robust expansion of B cells in culture can be challenging, as it typically requires the addition of serum, feeder cells, or specialized culture plates. The addition of serum can introduce unknown factors and result in lot-to-lot variability. Therefore, we have developed a first-to-market culture system that does not require the use of serum, feeder cells, or specialized culture plates to achieve robust in vitro expansion of mouse pan-B cells. Pan-B cells isolated from mouse splenocytes by immunomagnetic enrichment were cultured in serum-free ImmunoCultTM-XF B Cell Base Medium supplemented with ImmunoCultTM-ACF Mouse B Cell Expansion Supplement for > 9 days, starting with seeding densities of 100,000 cells/well and passaging every 3 days. Although pronounced donor variability was observed in the rate of expansion of viable cells, mean ~13-fold, ~150-fold, and ~200-fold rates were observed after 3, 6, and 9 days in culture, respectively (n=6 donors; range 94- to 328-fold at day 9). The expanded B cells also upregulated expression of activation and differentiation markers such as CD86 (mean 74%; n=6 donors; range 34 – 90%), CD138 (mean 68%; n=6 donors; range 57 – 73%), and CD267 (mean 79%; n=6 donors; range 49 – 91%) at day 9. The expansion supplement should facilitate mouse B cell studies for antibody discovery and diverse basic and translational applications.

A feeder cell-free activation and expansion strategy to generate memory-like NK cells sufficient for off-the-shelf multi-dose adoptive cell therapy.

Abstract Adoptive cell therapy (ACT) using NK cells is a promising armament in the fight against cancer. Cytokine induced memory like (CIML) NK cells have been shown in clinical studies to have potent antitumor activity with superior in vivo persistence. Currently, the expansion of NK cells for clinical development is mainly based on feeder cells, which imposes significant regulatory hurdles and increases the costs for manufacturing. We have developed fusion proteins, HCW9201 and HCW9206 comprising of IL-15/IL-18/IL-12 and IL15/IL-7/IL-21, respectively, capable of priming memory-like differentiation and expanding CIML NK cell products without using feeder cells. This “Kick and Expand” strategy allows greater than 100x expansion of CIML NK cells from donor PBMCs in as little as 14 days without the use of exogenous feeder cells. Continued expansion can yield sufficient CIML NK cells for cryopreservation and multiple ACT infusions. The NK cells generated have bona fide memory-like properties: enhanced antitumor activity across multiple cancer cell lines, higher metabolic capacity, stable epigenetic demethylation of the IFN-γ promoter and increased persistence in NSG mice, when compared to conventional NK cells. In conclusion, this “Kick and Expand” process supports generation of abundant CIML NK cells for multiple ACT infusions and provides simpler, more regulatory friendly, off-the-shelf platform for generating NK cell products, including those with chimeric antigen receptor (CAR) constructs.

Optimization of expansion techniques for adoptive NK cell transfer in dogs with cancer

Abstract Natural killer (NK) cells can recognize heterogeneous cancer cell targets without prior sensitization, making them promising prospects for use in immunotherapy. We have completed first-in-dog feasibility clinical trials in dogs with cancer using both autologous and allogeneic NK cells expanded from peripheral blood mononuclear cells (PBMCs). Previously, CD5 depletion of PBMCs has been used to enrich for a CD5dim expressing subset prior to NK co-culture with an irradiated feeder line, but this can limit the yield of the final NK product. The purpose of this study was to compare ex vivo culture conditions using standard CD5 depletion versus unmanipulated PBMCs plus feeder line co-culture with 100 IU/mL rhIL-2 in matched healthy donors, hypothesizing that PBMCs plus feeder cells would generate an equivalent or superior NK product to CD5 depletion. Cell count, fold change, and viability data were collected for both techniques in 12 dogs across five time points up to day 14. A mixed-effects model analysis showed no statistical difference in calculated cell counts, overall fold change, and viability (p>0.05 all) between PBMCs with feeders and CD5 depleted cells with feeders. PBMCs had a higher mean than CD5 depleted cells at day 14 in all three categories, reaching a peak mean of 677 million cells from 5 million PBMCs at day 0. Flow phenotype showed similar upregulation of NKp46 and Granzyme B expression. Killing assays against melanoma and osteosarcoma targets demonstrated comparable results among PBMCs plus feeders versus CD5 depleted NK cells (p>0.05). Overall, these findings support the use of unmanipulated PBMCs plus feeder line coculture as an equivalent method to CD5 depletion in the expansion of canine NK cells for adoptive immunotherapy. Supported by grants from National Institutes of Health/National Cancer Institute (U01 CA224166-01, 1R03CA252793, T32 CA251007-01)

