Point-of-care manufacturing of anti-CD19 CAR-T cells using a closed production platform: Experiences of an academic in Thailand.

Anti-CD19 chimeric antigen receptor (CAR)-T cell therapy has evolved as a standard of care for various forms of relapsed/refractory B cell malignancies in major developed countries. However, access to industry-driven CAR-T cell therapy is limited in developing countries, partly due to the centralized manufacturing system. Here, we demonstrated the feasibility of the point-of-care (POC) manufacturing of anti-CD19 CAR-T cells from heavily pretreated patients and healthy graft donors at an academic medical center in Thailand using a closed semi-automated production platform, CliniMACS Prodigy, and established in-process quality control and release testing to ensure their identity, purity, sterility, safety, and potency.

Anti-TIM3 chimeric antigen receptor-natural killer cells preferentially target primitive acute myeloid leukemia cells with minimal fratricide and exhaustion.

Acute myeloid leukemia (AML) is an aggressive and genetically heterogeneous disease with poor clinical outcomes. Refractory AML is common, and relapse remains a major challenge, attributable to the presence of therapy-resistant leukemic stem cells (LSCs), which possess self-renewal and repopulating capability. Targeting LSCs is currently the most promising avenue for long-term management of AML.

The dynamic expression of YAP is essential for the development of male germ cells derived from human embryonic stem cells.

YAP plays a vital role in controlling growth and differentiation in various cell lineages. Although the expression of YAP in mice testicular and spermatogenic cells suggests its role in mammalian spermatogenesis, the role of YAP in the development of human male germ cells has not yet been determined. Using an in vitro model and a gene editing approach, we generated human spermatogonia stem cell-like cells (hSSLCs) from human embryonic stem cells (hESCs) and investigated the role of YAP in human spermatogenesis.

and gene-edited human induced pluripotent stem cells for dissecting the functional roles of -GlcNAcylation in hematopoiesis.

Hematopoiesis continues throughout life to produce all types of blood cells from hematopoietic stem cells (HSCs). Metabolic state is a known regulator of HSC self-renewal and differentiation, but whether and how metabolic sensor -GlcNAcylation, which can be modulated via an inhibition of its cycling enzymes -GlcNAcase (OGA) and -GlcNAc transferase (OGT), contributes to hematopoiesis remains largely unknown. Herein, isogenic, single-cell clones of -depleted (OGAi) and -depleted (OGTi) human induced pluripotent stem cells (hiPSCs) were successfully generated from the master hiPSC line MUSIi012-A, which were reprogrammed from CD34 hematopoietic stem/progenitor cells (HSPCs) containing epigenetic memory.

Anti-TIM3 chimeric antigen receptor-natural killer cells from engineered induced pluripotent stem cells effectively target acute myeloid leukemia cells.

Background: Acute myeloid leukemia (AML) is a clonal malignant disorder which originates from a small number of leukemia-initiating cells or leukemic stem cells (LSCs)-the subpopulation that is also the root cause of relapsed/refractory AML. Chimeric antigen receptor (CAR)-T cell therapy has proved successful at combating certain hematologic malignancies, but has several hurdles that limit its widespread applications. CAR-natural killer (NK) cells do not carry the risk of inducing graft-versus-host disease (GvHD) frequently associated with allogeneic T cells, thereby overcoming time-consuming, autologous cell manufacturing, and have relatively safer clinical profiles than CAR-T cells.

Musashi-2 in cancer-associated fibroblasts promotes non-small cell lung cancer metastasis through paracrine IL-6-driven epithelial-mesenchymal transition.

Background: Lung cancer, the most common cause of cancer-related mortality worldwide, is predominantly associated with advanced/metastatic disease. The interaction between tumor cells and cancer-associated fibroblasts (CAFs) in tumor microenvironment is known to be essential for regulating tumor progression and metastasis, but the underlying mechanisms, particularly the role of RNA-binding protein Musashi-2 (MSI2) in CAFs in promoting non-small cell lung cancer (NSCLC) invasiveness and metastatic spread, remain obscure.

Methods: Genomic and proteomic database analyses were performed to evaluate the potential clinical significance of MSI2 in NSCLC tumor and stromal clinical specimens.

Cardiac glycoside ouabain efficiently targets leukemic stem cell apoptotic machinery independent of cell differentiation status.

Background: Acute myeloid leukemia (AML) is an aggressive hematologic malignancy characterized by an accumulation of immature leukemic myeloblasts initiating from leukemic stem cells (LSCs)-the subpopulation that is also considered the root cause of chemotherapy resistance. Repurposing cardiac glycosides to treat cancers has gained increasing attention and supporting evidence, but how cardiac glycosides effectively target LSCs, e.g.

