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Background: There is a significant demand for intermediate-scale bioreactors in academic and industrial institutions to produce cells for various applications in drug screening and/or cell therapy. However, the application of these bioreactors in cultivating hiPSC-derived immune cells and other blood cells is noticeably lacking. To address this gap, we have developed a xeno-free and chemically defined intermediate-scale bioreactor platform, which allows for the generation of standardized human iPSC-derived hematopoietic organoids and subsequent continuous production of macrophages (iPSC-Mac)., Methods: We describe a novel method for intermediate-scale immune cell manufacturing, specifically the continuous production of functionally and phenotypically relevant macrophages that are harvested on weekly basis for multiple weeks., Results: The continuous production of standardized human iPSC-derived macrophages (iPSC-Mac) from 3D hematopoietic organoids also termed hemanoids, is demonstrated. The hemanoids exhibit successive stage-specific embryonic development, recapitulating embryonic hematopoiesis. iPSC-Mac were efficiently and continuously produced from three different iPSC lines and exhibited a consistent and reproducible phenotype, as well as classical functionality and the ability to adapt towards pro- and anti-inflammatory activation stages. Single-cell transcriptomic analysis revealed high macrophage purity. Additionally, we show the ability to use the produced iPSC-Mac as a model for testing immunomodulatory drugs, exemplified by dexamethasone., Conclusions: The novel method demonstrates an easy-to-use intermediate-scale bioreactor platform that produces prime macrophages from human iPSCs. These macrophages are functionally active and require no downstream maturation steps, rendering them highly desirable for both therapeutic and non-therapeutic applications., (© 2024. The Author(s).)

Sialic acids are found as terminal sugars on glycan structures on cellular surfaces. T cells carry these sialoglycans abundantly, and they are thought to serve multiple functions in cell adhesion, cell migration, and protection from complement attack. We studied the role of sialoglycans on T cells in a mouse model with a T cell-specific deletion of cytidine monophosphate-sialic acid synthase (CMAS), the enzyme that is crucial for the synthesis of sialoglycans. These mice showed a T-cell deficiency in peripheral lymphoid organs. Many T cells with an undeleted Cmas allele were found in the periphery, suggesting that they escaped the Cre-mediated deletion. The remaining peripheral T cells of T cell-specific Cmas KO mice had a memory-like phenotype. Additional depletion of the complement factor C3 could not rescue the phenotype, showing that the T-cell defect was not caused by a host complement activity. Cmas -deficient T cells showed a high level of activated caspase 3, indicating an ongoing apoptosis. In bone marrow chimeric cellular transfer experiments, we observed a strong competitive disadvantage of Cmas -deficient T cells compared to wild-type T cells. These results show that sialoglycans on the surface of T cells are crucial for T-cell survival and maintenance. This function has not been recognized before and is similar to the function of sialoglycans on B cells., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Schmidt, Linder, Korn, Schellenberg, Meyer, Nimmerjahn, Werner, Abeln, Gerardy-Schahn, Münster-Kühnel and Nitschke.)

The complement system is a part of the innate immune system in the fluid phase and efficiently eliminates pathogens. However, its activation requires tight regulation on the host cell surface in order not to compromise cellular viability. Previously, we showed that loss of placental cell surface sialylation in mice in vivo leads to a maternal complement attack at the fetal-maternal interface, ultimately resulting in loss of pregnancy. To gain insight into the regulatory function of sialylation in complement activation, we here generated trophoblast stem cells (TSC) devoid of sialylation, which also revealed complement sensitivity and cell death in vitro. Glycolipid-analysis by multiplexed capillary gel electrophoresis coupled to laser-induced fluorescence detection (xCGE-LIF) allowed us to identify the monosialoganglioside GM1a as a key element of cell surface complement regulation. Exogenously administered GM1a integrated into the plasma membrane of trophoblasts, substantially increased binding of complement factor H (FH) and was sufficient to protect the cells from complement attack and cell death. GM1a treatment also rescued human endothelial cells and erythrocytes from complement attack in a concentration dependent manner. Furthermore, GM1a significantly reduced complement mediated hemolysis of erythrocytes from a patient with Paroxysmal nocturnal hemoglobinuria (PNH). This study demonstrates the complement regulatory potential of exogenously administered gangliosides and paves the way for sialoglycotherapeutics as a novel substance class for membrane-targeted complement regulators., (© 2023. Cell Death Differentiation Association (ADMC).)