Robust human regulatory T cell expansion with fusion proteins HCW9302 and HCW9213 circumvents need for magnetic-bead or feeder cell approaches for adoptive cell therapy

Abstract Regulatory T cells (CD4+CD25+FoxP3+) (Tregs) are a subset of CD4 T cells that suppress the activities of other immune cells and have applications in the treatment of autoimmune and inflammatory diseases. Their use as an adoptive cell therapy has been limited by the practicality of expanding and purifying clinically sufficient numbers of cells. HCW9213 and HCW9302 are fusion proteins based on HCW Biologics’ TOBI™ technology platform, consisting of anti-CD3/anti-CD28 antibody domains and IL-2 domains, respectively. When used in combination, these fusion proteins were capable of expanding human Treg cells in vitro without the use of anti-CD3/CD28 magnetic beads and/or feeder cells, improving the overall yield, process efficiency and overcoming regulatory hurdles in manufacturing. Tregs generated with these molecules displayed similar phenotypes and suppressive cytokine production as Tregs expanded with recombinant human IL-2. Using a proprietary anti-CD39 antibody to isolate CD39+ Tregs, we have also been able to generate a Treg population with twice the suppressive activity against CD4+ T responder cells as traditional CD4+CD25+CD127lo Tregs. Thus, using its novel fusion proteins, HCW Biologics has been able to develop a superior Treg cell product ideal for the use in adoptive cell transfer. Additionally, this Treg platform can potentially be further optimized with addition of disease-targeted chimeric antigen receptors (CAR).

Generation of large numbers of functional NK cells without feeders or serum

Abstract Natural killer (NK) cells are innate lymphocytes that modulate immune responses by secreting proinflammatory cytokines, and provide antitumor and antivirus activity. The ability to generate therapeutically relevant numbers of functional NK cells is critical for the development of advanced cellular immunotherapies. We have developed a streamlined culture workflow that enables expansion of functional NK cells without using either feeder cells or serum. Fresh human NK cells were isolated from peripheral blood using EasySep™, then cultured in ImmunoCult™ NK Cell Expansion Medium in plates coated with ImmunoCult™ NK Cell Expansion Coating Material. NK cells were fed, harvested, and replated on days 7 and 10 – 11 prior to phenotyping and functional assessment on day 14. The average frequency of CD56+CD3− NK cells was 88% (range 75 – 96%, n=34), 74% (range 49 – 92%) of which expressed CD16, with an average expansion of 88 fold (range 3 – 454). Expanded NK cells were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, or co-cultured with K562 cells. As detected by intracellular flow cytometry, the average frequency of interferon-γ+ among NK cells was 68% (range 41 – 90%, n=7) and 43% (range 12 – 55%) when stimulated by PMA/ionomycin or K562 cells, respectively. Similarly, the frequency of degranulated NK cells (CD107a+) was 81% (range 35 – 97%, n=7) and 64% (range 35 – 97%), respectively. The ability of expanded NK cells to kill target K562 cells was visualized by activated caspase in co-cultures with labeled K562 cells using the Incucyte® imaging system. At an effector:target ratio of 1:1, an average of 50% of K562 cells were killed (range 45 – 56, n=3). These results show large numbers of functional NK cells can be generated.

Keratinocyte Culture: Siriraj’s Experience

Objective: Cell-based therapy is gaining increasing prominence in medicine, where it has the potential to replace or repair damaged tissue using new engineered cells. Skin cell engineering, also known as keratinocyte culture or cultured epithelial autograft (CEA), is a promising field in cell-based therapy. CEA is now used in many parts of the world as an alternative treatment for some diseases that require large defects to be covered, such as severe and major burn patients and congenital melanocytic nevus. The use of CEA in conjunction with acellular skin substitution is rapidly expanding.Materials and Methods: This study is an initiative aimed at supporting the production and use of keratinocyte cultures at Siriraj Hospital. This is the first stage of developing sheet keratinocyte culture in vitro.Results: Our study yielded very promising results. As feeder cells, we used irradiated 3T3 murine fibroblasts, as per the standard protocol for keratinocyte culture. The growth duration was four weeks: 2 weeks for the 3T3 murine fibroblasts and 2 weeks for the keratinocytes. The keratinocytes grew rapidly and formed sheets with irradiated 3T3 murine fibroblasts. The retrieval of the cell sheets was straightforward thanks to the temperature-response cell culture dish and halo-ring cell recovery sheet. Flow cytometry revealed that the cells had a very high viability and purity. H&E staining revealed the sheets comprised two to four layers of stratified epithelial tissue.Conclusion: From this study, our method of manufacturing the CEA can offer a promising result. This can be use in the treatment which require large skin coverage. However, we aim to initiate animal and human trial phase next.

