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.

Generation of a human iPSC cell line (MUSIi017-A) from a donor with O negative blood type.

The Rh-negative type O blood group (O Rh-) is considered a universal donor for emergency blood transfusions. Due to the constant shortage of this rare blood group, the production of blood cells from iPSCs derived from the O Rh- donor could potentially serve as a limitless blood source for transfusions. In this report, we establish a MUSIi017-A iPSC line from peripheral blood mononuclear cells of a healthy donor with the O Rh- blood group.

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.

Inhibition of LATS kinases reduces tumorigenicity and increases the sensitivity of human chronic myelogenous leukemia cells to imatinib.

Chronic myelogenous leukemia (CML) is a clonal hematologic malignancy of the myeloid lineage caused by the oncogenic BCR/ABL fusion protein that promotes CML cell proliferation and protects them against drug-induced apoptosis. In this study, we determine LATS1 and LATS2 expression in CML cells derived from patients who are resistant to imatinib (IM) treatment. Significant upregulation of LATS1 and LATS2 was found in these CML patients compared to healthy donors.

An update on recent studies of extracellular vesicles and their role in hypercoagulability in thalassemia (Review).

Thromboembolic events are a significant clinical concern in thalassemia and hemoglobinopathies, highlighting the need for new strategies to treat and detect these specific hematologic complications. In recent years, extracellular vesicles (EVs) have garnered interest due to their role in cell-to-cell communication, including angiogenesis, immune responses and coagulation activation. Their multifaceted role depends on the cellular origin and cargo, making them potential diagnostic biomarkers and therapeutic agents.

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.

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.

Stem cell-derived exosomes from human exfoliated deciduous teeth promote angiogenesis in hyperglycemic-induced human umbilical vein endothelial cells.

Objective: To investigate the angiogenesis in human umbilical vein endothelial cells (HUVEC) under high glucose concentration, treated with exosomes derived from stem cells from human exfoliated deciduous teeth (SHED).

Methodology: SHED-derived exosomes were isolated by differential centrifugation and were characterized by nanoparticle tracking analysis, transmission electron microscopy, and flow cytometric assays. We conducted in vitro experiments to examine the angiogenesis in HUVEC under high glucose concentration.

Generation of RUNX1c-eGFP induced pluripotent stem cell, MUSIi012-A-4, using CRISPR/Cas9.

Runt-Related Transcription Factor 1c (RUNX1c) plays an important role in regulating the development of hematopoietic stem cells (HSC). Using CRISPR/Cas9 gene editing technology, we established a RUNX1c-eGFP reporter cell line from the MUSIi012-A cell line. The MUSIi012-A-4 cell line has normal stem cell morphology and karyotype, expresses pluripotency markers, and can be differentiated into all three germ layers in vitro and in vivo.

Derivation of MUSIi016-A iPSCs from peripheral blood with blood type O Rh positive.

MUSIi016-A, a human induced pluripotent stem cell (iPSC), generated from peripheral blood mononuclear cells of a healthy blood group O Rh positive donor was reprogrammed using Sendai viral vectors containing Yamanaka's factors. MUSIi016-A iPSC showed pluripotent stem cell characteristics, highly expressed pluripotent markers, and a capacity to differentiate into all three embryonic cell lineages. This iPSC can be used as a model for the generation of blood cells in vitro.

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.

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.

YAP and TAZ play a crucial role in human erythrocyte maturation and enucleation.

Background: Yes-associated protein (YAP) and WW domain-containing transcription regulator protein 1 (WWTR1, also known as TAZ) are two key transcription co-activators of the Hippo pathway. Both were originally characterized as organ size and cell proliferation regulators. Later studies demonstrated that the Hippo pathway may play a role in Drosophila and mammal hematopoiesis.

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).

Derivation of the MUSIe002-A human embryonic stem cell line.

The MUSIe002-A cell line was established from in vitro fertilization of human sperm and oocytes donated for research with informed consent. This cell line exhibited normal human embryonic stem cell (hESC) characteristics, including typical cell morphology, expression of all pluripotent stem cell markers, and potential to differentiate into three germ layers. A karyotyping analysis revealed 46 XY chromosome and cells that did not have mycoplasma contamination.

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.

Internalization of cell-derived microparticles triggers endothelial pro-inflammatory responses.

Background: Increased numbers of circulating microparticles (MPs) have long been documented in thalassemia and are considered as a contributing factor in developing the thromboembolic events (TEEs), which are associated with endothelial dysfunction. Indeed, the cellular and molecular mechanisms by which MPs and endothelial cells interact and their consequences remain poorly investigated.

Objective: The present study aims to compare the biological effects of MPs obtained from healthy subjects and β-thalassemia/HbE patients on endothelial pro-inflammatory responses.

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).

Generation of a serine/threonine-protein kinase LATS1 gene-edited iPSC MUSIi012-A-3.

In mammals, there are a number of kinases, including serine/threonine-protein kinase LATS1, that act as a core kinase of the Hippo pathway and that negatively regulate the Hippo effector protein YAP and its paralog TAZ. Using CRISPR/Cas9 technology, we established a stable LATS1 knockdown (LATS1-KD) iPSC from the MUSIi012-A cell line. The LATS1-KD iPSC MUSIi012-A-3 that was developed maintained both the normal karyotype and the pluripotent phenotype, and retained the ability to differentiate into all three embryonic germ layers.