Sialic acids (Sias) on the B cell membrane are involved in cell migration, in the control of the complement system and, as sialic acid-binding immunoglobulin-like lectin (Siglec) ligands, in the regulation of cellular signaling. We studied the role of sialoglycans on B cells in a mouse model with B cell-specific deletion of cytidine monophosphate sialic acid synthase (CMAS), the enzyme essential for the synthesis of sialoglycans. Surprisingly, these mice showed a severe B cell deficiency in secondary lymphoid organs. Additional depletion of the complement factor C3 rescued the phenotype only marginally, demonstrating a complement-independent mechanism. The B cell survival receptor BAFF receptor was not up-regulated, and levels of activated caspase 3 and processed caspase 8 were high in B cells of Cmas -deficient mice, indicating ongoing apoptosis. Overexpressed Bcl-2 could not rescue this phenotype, pointing to extrinsic apoptosis. These results show that sialoglycans on the B cell surface are crucial for B cell survival by counteracting several death-inducing pathways.

Among the enzymes of the biosynthesis of sialoglycoconjugates, uridine diphosphate-N-acetylglucosamine-2-epimerase/N-acetylmannosamine kinase (GNE), catalyzing the first essential step of the sialic acid (Sia) de novo biosynthesis, and cytidine monophosphate (CMP)-Sia synthase (CMAS), activating Sia to CMP-Sia, are particularly important. The knockout of either of these enzymes in mice is embryonically lethal. While the lethality of Cmas-/- mice has been attributed to a maternal complement attack against asialo fetal placental cells, the cause of lethality in Gne-deficient embryos has remained elusive. Here, we advanced the significance of sialylation for embryonic development through detailed histological analyses of Gne-/- embryos and placentae. We found that Gne-/- embryonic and extraembryonic tissues are hyposialylated rather than being completely deficient of sialoglycans, which holds true for Cmas-/- embryos. Residual sialylation of Gne-/- cells can be explained by scavenging free Sia from sialylated maternal serum glycoconjugates via the lysosomal salvage pathway. The placental architecture of Gne-/- mice was unaffected, but severe hemorrhages in the neuroepithelium with extensive bleeding into the cephalic ventricles were present at E12.5 in the mutants. At E13.5, the vast majority of Gne-/- embryos were asystolic. This phenotype persisted when Gne-/- mice were backcrossed to a complement component 3-deficient background, confirming distinct pathomechanisms of Cmas-/- and Gne-/- mice. We conclude that the low level of sialylation observed in Gne-/- mice is sufficient both for immune homeostasis at the fetal-maternal interface and for embryonic development until E12.5. However, formation of the neural microvasculature is the first critical process, depending on a higher degree of sialylation during development of the embryo proper., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Primary or secondary immunodeficiencies are characterized by disruption of cellular and humoral immunity. Respiratory infections are a major cause of morbidity and mortality among immunodeficient or immunocompromised patients, with Staphylococcus aureus being a common offending organism. We propose here an adoptive macrophage transfer approach aiming to enhance impaired pulmonary immunity against S aureus. Our studies, using human-induced pluripotent stem cell-derived macrophages (iMφs), demonstrate efficient antimicrobial potential against methicillin-sensitive and methicillin-resistant clinical isolates of S aureus. Using an S aureus airway infection model in immunodeficient mice, we demonstrate that the adoptive transfer of iMφs is able to reduce the bacterial load more than 10-fold within 20 hours. This effect was associated with reduced granulocyte infiltration and less damage in lung tissue of transplanted animals. Whole transcriptome analysis of iMφs compared with monocyte-derived macrophages indicates a more profound upregulation of inflammatory genes early after infection and faster normalization 24 hours postinfection. Our data demonstrate high therapeutic efficacy of iMφ-based immunotherapy against S aureus infections and offer an alternative treatment strategy for immunodeficient or immunocompromised patients., (© 2021 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NC-ND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved.)

