2B4 inhibits the apoptosis of natural killer cells through phosphorylated extracellular signal-related kinase/B-cell lymphoma 2 signal pathway.

BACKGROUND AIMS: Decades after the identification of natural killer (NK) cells as potential effector cells against malignantly transformed cells, an increasing amount of research suggests that NK cells are a prospective choice of immunocytes for cancer immunotherapy in addition to T lymphocytes for cancer immunotherapy. Recent studies have led to a breakthrough in the combination of hematopoietic stem-cell transplantation with allogeneic NK cells infusion for the treatment of malignant tumors. However, the short lifespan of NK cells in patients is the major impediment, limiting their efficacy. Therefore, prolonging the survival of NK cells will promote the application of NK-cell immunotherapy. As we have known, NK cells use a "missing-self" mechanism to lyse target cells and exert their functions through a wide array of activating, co-stimulatory and inhibitory receptors. Our previous study has suggested that CD244 (2B4), one of the co-stimulatory receptors, can improve the function of chimeric antigen receptor NK cells. However, the underlying mechanism of how 2B4 engages in the function of NK cells requires further investigation. Overall, we established a feeder cell with the expression of CD48, the ligand of 2B4, to investigate the function of 2B4-CD48 axis in NK cells, and meanwhile, to explore whether the newly generated feeder cell can improve the function of ex vivo-expanded NK cells. METHODS: First, K562 cells overexpressing 4-1BBL and membrane-bound IL-21 (mbIL-21) were constructed (K562-41BBL-mbIL-21) and were sorted to generate the single clone. These widely used feeder cells (K562-41BBL-mbIL-21) were named as Basic Feeder hereinafter. Based on the Basic feeder, CD48 was overexpressed and named as CD48 Feeder. Then, the genetically modified feeder cells were used to expand primary NK cells from peripheral blood or umbilical cord blood. In vitro experiments were performed to compare proliferation ability, cytotoxicity, survival and activation/inhibition phenotypes of NK cells stimulated via different feeder cells. K562 cells were injected into nude mice subcutaneously with tail vein injection of NK cells from different feeder system for the detection of NK in vivo persistence and function. RESULTS: Compared with Basic Feeders, CD48 Feeders can promote the proliferation of primary NK cells from peripheral blood and umbilical cord blood and reduce NK cell apoptosis by activating the p-ERK/BCL2 pathway both in vitro and in vivo without affecting overall phenotypes. Furthermore, NK cells expanded via CD48 Feeders showed stronger anti-tumor capability and infiltration ability into the tumor microenvironment. CONCLUSIONS: In this preclinical study, the engagement of the 2B4-CD48 axis can inhibit the apoptosis of NK cells through the p-ERK/BCL2 signal pathway, leading to an improvement in therapeutic efficiency.

Optimization of bovine embryonic fibroblast feeder layer prepared by Mitomycin C.

Feeder cells play important roles in In-vitro culture of stem cells. However, the preparation protocol of feeder cells produced by bovine embryonic fibroblast cells (bEFs) is still lack. In this study, the preparation of bEF-feeder by Mitomycin C was optimized with different concentrations and treatment time. The cell viability of bEFs was detected by CCK8 and 5-Ethynyl-2'-deoxyuridine. The growth of bESCs in each bEFs-feeder group was assessed by alkaline phosphatase staining and CCK8. Quantitative real time PCR was used to detect the mRNA expression of pluripotency-related genes of bESCs. Results showed that the proliferation of bEFs was significantly repressed while bEFs were treated with 14 ug/mL or 16 ug/mL Mitomycin C for 3 h, and the cell viability within 2-4 days after treatment was consistent with the 1st day. The numbers of bESCs clones in bEF-feeder treated with 14 µg/mL Mitomycin C for 3 h or 16 µg/mL Mitomycin C for 3 h were significantly higher than that in bEF-feeder treated with 8 µg/mL Mitomycin C for 8 h or bEFs treated with 6 µg/mL Mitomycin C for 9 h. The mRNA expression of pluripotency-related genes in bESCs cultured by bEF-feeder were higher than the MEF-feeder, the clone morphology of bESCs cultured in bEF-feeder was rounder and sharper than the MEF-feeder. In conclusion, the bEF-feeder prepared with 14 µg/mL Mitomycin C for 3 h or 16 µg/mL Mitomycin C for 3 h could effectively maintains the growth of bESCs, and bEF-feeder is more suitable for bESCs culture than the MEF-feeder.