Comparison of the characteristics of NK cells after two different methods of expansion and observation of the clinical efficacy in patients who relapsed post allogeneic hematopoietic stem cell transplantation

目的比较不同扩增体系NK细胞生物学特性的差异以及治疗异基因造血干细胞移植后（allo-HSCT）白血病复发的疗效。方法分别采用CD3/CD52单抗扩增法和饲养层细胞扩增法诱导供者来源NK细胞大量扩增，检测扩增前后NK细胞表型、因子分泌、细胞毒的变化规律；选取16例allo-HSCT后复发白血病患者，8例输注CD3/CD52单抗扩增NK细胞，8例输注饲养层细胞扩增NK细胞，观察患者的治疗反应和长期生存情况。结果①与CD3/CD52单抗扩增体系相比，饲养层细胞扩增体系NK细胞纯度较高、NK细胞表面活化性受体DNAM-1及NKp30表达较高、抑制性受体CTLA-4表达较低，而两种NK细胞NKG2D/CD25/CD69/Trail/PD-1/TIM-3/TIGIT表达差异无统计学意义。②两种扩增体系NK细胞Ki-67指数均明显增加，以饲养层细胞扩增NK细胞尤为明显；饲养层细胞扩增NK细胞穿孔素及颗粒酶B表达水平均明显高于CD3/CD52单抗扩增NK细胞。③16例allo-HSCT后复发白血病患者NK细胞输注过程中均未观察到明显不良反应。NK细胞输注后中位随访时间为2554（917～2583）d，CD3/CD52单抗扩增组中3例患者无白血病存活，5例死亡；饲养层细胞扩增组中6例患者长期存活，2例死亡。5例患者在NK细胞输注前存在移植物抗宿主病，NK细胞输注后移植物抗宿主病未加重甚至缓解。结论CD3/CD52单抗扩增与饲养层细胞扩增体系NK细胞的生物学特性具有明显差异；NK细胞输注对allo-HSCT后复发白血病患者的疗效仍需要进一步验证。

Notch signaling is required for generation of conventional type 1 dendritic cells from human induced pluripotent stem cells

Abstract Compelling evidence shows a critical role of conventional type 1 dendritic cells (cDC1) in T-cell mediated antitumor immunity; however, obtaining large numbers of cDC1 is difficult. Theoretically, this limitation can be overcome by using induced pluripotent stem cells (iPSC) that could provide an unlimited source of autologous cDC1. However, generation of cDC1 from human iPSC remains elusive. Here, we hypothesized that Notch signaling would facilitate generation of cDC1 from human iPSC. We found that human iPSC gave rise to CD141+XCR1+CLEC9A+HLA-DR+ cells on OP9 feeder cells expressing the Notch ligand delta-like 1 (OP9-DL1) while the majority of iPSC-derived cells differentiated on OP9 cells were monocytic cells. Single-cell RNA sequencing analyses confirmed the presence of cDC1 as well as identified the heterogeneity of iPSC-derived CD1c+ cells; cDC2A, cDC2B, CD5+cDC2, and DC3. Inhibition of Notch signaling during co-culture of iPSC-derived CD34+ hematopoietic progenitor cells with OP9-DL1 cells abrogated generation of cDC1, and made expression of CD5, CLEC4A, CLEC10A and CD163 in CD1c+ DC similar to those differentiated on OP9 cells. Notch-activated human iPSC-derived XCR1+CLEC9A+HLA-DR+CD11c+ cells exhibited similar gene expression profile with peripheral blood cDC1, and phagocytotic, T-cell proliferative, and cytokine producing functions. Our study demonstrates that Notch signaling is required for the differentiation of cDC1-like cells, and facilitates generation of various cDC subsets from human iPSC. These findings provide insights into the future development of personalized treatment with unlimited numbers of autologous cDC1 from human iPSC. This work was supported by National Cancer Institute (NCI) grant P30CA016056 involving the use of Roswell Park’s Flow and Image Cytometry, Bioinformatics, and Genomic Shared Resources. This work was supported by the Melanoma Research Alliance and the Sarcoma Foundation of America (F. Ito), Uehara Memorial Foundation (T. Oba), and National Cancer Institute (NCI) grant, U24CA232979 (M. Long and S. Liu), K08CA197966 and R01CA255240-01A1 (F. Ito).

Analysis of the diagnosis of Japanese patients with primary ciliary dyskinesia using a conditional reprogramming culture.