Role of YAP in hematopoietic differentiation and erythroid lineage specification of human-induced pluripotent stem cells.

Background: In vitro production of hematopoietic stem/progenitor cells (HSPCs) from human-induced pluripotent stem cells (hiPSCs) provides opportunities for fundamental research, disease modeling, and large-scale production of HLA-matched HSPCs for therapeutic applications. However, a comprehensive understanding of the signaling mechanisms that regulate human hematopoiesis is needed to develop a more effective procedure for deriving HSPCs from hiPSCs.

Methods: In this study, we investigate the role of YAP during the hematopoietic differentiation of hiPSCs to HSPCs and erythrocytes using the isogenic YAP-overexpressing (YAP-S5A) and YAP-depleting (YAP-KD) hiPSCs to eliminate the effects of a genetic background variation.

Generation and Functional Characterization of Anti-CD19 Chimeric Antigen Receptor-Natural Killer Cells from Human Induced Pluripotent Stem Cells.

Natural killer (NK) cells are a part of innate immunity that can be activated rapidly in response to malignant transformed cells without prior sensitization. Engineering NK cells to express chimeric antigen receptors (CARs) allows them to be directed against corresponding target tumor antigens. CAR-NK cells are regarded as a promising candidate for cellular immunotherapy alternatives to conventional CAR-T cells, due to the relatively low risk of graft-versus-host disease and safer clinical profile.

CRISPR/Cas9 mediated approach to generate YAP-depleted human embryonic stem cell line (MUSIe002-A-1).

Yes-associated protein (YAP), an important effector protein of the Hippo signaling pathway, acts as a molecular switch in controlling cell proliferation and apoptosis. In this study, a YAP-targeted isogenic sub-clone of the MUSIe002-A was generated, designated as MUSIe002-A-1. The MUSIe002-1 cell line had normal pluripotent stem cell characteristics and karyotype.

Bcl-2 Family Members Bcl-xL and Bax Cooperatively Contribute to Bortezomib Resistance in Mantle Cell Lymphoma.

Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin lymphoma with poor prognosis, due to the inevitable development of drug resistance. Despite being the first-in-class proteasome inhibitor for relapsed/refractory MCL, resistance to bortezomib (BTZ) in MCL patients remains a major hurdle of effective therapy, and relapse following BTZ is frequent. Understanding the mechanisms underlying BTZ resistance is, therefore, important for improving the clinical outcome and developing novel therapeutic strategies.

Episomal vector-based generation of human induced pluripotent stem cell line MUSIi020-A from peripheral blood T-cells.

Human induced pluripotent stem cell (iPSC) line MUSIi020-A was generated from T cells isolated from peripheral blood of a healthy 37-year-old female and reprogrammed using episomal plasmid vectors. The established transgene-free MUSIi020-A, which retained a normal karyotype, displayed pluripotency as characterized by expression of pluripotency markers and by in vitro spontaneous differentiation toward three embryonic germ layers. This cell line may represent a valuable tool for studying T cell development and a potential cell source for cancer immunotherapy.

Inhibition of O-GlcNAcase Inhibits Hematopoietic and Leukemic Stem Cell Self-Renewal and Drives Dendritic Cell Differentiation via STAT3/5 Signaling.

Myeloid differentiation blockage at immature and self-renewing stages is a common hallmark across all subtypes of acute myeloid leukemia (AML), despite their genetic heterogeneity. Metabolic state is an important regulator of hematopoietic stem cell (HSC) self-renewal and lineage-specific differentiation as well as several aggressive cancers. However, how O-GlcNAcylation, a nutrient-sensitive posttranslational modification of proteins, contributes to both normal myelopoiesis and AML pathogenesis remains largely unknown.

Metabolic sensor O-GlcNAcylation regulates erythroid differentiation and globin production via BCL11A.

Background: Human erythropoiesis is a tightly regulated, multistep process encompassing the differentiation of hematopoietic stem cells (HSCs) toward mature erythrocytes. Cellular metabolism is an important regulator of cell fate determination during the differentiation of HSCs. However, how O-GlcNAcylation, a posttranslational modification of proteins that is an ideal metabolic sensor, contributes to the commitment of HSCs to the erythroid lineage and to the terminal erythroid differentiation has not been addressed.

CDK12 Is Necessary to Promote Epidermal Differentiation Through Transcription Elongation.