Cellular therapy: Adoptive immunotherapy with expanded natural killer cells.

Adoptive immunotherapy with natural killer cells was pioneered 30 years ago in human clinical trials with the development of cytokine-induced killer cells-unfractionated peripheral blood mononuclear cell (PBMC) populations activated overnight with IL-2. Higher doses were subsequently made possible through the advent of steady-state apheresis, allowing the collection of PBMC numbers equivalent to an entire adult blood volume, and increased purity made feasible through magnetic CD3-depletion and/or CD56-selection methods. Still, these approaches rarely achieved clinical dosing above a single infusion of 108 NK cells/kg, except with substantial donor-recipient size mismatch (eg, parents donating cells to children). To address this shortcoming, leukemia cell lines with NK cell-like function or ex vivo expansion approaches centered on the homeostatic cytokine IL-15 were developed. Here, we describe the development of an ex vivo expansion system based on a feeder cell expressing membrane-bound IL-21 that enables log-phase growth of primary NK cells for many weeks without inducing senescence, and describe the biology, correlative science, and translation to clinical trials for patients with leukemia, brain tumors, and solid tumors.

Selective Expansion of NKG2C+ Adaptive NK Cells Using K562 Cells Expressing HLA-E.

Adaptive natural killer (NK) cells expressing self-specific inhibitory killer-cell immunoglobulin-like receptors (KIRs) can be expanded in vivo in response to human cytomegalovirus (HCMV) infection. Developing a method to preferentially expand this subset is essential for effective targeting of allogeneic cancer cells. A previous study developed an in vitro method to generate single KIR+ NK cells for enhanced targeting of the primary acute lymphoblastic leukemia cells; however, the expansion rate was quite low. Here, we present an effective expansion method using genetically modified K562-HLA-E feeder cells for long-term proliferation of adaptive NK cells displaying highly differentiated phenotype and comparable cytotoxicity, CD107a, and interferon-γ (IFN-γ) production. More importantly, our expansion method achieved more than a 10,000-fold expansion of adaptive NK cells after 6 weeks of culture, providing a high yield of alloreactive NK cells for cell therapy against cancer.

Novel Human Prostate Epithelial Cell Cultures.

Prostate cancer is the most common male cancer in the USA and the second leading cause of male cancer death in the USA. African American men have higher incidence and mortality rate from prostate cancer compared to Caucasian men in North America, indicating the prostate cancer is a major public health problem in this population. Studies of prostate cancer have been hampered by various factors including (1) restricted access to tissues, (2) difficulties in propagating premalignant lesions and primary prostate tumors in vitro, and (3) limited availability of prostate cell lines for in vitro studies. There is no commercially available pair of non-malignant and tumor cells derived from the same prostate cancer patient. Primary prostate epithelial cells grow for a finite life span and then senesce. Immortalization is defined by continuous growth of otherwise senescing cells and is believed to represent an early stage in tumor progression. To examine these early stages, we have developed in vitro models of prostate epithelial cell immortalization. Generation of human primary epithelial (HPE) cells has been achieved using the serum-free keratinocyte growth medium. Retrovirus containing human telomerase reverse transcriptase (hTERT) was also used for the generation of primary non-malignant and malignant tumor cells. In addition, we have established the first immortalized cell lines of a pair of non-malignant and malignant tumors derived from an African American prostate cancer patient. Interestingly, we have found that the Rock inhibitor and feeder cells induced the conditioned reprogramming (CR) of epithelial cells-normal and tumor epithelial cells from many tissues to proliferate indefinitely in vitro, without transduction of viral or cellular genes. More recently, using CR, we have established normal and tumor cultures respectively from a patient prostatectomy. These CR cells grow indefinitely in vitro and retain stable karyology. The tumor-derived CR cells produced tumors in SCID mice. The use of novel pair of non-malignant and malignant tumor cells derived from the same patient provides a unique in vitro model for studies of early prostate cancer and for testing preventive and therapeutic regimens.