Primary ciliary dyskinesia (PCD) is diagnosed through multiple methods, including transmission electron microscopy (TEM), a high-speed video microscopy analysis (HSVA), immunofluorescence (IF), and genetic testing. A primary cell culture has been recommended to avoid the misdiagnosis of secondary ciliary dyskinesia derived from infection or inflammation and improve diagnostic accuracy. However, primary cells fail to differentiate into ciliated cells through repeated passages. The conditional reprogramming culture (CRC) method, a combination of a Rho-kinase inhibitor and fibroblast feeder cells, has been applied to cystic fibrosis. The goal of this study was to evaluate the value of CRC in diagnosing PCD in Japanese patients. Eleven patients clinically suspected of having PCD were included. Airway epithelial cells were obtained from an endobronchial forceps biopsy and cultured at the air-liquid interface (ALI) combined with CRC. Ciliary movement, ultrastructure, and mutated ciliary protein evaluation were performed using HSVA, TEM, and IF, respectively. Genetic testing was performed on some patients. CRC yielded dense and well-differentiated ciliated cells with a high success rate (∼90%). In patients with PCD, the ciliary ultrastructure phenotype (outer dynein arm defects or normal ultrastructure) and IF findings (absence of the mutated ciliary protein) were confirmed after CRC. In DNAH11-mutant cases with normal ultrastructure by TEM, the HSVA revealed stiff and hyperfrequent ciliary beating with low bending capacity in CRC-expanded cells, thereby supporting the diagnosis. CRC could be a potential tool for improving diagnostic accuracy and contributing to future clinical and basic research in PCD.

Feeding role of mouse embryonic fibroblast cells is influenced by genetic background, cell passage and day of isolation.

Mouse embryonic fibroblast (MEF) cells are commonly used as feeder cells to maintain the pluripotent state of stem cells. MEFs produce growth factors and provide adhesion molecules and extracellular matrix (ECM) compounds for cellular binding. In the present study, we compared the expression levels of Fgf2, Bmp4, ActivinA, Lif and Tgfb1 genes at the mRNA level and the level of Fgf2 protein secretion and Lif cytokine secretion at passages one, three and five of MEFs isolated from 13.5-day-old and 15.5-day-old embryos of NMRI and C57BL/6 mice using real-time PCR and enzyme-linked immunosorbent assay. We observed differences in the expression levels of the studied genes and secretion of the two growth factors in the three passages of MEFs isolated from 13.5-day-old and 15.5-day-old embryos, respectively. These differences were also observed between the NMRI and C57BL/6 strains. The results of this study suggested that researchers should use mice embryos that have different genetic backgrounds and ages, in addition to different MEF passages, when producing MEFs based on the application and type of their study.

A Role for Exchange of Extracellular Vesicles in Porcine Spermatogonial Co-Culture.

Spermatogonial stem cells (SSCs) provide the basis for lifelong male fertility through self-renewal and differentiation. Prepubertal male cancer patients may be rendered infertile by gonadotoxic chemotherapy and, unlike sexually mature men, cannot store sperm. Alternatively, testicular biopsies taken prior to treatment may be used to restore fertility in adulthood. Testicular SSC populations are limited, and in vitro culture systems are required to increase numbers of SSCs for treatment, demanding culture systems for SSC propagation. Using the pig as a non-rodent model, we developed culture systems to expand spermatogonia from immature testis tissue, comparing different feeders (Sertoli cells, peritubular myoid cells (PMCs) and pig fetal fibroblasts (PFFs)). Spermatogonia co-cultured with Sertoli cells, PMCs and PFFs had comparable rates of proliferation and apoptosis. To elucidate the mechanism behind the beneficial nature of feeder layers, we investigated the role of extracellular vesicles in crosstalk between spermatogonia and feeder cells. Sertoli cell-released exosomes are incorporated by spermatogonia, and inhibition of exosomal release reduces spermatogonial proliferation. Together, these results show that PMCs, PFFs and Sertoli cells promote spermatogonial proliferation in co-culture, with exosomal exchange representing one possible mechanism. Further characterization of exosomal cargo may ultimately allow the development of feeder-free culture systems for clinical use.

3D-organoid culture supports differentiation of human CAR+ iPSCs into highly functional CAR Tcells.