Proper differentiation of the epidermis is essential to prevent water loss and to protect the body from the outside environment. Perturbations in this process can lead to a variety of skin diseases that impacts 1 in 5 people. While transcription factors that control epidermal differentiation have been well characterized, other aspects of transcription control such as elongation are poorly understood.

Distinctive Roles of YAP and TAZ in Human Endothelial Progenitor Cells Growth and Functions.

The hippo signaling pathway plays an essential role in controlling organ size and balancing tissue homeostasis. Its two main effectors, yes-associated protein (YAP) and WW domain-containing transcription regulator 1, WWTR1 or TAZ, have also been shown to regulate endothelial cell functions and angiogenesis. In this study, the functions of YAP and TAZ in human endothelial progenitor cells (EPCs) were investigated by a loss-of-function study using CRISPR/Cas9-mediated gene knockdown (KD).

Induced Pluripotent Stem Cells as a Tool for Modeling Hematologic Disorders and as a Potential Source for Cell-Based Therapies.

The breakthrough in human induced pluripotent stem cells (hiPSCs) has revolutionized the field of biomedical and pharmaceutical research and opened up vast opportunities for drug discovery and regenerative medicine, especially when combined with gene-editing technology. Numerous healthy and patient-derived hiPSCs for human disease modeling have been established, enabling mechanistic studies of pathogenesis, platforms for preclinical drug screening, and the development of novel therapeutic targets/approaches. Additionally, hiPSCs hold great promise for cell-based therapy, serving as an attractive cell source for generating stem/progenitor cells or functional differentiated cells for degenerative diseases, due to their unlimited proliferative capacity, pluripotency, and ethical acceptability.

Selective Cytotoxicity of Single and Dual Anti-CD19 and Anti-CD138 Chimeric Antigen Receptor-Natural Killer Cells against Hematologic Malignancies.

Natural killer (NK) cells are part of the first line of defense that rapidly respond to malignant transformed cells. Chimeric antigen receptor- (CAR-) engineered NK cells, although are still at the preliminary stage, have been shown to be alternative to CAR-T cells, mainly due to the absence of graft-versus-host disease and safer clinical profile. Allogeneic human NK cell line NK-92 cells, equipped by CAR, are being developed for clinical applications.

Episomal vector reprogramming of human umbilical cord blood natural killer cells to an induced pluripotent stem cell line MUSIi013-A.

Natural killer (NK) cells were isolated from human umbilical cord blood from a healthy newborn and reprogrammed by episomal vectors carrying reprograming factors L-MYC, LIN28, OCT4, SOX2, KLF4, EBNA-1, and shRNA against p53 delivered using nucleofection. The obtained MUSIi013-A human induced pluripotent stem cell (iPSC) line highly expressed pluripotency markers, had the capacity to differentiate into derivatives of the three germ layers, while retained a normal karyotype. This cell line may be a useful tool to study epigenic memory that may predispose hiPSCs to enhanced NK differentiation.

O-GlcNAcylation homeostasis controlled by calcium influx channels regulates multiple myeloma dissemination.

Background: Multiple myeloma (MM) cell motility is a critical step during MM dissemination throughout the body, but how it is regulated remains largely unknown. As hypercalcemia is an important clinical feature of MM, high calcium (Ca) and altered Ca signaling could be a key contributing factor to the pathological process.

Methods: Bioinformatics analyses were employed to assess the clinical significance of Ca influx channels in clinical specimens of smoldering and symptomatic MM.

Metabolic sensor O-GlcNAcylation regulates megakaryopoiesis and thrombopoiesis through c-Myc stabilization and integrin perturbation.

Metabolic state of hematopoietic stem cells (HSCs) is an important regulator of self-renewal and lineage-specific differentiation. Posttranslational modification of proteins via O-GlcNAcylation is an ideal metabolic sensor, but how it contributes to megakaryopoiesis and thrombopoiesis remains unknown. Here, we reveal for the first time that cellular O-GlcNAcylation levels decline along the course of megakaryocyte (MK) differentiation from human-derived hematopoietic stem and progenitor cells (HSPCs).

A novel TRPM7/O-GlcNAc axis mediates tumour cell motility and metastasis by stabilising c-Myc and caveolin-1 in lung carcinoma.

Background: Calcium is an essential signal transduction element that has been associated with aggressive behaviours in several cancers. Cell motility is a prerequisite for metastasis, the major cause of lung cancer death, yet its association with calcium signalling and underlying regulatory axis remains an unexplored area.

Methods: Bioinformatics database analyses were employed to assess correlations between calcium influx channels and clinical outcomes in non-small cell lung cancer (NSCLC).