Influence of autologous dendritic cells on the in-vitro expansion and functions of peripheral blood NK cells.

CONTEXT: Allogeneic reactive NK cells were previously shown to exert a graft-versus-leukemia (GVL) effect during allogeneic hematopoietic stem cell transplantation, as well as reduce the incidence of graft-versus-host disease (GVHD). OBJECTIVE: We used autologous immature DCs as feeder cells for the in-vitro expansion of NK cells and studied the function of the NK cell cultures. MATERIALS AND METHODS: NK cells were cultured for 15 days in the presence of autologous, immature DCs. Fold expansion, killing activity and expression of IFN-γ, perforin and granzyme B were evaluated. RESULTS: The highest NK cell expansion efficiency was observed when the ratio of NK cells:DCs was 2:1 and when cells were cultured in a contact-dependent manner. The killing activity of NK cells was highest when the NK:DC ratio was 10:1. NK cell cultures exhibited a significant upregulation in the mRNA expression of IFN-γ, perforin and granzyme B when the ratio of NK cells to DCs was 10:1. DISCUSSION: We successfully amplified NK cells using autologous immature DCs derived from human peripheral monocytes after induction as feeder cells. The use of autologous immature DCs for ex-vivo expansion of NK cells can be clinically applied to overcome limitations, such as the small number of NK cells in peripheral blood, and the high cost of NK cell sorting. Transfusion of allogeneic reactive NK cells has been suggested as a potential adjunctive therapeutic strategy after transplantation. CONCLUSION: Autologous immature DCs can be used as feeder cells for ex-vivo expansion of functional NK cells.

An Extended Flow Cytometry Evaluation of ex Vivo Expanded NK Cells Using K562.Clone1, a Feeder Cell Line Manufactured in Brazil.

Natural killer (NK) cells play a crucial role in the immune system's response against cancer. However, the challenge of obtaining the required quantity of NK cells for effective therapeutic response necessitates the development of strategies for their ex vivo expansion. This study aimed to develop a novel feeder cell line, K562.Clone1, capable of promoting the ex vivo expansion of NK cells while preserving their cytotoxic potential. he K562 leukemic cell line was transduced with mbIL-21 and 4-1BBL proteins to generate K562.Clone1 cells. NK cells were then co-cultured with these feeder cells, and their expansion rate was monitored over 14 days. The cytotoxic potential of the expanded NK cells was evaluated against acute myeloid leukemia blasts and tumor cell lines of leukemia and glial origin. Statistical analysis was performed to determine the significance of the results. The K562.Clone1 co-cultured with peripheral NK showed a significant increase in cell count, with an approximate 94-fold expansion over 14 days. Expanded NK cells demonstrated cytotoxicity against the tested tumor cell lines, indicating preservation of their cytotoxic characteristics. Additionally, the CD56, CD16, inhibitory KIRs, and activation receptors were conserved and present in a well-balanced manner. The study successfully developed a feeder cell line, K562.Clone1, that effectively promotes the expansion of NK cells ex vivo while maintaining their cytotoxic potential. This development could significantly contribute to the advancement of NK cell therapy, especially in Brazil.

Lighting the way: an economical alternative to feeder cell irradiation for T-cell expansion.