The periodontal pathogen Tannerella forsythia utilizes host sialic acids as a nutrient source. To also make O-acetylated sialyl residues susceptible to the action of its sialidase and sialic acid uptake system, Tannerella produces NanS, an O-acetylesterase with two putative catalytic domains. Here, we analyzed NanS by homology modeling, predicted a catalytic serine-histidine-aspartate triad for each catalytic domain and performed individual domain inactivation by single alanine exchanges of the triad nucleophiles S32 and S311. Subsequent functional analyses revealed that both domains possess sialyl-O-acetylesterase activity, but differ in their regioselectivity with respect to position O9 and O7 of sialic acid. The 7-O-acetylesterase activity inherent to the C-terminal domain of NanS is unique among sialyl-O-acetylesterases and fills the current gap in tools targeting 7-O-acetylation. Application of the O7-specific variant NanS-S32A allowed us to evidence the presence of cellular 7,9-di-O-acetylated sialoglycans by monitoring the gain in 9-O-acetylation upon selective removal of acetyl groups from O7. Moreover, we established de-7,9-O-acetylation by wild-type NanS as an easy and efficient method to validate the specific binding of three viral lectins commonly used for the recognition of (7),9-O-acetylated sialoglycans. Their binding critically depends on an acetyl group in O9, yet de-7,9-O-acetylation proved advantageous over de-9-O-acetylation as the additional removal of the 7-O-acetyl group eliminated ligand formation by 7,9-ester migration. Together, our data show that NanS gained dual functionality through recruitment of two esterase modules with complementary activities. This enables Tannerella to scavenge 7,9-di-O-acetylated sialyl residues and provides a novel, O7-specific tool for studying sialic acid O-acetylation., (© The Author(s) 2021. Published by Oxford University Press. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Hematopoietic development is spatiotemporally tightly regulated by defined cell-intrinsic and extrinsic modifiers. The role of cytokines has been intensively studied in adult hematopoiesis; however, their role in embryonic hematopoietic specification remains largely unexplored. Here, we used induced pluripotent stem cell (iPSC) technology and established a 3-dimensional, organoid-like differentiation system (hemanoid) maintaining the structural cellular integrity to evaluate the effect of cytokines on embryonic hematopoietic development. We show, that defined stages of early human hematopoietic development were recapitulated within the generated hemanoids. We identified KDR+/CD34high/CD144+/CD43-/CD45- hemato-endothelial progenitor cells (HEPs) forming organized, vasculature-like structures and giving rise to CD34low/CD144-/CD43+/CD45+ hematopoietic progenitor cells. We demonstrate that the endothelial to hematopoietic transition of HEPs is dependent on the presence of interleukin 3 (IL-3). Inhibition of IL-3 signalling blocked hematopoietic differentiation and arrested the cells in the HEP stage. Thus, our data suggest an important role for IL-3 in early human hematopoiesis by supporting the endothelial to hematopoietic transition of hemato-endothelial progenitor cells and highlight the potential of a hemanoid-based model to study human hematopoietic development.

Background: The etiology of steroid-resistant nephrotic syndrome, which manifests as FSGS, is not completely understood. Aberrant glycosylation is an often underestimated factor for pathologic processes, and structural changes in the glomerular endothelial glycocalyx have been correlated with models of nephrotic syndrome. Glycans are frequently capped by sialic acid (Sia), and sialylation's crucial role for kidney function is well known. Human podocytes are highly sialylated; however, sialylation's role in podocyte homeostasis remains unclear., Methods: We generated a podocyte-specific sialylation-deficient mouse model ( PCmas -/- ) by targeting CMP-Sia synthetase, and used histologic and ultrastructural analysis to decipher the phenotype. We applied CRISPR/Cas9 technology to generate immortalized sialylation-deficient podocytes (asialo-podocytes) for functional studies., Results: Progressive loss of sialylation in PCmas -/- mice resulted in onset of proteinuria around postnatal day 28, accompanied by foot process effacement and loss of slit diaphragms. Podocyte injury led to severe glomerular defects, including expanded capillary lumen, mesangial hypercellularity, synechiae formation, and podocyte loss. In vivo , loss of sialylation resulted in mislocalization of slit diaphragm components, whereas podocalyxin localization was preserved. In vitro , asialo-podocytes were viable, able to proliferate and differentiate, but showed impaired adhesion to collagen IV., Conclusions: Loss of cell-surface sialylation in mice resulted in disturbance of podocyte homeostasis and FSGS development. Impaired podocyte adhesion to the glomerular basement membrane most likely contributed to disease development. Our data support the notion that loss of sialylation might be part of the complex process causing FSGS. Sialylation, such as through a Sia supplementation therapy, might provide a new therapeutic strategy to cure or delay FSGS and potentially other glomerulopathies., (Copyright © 2019 by the American Society of Nephrology.)