Unlimited generation of chimeric antigen receptor (CAR) Tcells from human-induced pluripotent stem cells (iPSCs) is an attractive approach for "off-the-shelf" CAR Tcell immunotherapy. Approaches to efficiently differentiate iPSCs into canonical αβ Tcell lineages, while maintaining CAR expression and functionality, however, have been challenging. We report that iPSCs reprogramed from CD62L+ naive and memory Tcells followed by CD19-CAR engineering and 3D-organoid system differentiation confers products with conventional CD8αβ-positive CAR Tcell characteristics. Expanded iPSC CD19-CAR Tcells showed comparable antigen-specific activation, degranulation, cytotoxicity, and cytokine secretion compared with conventional CD19-CAR Tcells and maintained homogeneous expression of the TCR derived from the initial clone. iPSC CD19-CAR Tcells also mediated potent antitumor activity invivo, prolonging survival of mice with CD19+ human tumor xenografts. Our study establishes feasible methodologies to generate highly functional CAR Tcells from iPSCs to support the development of "off-the-shelf" manufacturing strategies.

The In Vitro Differentiation of Human CD141+CLEC9A+ Dendritic Cells from Mobilized Peripheral Blood CD34+ Hematopoietic Stem Cells.

As shown in various preclinical studies, conventional type-1 dendritic cells, or cDC1s, play a critical role in the immunological rejection of tumors and in the defense against pathogens. This indispensability stems from their potent capacity to activate cytotoxic T cells, especially via the cross-presentation of exogenous antigens. For this reason, cDC1s have become an attractive target for immunotherapy. Here we report a simplified method for generating large numbers of cDC1-like cells in vitro from mobilized human peripheral blood CD34+ hematopoietic stem cells using FMS-like tyrosine kinase 3 ligand (FLT3L) and granulocyte-macrophage colony-stimulating factor (GM-CSF). An important aspect of this Protocol is the growth of cells on a non-tissue culture-treated surface rather than on a tissue culture-treated surface since the latter suppresses cDC1-marker expression. The resulting CD11c+ DCs express high levels of cDC1-specific markers such as CD141, CLEC9A, TLR3, and several DC maturation markers. Compared to alternative differentiation methods, this method generates large numbers of cDC1-like cells without the need for immortalized feeder cells and should prove useful for studying cDC1 immunobiology and clinical applications of this DC subset. © 2022 Wiley Periodicals LLC. Basic Protocol: Generation of human CD141+CLEC9A+ dendritic cells from mobilized peripheral blood CD34+ hematopoietic stem cells Support Protocol: Flow cytometric immunophenotyping of CD141+ dendritic cells.

Differential effects on natural killer cell production by membrane-bound cytokine stimulations.

Robust manufacturing production of natural killer (NK) cells has been challenging in allogeneic NK cell-based therapy. Here, we compared the impact of cytokines on NK cell expansion by developing recombinant K562 feeder cell lines expressing membrane-bound cytokines, mIL15, mIL21, and 41BBL, individually or in combination. We found that 41BBL played a dominant role in promoting up to 500,000-fold of NK cell expansion after a 21-day culture process without inducing exhaustion. However, 41BBL stimulation reduced the overall cytotoxic activity of NK cells when combined with mIL15 and/or mIL21. Additionally, long-term stimulation with mIL15 and/or mIL21, but not 41BBL, increased CD56 expression and the CD56bright population, which is unexpectedly correlated with NK cell cytotoxicity. By conducting single-cell sequencing, we identified distinct subpopulations of NK cells induced by different cytokines, including an adaptive-like CD56bright CD16- CD49a+ subset induced by mIL15. Through gene expression analysis, we found that different cytokines modulated signaling pathways and target genes involved in cell cycle, senescence, self-renewal, migration, and maturation in different ways. Together, our study demonstrates that cytokine signaling pathways play distinct roles in NK cell expansion and differentiation, which sheds light on NK cell process designs to improve productivity and product quality.

A Trichinella spiralis new born larvae-specific protein, Ts-NBL1, interacts with host\u2019s cell vimentin

The parasitic nematode Trichinella has a special relationship with its host as it has a unique intracellular location within the feeder cell which is a structure derived from skeletal muscle fiber. It has been proposed that “parakines” secreted by Trichinella larvae serve as messengers to implement communication between the parasite and the muscle cells through a molecular cross-talk to ensure permanent coexistence within the host. The Ts-NBL1 protein is considered to be a potential key “parakine” involved in the early invasion of the muscle fiber and its transformation into a feeder cell during Trichinella spiralis infection. This study used for the first time yeast two-hybrid (Y2H) technology in Trichinella to identify Ts-NBL1 interacting proteins. GST co-affinity purification experiments confirmed vimentin as an important interactor. The discovery of the new host proteins interacting with Ts-NBL1 will help to suggest that Ts-NBL1 contributes to participate in the capsule formation of feeder cells and provide ideas for understanding the molecular and cellular mechanisms involved in the survival of Trichinella in the host.