A robust T-cell expansion process involves co-culturing T-cells with non-proliferating feeder cells combined with anti-CD3 antibody and IL-2. Although ionizing irradiation effectively inhibits feeder cell proliferation, the high operating costs limit cell therapy research to well-funded institutions. UVC, known for causing DNA damage-induced cell death and commonly used for environmental sterilization, presents a cost-effective alternative to ionizing irradiation for generating non-proliferating feeder cells. UVC irradiation of K562 artificial antigen presenting cells (aAPCs) resulted in significant DNA damage, evidenced by increased γ-H2AX phosphorylation within 15 minutes and elevated 8-OHdG levels at 24 hours. This indicates the occurrence of DNA double-strand breaks and oxidative damage. Following UVC irradiation, glucose uptake and ATP production were significantly reduced, whereas aCD3 retention at the surface of the cell increased twofold. Selective inhibition of glucose uptake and ATP production similarly enhanced aCD3 retention by approximately 10-fold and 6-fold, respectively. This suggests that UVC-induced energy deprivation dampens aCD3 internalization, potentially enhancing T-cell activation through prolonged aCD3 and T-cell receptor interaction. Tumor-infiltrating lymphocytes (TILs) expanded with UVC-irradiated PBMCs demonstrated comparable viability, expansion, immunophenotype, and effector function to those expanded with ionizing irradiation. UVC irradiation was equally effective in suppressing feeder cell proliferation and facilitating the expansion of functionally potent T-cells compared to traditional ionizing irradiation. Implementing UVC irradiation in T-cell expansion can significantly reduce costs, enhancing the accessibility and feasibility of cell therapy research across various institutions.

Obtaining Gene-Modified HLA-E-Expressing Feeder Cells for Stimulation of Natural Killer Cells.

Human cytomegalovirus (HCMV)-specific adaptive NK cells are capable of recognizing viral peptides presented by HLA-E on infected cells via the NKG2C receptor. Using retroviral transduction, we have generated a K562-cell-based line expressing HLA-E in the presence of the HLA-E-stabilizing peptide, which has previously shown the capacity to enhance adaptive NK cell response. The obtained K562-21E cell line was employed to investigate proliferative responses of the CD57- NK cell subset of HCMV-seropositive and seronegative donors. Stimulation of CD57- NK cells with K562-21E/peptide resulted in an increased cell expansion during the 12-day culturing period, regardless of the serological HCMV status of the donor. The enhanced proliferation in response to the peptide was associated with a greater proportion of CD56brightHLA-DR+ NK cells. In later stages of cultivation, the greatest proliferative response to K562-21E/peptide was shown for a highly HCMV-seropositive donor. These expanded NK cells were characterized by the accumulation of CD57-KIR2DL2/3+NKG2C+NKG2A- cells, which are hypothesized to represent adaptive NK cell progenitors. The K562-21E feeder cells can be applied both for the accumulation of NK cells as therapeutic effectors, and for the study of NK cell maturation into the adaptive state after the HLA-E peptide presentation.

Building a Better Defense: Expanding and Improving Natural Killer Cells for Adoptive Cell Therapy.

Natural killer (NK) cells have gained attention as a promising adoptive cell therapy platform for their potential to improve cancer treatments. NK cells offer distinct advantages over T-cells, including major histocompatibility complex class I (MHC-I)-independent tumor recognition and low risk of toxicity, even in an allogeneic setting. Despite this tremendous potential, challenges persist, such as limited in vivo persistence, reduced tumor infiltration, and low absolute NK cell numbers. This review outlines several strategies aiming to overcome these challenges. The developed strategies include optimizing NK cell expansion methods and improving NK cell antitumor responses by cytokine stimulation and genetic manipulations. Using K562 cells expressing membrane IL-15 or IL-21 with or without additional activating ligands like 4-1BBL allows "massive" NK cell expansion and makes multiple cell dosing and "off-the-shelf" efforts feasible. Further improvements in NK cell function can be reached by inducing memory-like NK cells, developing chimeric antigen receptor (CAR)-NK cells, or isolating NK-cell-based tumor-infiltrating lymphocytes (TILs). Memory-like NK cells demonstrate higher in vivo persistence and cytotoxicity, with early clinical trials demonstrating safety and promising efficacy. Recent trials using CAR-NK cells have also demonstrated a lack of any major toxicity, including cytokine release syndrome, and, yet, promising clinical activity. Recent data support that the presence of TIL-NK cells is associated with improved overall patient survival in different types of solid tumors such as head and neck, colorectal, breast, and gastric carcinomas, among the most significant. In conclusion, this review presents insights into the diverse strategies available for NK cell expansion, including the roles played by various cytokines, feeder cells, and culture material in influencing the activation phenotype, telomere length, and cytotoxic potential of expanded NK cells. Notably, genetically modified K562 cells have demonstrated significant efficacy in promoting NK cell expansion. Furthermore, culturing NK cells with IL-2 and IL-15 has been shown to improve expansion rates, while the presence of IL-12 and IL-21 has been linked to enhanced cytotoxic function. Overall, this review provides an overview of NK cell expansion methodologies, highlighting the current landscape of clinical trials and the key advancements to enhance NK-cell-based adoptive cell therapy.