The negatively charged sugar sialic acid (Sia) occupies the outermost position in the bulk of cell surface glycans. Lack of sialylated glycans due to genetic ablation of the Sia-activating enzyme CMP-sialic acid synthase (CMAS) resulted in embryonic lethality around day 9.5 post coitum (E9.5) in mice. Developmental failure was caused by complement activation on trophoblasts in Cmas-/- implants and was accompanied by infiltration of maternal neutrophils at the fetal-maternal interface, intrauterine growth restriction, impaired placental development, and a thickened Reichert's membrane. This phenotype, which shared features with complement receptor 1-related protein Y (Crry) depletion, was rescued in E8.5 Cmas-/- mice upon injection of cobra venom factor, resulting in exhaustion of the maternal complement component C3. Here we show that Sia is dispensable for early development of the embryo proper but pivotal for fetal-maternal immune homeostasis during pregnancy, i.e., for protecting the allograft implant against attack by the maternal innate immune system. Finally, embryos devoid of cell surface sialylation suffered from malnutrition due to inadequate placentation as a secondary effect.

The negatively charged nonulose sialic acid (Sia) is essential for murine development in vivo. In order to elucidate the impact of sialylation on differentiation processes in the absence of maternal influences, we generated mouse embryonic stem cell (mESC) lines that lack CMP-Sia synthetase (CMAS) and thereby the ability to activate Sia to CMP-Sia. Loss of CMAS activity resulted in an asialo cell surface accompanied by an increase in glycoconjugates with terminal galactosyl and oligo-LacNAc residues, as well as intracellular accumulation of free Sia. Remarkably, these changes did not impact intracellular metabolites or the morphology and transcriptome of pluripotent mESC lines. Moreover, the capacity of Cmas -/- mESCs for undirected differentiation into embryoid bodies, germ layer formation and even the generation of beating cardiomyocytes provides first and conclusive evidence that pluripotency and differentiation of mESC in vitro can proceed in the absence of (poly)sialoglycans., (© 2017 The Authors. Published by Wiley-VCH Verlag GmbH & Co. KGaA.)

The healthcare industry is changing, posing challenges to Medicare-certified home healthcare agencies (HHAs). With healthcare reform and the 2011 prospective payment system changes, including reduction in reimbursement to agencies, it is imperative to assess an organization's wound care program. HHAs must provide quality care at lower costs, using evidence-based medicine that may include nontraditional approaches to providing care.

The polysialic acid (polySia) modification of the neural cell adhesion molecule NCAM is a key regulator of cell migration. Yet its role in NCAM-dependent or NCAM-independent modulation of motility and cell-matrix adhesion is largely unresolved. Here, we demonstrate that loss of polySia attenuates tumour cell migration and augments the number of focal adhesions in a cell-cell contact- and NCAM-dependent manner. In the presence or absence of polySia, NCAM never colocalised with focal adhesions but was enriched at cell-cell contacts. Focal adhesion of polySia- and NCAM-negative cells was enhanced by incubation with soluble NCAM or by removing polySia from heterotypic contacts with polySia-NCAM-positive cells. Focal adhesion was compromised by the src-family kinase inhibitor PP2, whereas loss of polySia or exposure to NCAM promoted the association of p59(Fyn) with the focal adhesion scaffolding protein paxillin. Unlike other NCAM responses, NCAM-induced focal adhesion was not prevented by inhibiting FGF receptor activity and could be evoked by NCAM fragments comprising immunoglobulin domains three and four but not by the NCAM fibronectin domains alone or by an NCAM-derived peptide known to interact with and activate FGF receptors. Together, these data indicate that polySia regulates cell motility through NCAM-induced but FGF-receptor-independent signalling to focal adhesions., (@ 2011. Published by The Company of Biologists Ltd)

The primary intent behind Medicaid was to mainstream the poor and enable them to receive the same level and quality of care enjoyed by the middle class. This study compares the hospital utilization (total charges, length of stay, charges per day) and mortality levels among beneficiaries of Arizona's experimental Medicaid program with those of privately insured patients. The analysis is based on 121,874 discharges of patients with 11 different conditions from nonfederal general hospitals in Arizona during 1989 and 1990. After controlling for severity of illness and the specific hospital used, as well as several patient, hospital, and physician factors, we find that AHCCCS patients with medical, surgical, and pediatric diagnoses exhibit few significant differences in utilization and mortality compared to patients with private insurance. However, AHCCCS patients undergoing vaginal delivery exhibit significantly lower charges and length of stay, suggesting they underuse these services. AHCCCS women undergoing cesarean section exhibit higher charges and longer stays. We conclude that Arizona's Medicaid program provides hospital care equivalent to that received by privately-insured patients for many but not all conditions.