Toll-like receptors (TLRs) play a crucial role in the innate immune response, mediating cellular interactions with the microenvironment and influencing periodontal disease progression. This in vitro study aimed to comprehensively characterize the TLR expression profile of periodontal ligament mesenchymal stem/progenitor cells (PDLSCs) and investigate its modulation by inflammatory stimuli associated with periodontal disease. PDLSCs (n = 6) were isolated, selected using anti-STRO-1 antibodies, and cultured to evaluate their colony-forming abilities and stem/progenitor characteristics. Baseline and inflammation-induced TLR expressions were evaluated using RT-PCR and protein analyses following cytokine-mediated stimulation. PDLSCs exhibited the expected stem cell characteristics and expressed multiple TLRs under both conditions. Notably, inflammatory stimulation significantly upregulated TLR1 and TLR2 while downregulating TLR10 (p < 0.05). These findings provide a comprehensive characterization of TLR expression in PDLSCs and demonstrate how inflammation modulates their innate immune profile. The observed shifts in TLR expression may influence PDLSC responses to microbial pathogens and impact their immunomodulatory and regenerative properties in periodontal tissues. Understanding these interactions could contribute to developing targeted strategies for improving PDLSC-based therapies in periodontal disease.

Even after chimeric antigen receptor (CAR)-based immunotherapy has dramatically changed therapeutic approaches for malignancies, balancing therapeutic efficacy with labor and financial cost remains a major problem for immunotherapy. Current study developed a cost-effective and enhanced approach to chimeric antigen receptor (CAR)-macrophage therapy for cancer and demonstrated its therapeutic effects by repeated administration of anti-HER2 CAR macrophages generated from human pluripotent stem cell (PSC)-derived immortalized myeloid cell lines (ML). These ML-derived CAR macrophages (CAR-ML-MPs) exhibit potent antigen-specific killing activity against HER2-expressing tumor cells by phagocytosis in vitro and effectively inhibit tumor progression in vivo , which is enhanced by repeated administration. CAR-ML-MPs provide a promising off-the-shelf cellular resource for tumor adoptive cell immunotherapy, solving the cost and time problems associated with conventional CAR-based immunotherapy.

Mucopolysaccharidosis type II (OMIM 309900) is a lysosomal storage disorder caused by iduronate 2-sulfatase (IDS) deficiency and accumulation of glycosaminoglycans, leading to progressive neurodegeneration. As intravenously infused enzyme replacement therapy cannot cross the blood-brain barrier (BBB), it fails to treat brain pathology, highlighting the unmet medical need to develop alternative therapies. Here, we test modified versions of hematopoietic stem and progenitor cell (HSPC)-mediated lentiviral gene therapy (LVGT) using IDS tagging in combination with the ubiquitous MND promoter to optimize efficacy in brain and to investigate its mechanism of action. We find that IDS tagging with IGF2 or ApoE2, but not RAP12x2, improves correction of brain heparan sulfate and neuroinflammation at clinically relevant vector copy numbers. HSPC-derived cells engrafted in brain show efficiencies highest in perivascular areas, lower in choroid plexus and meninges, and lowest in parenchyma. Importantly, the efficacy of correction was independent of the number of brain-engrafted cells. These results indicate that tagged versions of IDS can outperform untagged IDS in HSPC-LVGT for the correction of brain pathology in MPS II, and they imply both cell-mediated and tag-mediated correction mechanisms, including passage across the BBB and increased uptake, highlighting their potential for clinical translation.
© 2023 The Authors.

Xenotransplantation is frequently used to study normal and malignant hematopoiesis of human cells. However, conventional mouse xenotransplantation models lack essential human-specific bone-marrow (BM)-microenvironment-derived survival, proliferation, and self-renewal signals for engraftment of normal and malignant blood cells. As a consequence, many human leukemias and other hematologic disorders do not robustly engraft in these conventional models. Here, we describe a complete workflow for the generation of humanized ossicles with an accessible BM microenvironment that faithfully recapitulates normal BM niche morphology and function. The ossicles, therefore, allow for accelerated and superior engraftment of primary patient-derived acute myeloid leukemia (AML) and other hematologic malignancies such as myelofibrosis (MF) in mice. The humanized ossicles are formed by in situ differentiation of BM-derived mesenchymal stromal cells (MSCs). Human hematopoietic cells can subsequently be transplanted directly into the ossicle marrow space or by intravenous injection. Using this method, a humanized engraftable BM microenvironment can be formed within 6-10 weeks. Engraftment of human hematopoietic cells can be evaluated by flow cytometry 8-16 weeks after transplantation. This protocol describes a robust and reproducible in vivo methodology for the study of normal and malignant human hematopoiesis in a more physiologic setting.

Extracellular vesicles (EVs) released by mesenchymal stromal cells (MSCs) may contribute to biological processes such as tissue regeneration, immunomodulation and neuroprotection. Evaluation of their therapeutic potential and application in future clinical trials demands thorough characterization of EV content and production under defined medium conditions, devoid of xenogenic substances and serum-derived vesicles. Addressing the apparent need for such a growth medium, we have developed a medium formulation based on pooled human platelet lysate (pHPL), free from animal-derived xenogenic additives and depleted of EVs.
Depletion of EVs from complete growth medium was achieved by centrifugation at 120 000 g for 3 h, which reduced RNA-containing pHPL EVs to below the detection limit.
Bone marrow (BM)-derived MSCs propagated in this medium retained the characteristic surface marker expression, cell morphology, viability and in vitro osteogenic and adipogenic differentiation potential. The proliferation rate was not significantly affected after 48 h but was decreased by 13% after 96 h. EVs collected from BM-MSCs cultured in EV-depleted medium revealed a similar RNA pattern as EVs generated in standard pHPL EV-containing medium but displayed a more clearly defined pattern of proteins characteristic for EVs. Reduction of pHPL content from 10% to 2% or serum-/pHPL-free conditions strongly altered MSC characteristics and RNA content of released EV.
The 10% pHPL-based EV-depleted medium is appropriate for purification of exclusively human MSC-derived EVs. With this Good Manufacturing Practice-grade protocol, characterization and establishment of protein and RNA profiles from MSC-derived EVs can now be achieved to identify active components in therapeutic EVs for future clinical application.
Copyright © 2017 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.