Feeder cell training shapes the phenotype and function of in vitro expanded natural killer cells.

Natural killer (NK) cells are candidates for adoptive cell therapy, and the protocols for their activation and expansion profoundly influence their function and fate. The complexity of NK cell origin and feeder cell cues impacts the heterogeneity of expanded NK (eNK) cells. To explore this, we compared the phenotype and function of peripheral blood-derived NK (PB-NK) and umbilical cord blood-derived NK (UCB-NK) cells activated by common feeder cell lines, including K562, PLH, and 221.AEH. After first encounter, most PB-NK cells showed degranulation independently of cytokines production. Meanwhile, most UCB-NK cells did both. We observed that each feeder cell line uniquely influenced the activation, expansion, and ultimate fate of PB eNK and UCB eNK cells, determining whether they became cytokine producers or killer cells. In addition, they also affected the functional performance of NK cell subsets after expansion, that is, expanded conventional NK (ecNK) and expanded FcRγ- NK (eg-NK) cells. Hence, the regulation of eNK cell function largely depends on the NK cell source and the chosen expansion system. These results underscore the significance of selecting feeder cells for NK cell expansion from various sources, notably for customized adoptive cell therapies to yield cytokine-producing or cytotoxic eNK cells.

X-ray-irradiated K562 feeder cells for expansion of functional CAR-T cells.

Immunotherapy, particularly CAR-T therapy has recently emerged as an innovator for cancer treatment. Gamma-irradiated K562 cells is a common and effective method to stimulated CAR-T cells prior to treatment. However, high cost and limited equipment of gamma-irradiation is drawback of this method. This requires the establishment of CAR-T-expanding alternatives, such as X-ray-irradiated K562 cells. X-ray irradiation was used to deactivate K562 cells. The post-irradiative cell survival was investigated by counting of the number of cells, staining with Trypan Blue and PI. FACS analysis was applied to detect the expression of cell surface markers. The production of CD19-CAR-T cells were executed from fresh blood donor by CD19-CAR-plasmid transfection, followed by the stimulation with X-ray-irradiated K562 feeder cells. The function of produced CAR-T cells was checked by their ability to kill Daudi cells. X-ray-irradiation inhibited the propagation and viability of K562 cells in a dose- and time-dependent manner. Interestingly, CAR-T-stimulating effectors were remained on the surface of X-ray-irradiated K562 cells. CD-19-CAR-T cells were produced successfully, suggested by number of CAR-positive cells in transfected and stimulated population, compared to un-transfected group. Lastly, our data showed that engineered CAR-T cells effectively killed Daudi cells. Our data demonstrated the efficacy of X-ray on deactivation K562 feeder cells which subsequently stimulated and expanded functional CAR-T cells. Thus, X-ray can be used as an alternative to inactivate K562 cells prior to using as a feeder of CAR-T cells.

Feeder cells treated with ethanol can be used to maintain self-renewal and pluripotency of human pluripotent stem cells.

Feeder cells play an important role in the culture of human pluripotent stem cells (hPSCs) in vitro. Previously, we used methanol as a fixative to prepare feeder cells for the cultivation of pluripotent stem cells (PSCs), and this method could maintain the self-renewal and pluripotency of PSCs. However, methanol is toxic, and so here we examined whether ethanol could be used to prepare feeder cells as a fixative for hPSC culturing. Primed, naïve, and extended human embryonic stem cells and induced pluripotent stem cells can maintain self-renewal and undifferentiated potential on feeder cells treated with ethanol for an extended period. RNA sequencing analysis showed that the expression of collagen-related genes in hPSCs cultured on feeder cells treated with ethanol was significantly lower as compared with hPSCs cultured on feeder cells treated with mitomycin C. Therefore, we speculate that the signaling pathway mediated by collagen-related genes may, at least in part, contribute to the maintenance of self-renewal and pluripotency of PSCs induced by feeder cells treated with chemicals.

Natural Killer Cell Expansion and Cytotoxicity Differ Depending on the Culture Medium Used.

Background: Adoptive cell therapy using umbilical cord blood (UCB)-derived allogeneic natural killer (NK) cells has shown encouraging results. However, because of the insufficient availability of NK cells and limited UCB volume, more effective culture methods are required. NK cell expansion and functionality are largely affected by the culture medium. While human serum is a major affecting component in culture media, the way it regulates NK cell functionality remains elusive. We elucidated the effects of different culture media and human serum supplementation on UCB NK cell expansion and functionality. Methods: UCB NK cells were cultured under stimulation with K562-OX40L-mbIL-18/21 feeder cells and IL-2 and IL-15 in serum-containing and serum-free culture media. The effects of the culture media and human serum supplementation on NK cell expansion and cytotoxicity were evaluated by analyzing the expansion rate, activating and inhibitory receptor levels, and the cytotoxicity of the UCB NK cells. Results: The optimal medium for NK cell expansion was Dulbecco's modified Eagle's medium/Ham's F12 with supplements and that for cytotoxicity was AIM V supplemented with Immune Cell Serum Replacement. Shifting media is an advantageous strategy for obtaining several highly functional UCB NK cells. Live cell imaging and killing time measurement revealed that human serum enhanced NK cell proliferation but delayed target recognition, resulting in reduced cytotoxicity. Conclusions: Culture medium supplementation with human serum strongly affects UCB NK cell expansion and functionality. Thus, culture media should be carefully selected to ensure both NK cell quantity and quality for adoptive cell therapy.

Feeder cell-dependent primary culture of single blastula-derived embryonic cell lines from marine medaka (Oryzias dancena).

Fish embryonic stem cells (ESCs) are derived from blastomeres that have been cultured from blastula embryos. The most widely used method for derivation of fish ESCs is the culture of blastomeres that have been isolated from approximately 10 blastula embryos under feeder-free conditions. However, this method leads to intercellular genetic heterogeneity among the cultured cells, which is a major obstacle to the development of stable ESC culture conditions. In this study, to establish ESC lines with intercellular genetic homogeneity at the early stage of culture, we attempted to derive embryonic cell lines from single blastula-derived blastomeres of marine medaka (Oryzias dancena) in a feeder cell culture system. Using basic fibroblast growth factor-expressing feeder cells during primary culture, we successfully established 22 single blastula-derived embryonic cell lines that could be subcultured more than 20 times. In contrast, we were unable to efficiently derive cell lines using wild-type feeder cells and under feeder-free conditions. The established cell lines exhibited ESC-like cell characteristics in terms of alkaline phosphatase activity, pluripotency-related gene expression, and embryoid body formation. The results of this study will contribute to the development of methods for derivation of fish ESCs.

The Role of Genetically Modified Human Feeder Cells in Maintaining the Integrity of Primary Cultured Human Deciduous Dental Pulp Cells.

Tissue-specific stem cells exist in tissues and organs, such as skin and bone marrow. However, their pluripotency is limited compared to embryonic stem cells. Culturing primary cells on plastic tissue culture dishes can result in the loss of multipotency, because of the inability of tissue-specific stem cells to survive in feeder-less dishes. Recent findings suggest that culturing primary cells in medium containing feeder cells, particularly genetically modified feeder cells expressing growth factors, may be beneficial for their survival and proliferation. Therefore, the aim of this study was to elucidate the role of genetically modified human feeder cells expressing growth factors in maintaining the integrity of primary cultured human deciduous dental pulp cells. Feeder cells expressing leukemia inhibitory factor, bone morphogenetic protein 4, and basic fibroblast growth factor were successfully engineered, as evidenced by PCR. Co-culturing with mitomycin-C-treated feeder cells enhanced the proliferation of newly isolated human deciduous dental pulp cells, promoted their differentiation into adipocytes and neurons, and maintained their stemness properties. Our findings suggest that genetically modified human feeder cells may be used to maintain the integrity of primary cultured human deciduous dental pulp cells.

Harnessing natural killer cells for the treatment of multiple myeloma.

Multiple myeloma (MM) is hematologic malignancy that is associated with profound immune alterations. Myeloma cells are susceptible to killing by natural killer (NK) cells but acquire the ability to elude NK cell surveillance by avoiding recognition and suppressing NK cell function. Major advances in the treatment of multiple myeloma have been achieved by effective new drugs that redirect NK cells and enhance their function. Despite significant progress, myeloma remains incurable and novel treatment approaches are required. Strategies to take advantage of the intrinsic antitumor properties of NK cells to treat MM represent a novel immunotherapeutic approach. One such strategy is adoptive NK cell therapy that consist of infusions of NK cells that have been propagated ex vivo. Adoptive NK cell therapy encompasses contemporary genetic engineering strategies such as chimeric antigen receptor (CAR)-engineered NK cells. An alternative approach involves the use of pharmacologic agents to enhance NK cell activity against myeloma. NK cell-modulating therapies can be used to bolster the function of endogenous NK cells or to enhance the efficacy of adoptively infused NK cells. Here, we review the mechanistic complexities influencing NK cell activity in MM and highlight a variety of innovative NK cell-based strategies being developed for the treatment of MM.

Preparation of Mouse Embryonic Fibroblasts as Feeders for iPSC Generation and Maintenance.

Mouse embryonic fibroblasts (MEFs) can be used in co-culture to support generation of induced pluripotent stem cells (iPSCs) and the normal growth and proliferation of human pluripotent stem cells (hPSCs). Here, we describe the necessary steps to derive, expand, harvest, inactivate, plate, and use MEFs as feeders for iPSC generation and maintenance.

Generation of Knockout Human Primary Keratinocytes by CRISPR/Cas9.

The culture of epidermal human primary keratinocytes (HPKs) represents a well-established model in biological and dermatological research. In addition, HPKs are used in three-dimensional organotypic cultures (OTCs), and gene therapeutic approaches have been reported for the treatment of patients suffering from epidermolysis bullosa, a severe blistering disease that can result in postnatal lethality. Therefore, there is a strong need for the development of techniques for the stable and specific genetic manipulation of HPKs, for example, by genome editing via the CRISPR/Cas9 approach. However, the main disadvantage of working with HPKs is the fact that these cells are prone to terminal differentiation and proliferate only for few passages in monoculture. As it is well known that the co-culture of HPKs with fibroblasts strongly increases the lifetime of the epidermal cells, we developed a protocol for the stable modification of HPKs by CRISPR/Cas9 via lentiviral transduction in the presence of 3T3-J2 fibroblasts as feeder cells. Selection of transduced HPKs is achieved with antibiotics in co-culture with antibiotic-resistant feeder cells. Modified HPKs generated by our protocol have the potential to generate epidermis-like structures in OTCs.

Primary Culture of Cornea-Limbal Epithelial Cells In Vitro.

The cultivation of corneal-limbal cells in vitro represents an excellent means to generate models to study cornea function and disease processes. These in vitro expanded cornea-limbal epithelial cell cultures are rich in stem cells for cornea, and hence can be used as a cell therapy for cornea-limbal deficiency. This chapter details the primary culture of these cornea-limbal cells, which can be used as model for further studies of the cornea surface.