Activated mesenchymal stem/stromal cells promote myeloid cell differentiation via CCL2/CCR2 signaling.

Myeloid cells, which originate from hematopoietic stem/progenitor cells (HSPCs), play a crucial role in mitigating infections. This study aimed to explore the impact of mesenchymal stem/stromal cells (MSCs) on the differentiation of HSPCs and progenitors through the C-C motif chemokine CCL2/CCR2 signaling pathway. Murine MSCs, identified as PDGFRα+Sca-1+ cells (PαS cells), were found to secrete CCL2, particularly in response to lipopolysaccharide stimulation. MSC-secreted CCL2 promoted the differentiation of granulocyte/macrophage progenitors into the myeloid lineage. MSC-derived CCL2 plays an important role in the early phase of myeloid cell differentiation in vivo. Single-cell RNA sequencing analysis confirmed that CCL2-mediated cell fate determination was also observed in human bone marrow cells. These findings provide valuable insights for investigating the in vivo effects of MSC transplantation.

Novel artificial nerve transplantation of human iPSC-derived neurite bundles enhanced nerve regeneration after peripheral nerve injury.

BACKGROUND: Severe peripheral nerve damage always requires surgical treatment. Autologous nerve transplantation is a standard treatment, but it is not sufficient due to length limitations and extended surgical time. Even with the available artificial nerves, there is still large room for improvement in their therapeutic effects. Novel treatments for peripheral nerve injury are greatly expected. METHODS: Using a specialized microfluidic device, we generated artificial neurite bundles from human iPSC-derived motor and sensory nerve organoids. We developed a new technology to isolate cell-free neurite bundles from spheroids. Transplantation therapy was carried out for large nerve defects in rat sciatic nerve with novel artificial nerve conduit filled with lineally assembled sets of human neurite bundles. Quantitative comparisons were performed over time to search for the artificial nerve with the therapeutic effect, evaluating the recovery of motor and sensory functions and histological regeneration. In addition, a multidimensional unbiased gene expression profiling was carried out by using next-generation sequencing. RESULT: After transplantation, the neurite bundle-derived artificial nerves exerted significant therapeutic effects, both functionally and histologically. Remarkably, therapeutic efficacy was achieved without immunosuppression, even in xenotransplantation. Transplanted neurite bundles fully dissolved after several weeks, with no tumor formation or cell proliferation, confirming their biosafety. Posttransplant gene expression analysis highlighted the immune system's role in recovery. CONCLUSION: The combination of newly developed microfluidic devices and iPSC technology enables the preparation of artificial nerves from organoid-derived neurite bundles in advance for future treatment of peripheral nerve injury patients. A promising, safe, and effective peripheral nerve treatment is now ready for clinical application.

Quality Control of Stem Cell-Based Cultured Meat According to Specific Differentiation Abilities.

The demand for stem cell-based cultured meat as an alternative protein source is increasing in response to global food scarcity. However, the definition of quality controls, including appropriate growth factors and cell characteristics, remains incomplete. Cluster of differentiation (CD) 29 is ubiquitously expressed in bovine muscle tissue and is a marker of progenitor cells in cultured meat. However, CD29+ cells are naturally heterogeneous, and this quality control issue must be resolved. In this study, the aim was to identify the subpopulation of the CD29+ cell population with potential utility in cultured meat production. The CD29+ cell population exhibited heterogeneity, discernible through the CD44 and CD344 markers. CD29+CD44-CD344- cells displayed the ability for long-term culture, demonstrating high adipogenic potential and substantial lipid droplet accumulation, even within 3D cultures. Conversely, CD29+CD44+ cells exhibited rapid proliferation but were not viable for prolonged culture. Using cells suitable for adipocyte and muscle differentiation, we successfully designed meat buds, especially those rich in fat. Collectively, the identification and comprehension of distinct cell populations within bovine tissues contribute to quality control predictions in meat production. They also aid in establishing a stable and reliable cultured meat production technique.

Secretory GFP reconstitution labeling of neighboring cells interrogates cell-cell interactions in metastatic niches.

Cancer cells inevitably interact with neighboring host tissue-resident cells during the process of metastatic colonization, establishing a metastatic niche to fuel their survival, growth, and invasion. However, the underlying mechanisms in the metastatic niche are yet to be fully elucidated owing to the lack of methodologies for comprehensively studying the mechanisms of cell-cell interactions in the niche. Here, we improve a split green fluorescent protein (GFP)-based genetically encoded system to develop secretory glycosylphosphatidylinositol-anchored reconstitution-activated proteins to highlight intercellular connections (sGRAPHIC) for efficient fluorescent labeling of tissue-resident cells that neighbor on and putatively interact with cancer cells in deep tissues. The sGRAPHIC system enables the isolation of metastatic niche-associated tissue-resident cells for their characterization using a single-cell RNA sequencing platform. We use this sGRAPHIC-leveraged transcriptomic platform to uncover gene expression patterns in metastatic niche-associated hepatocytes in a murine model of liver metastasis. Among the marker genes of metastatic niche-associated hepatocytes, we identify Lgals3, encoding galectin-3, as a potential pro-metastatic factor that accelerates metastatic growth and invasion.

Conservation of Markers and Stemness in Adipose Stem and Progenitor Cells between Cattle and Other Species.

Adipose stem and progenitor cells (ASPCs) have been isolated from humans and animals for use in regenerative medicine and therapy. However, knowledge of ASPCs in other species is limited. Particularly, ASPCs in livestock are expected to enhance the fat content and meat composition. In this study, we isolated bovine ASPCs using cell surface markers. Specifically, we focused on ASPC markers in humans and experimental animals, namely CD26, CD146, and CD54. Stromal vascular fraction cells from bovine fat were separated using flow cytometry before primary culture. We evaluated the self-renewal and adipogenic potential of each fraction. We identified four cell populations: CD26-CD146+CD54+, CD26-CD146+CD54-, CD26-CD146-, and CD26+CD146-. Among them, the CD26-CD146+ fraction, particularly CD54+, demonstrated the properties of preadipocytes (PreAs), characterized by slow proliferation and a high adipogenic capacity. In conclusion, we could collect and characterize possible PreAs as CD26-CD146+CD54+ or CD26-CD146+CD54-, which are expected for in vitro bovine adipogenic assays in the future.

Mesenchymal properties of iPSC-derived neural progenitors that generate undesired grafts after transplantation.

Although neural stem/progenitor cells derived from human induced pluripotent stem cells (hiPSC-NS/PCs) are expected to be a cell source for cell-based therapy, tumorigenesis of hiPSC-NS/PCs is a potential problem for clinical applications. Therefore, to understand the mechanisms of tumorigenicity in NS/PCs, we clarified the cell populations of NS/PCs. We established single cell-derived NS/PC clones (scNS/PCs) from hiPSC-NS/PCs that generated undesired grafts. Additionally, we performed bioassays on scNS/PCs, which classified cell types within parental hiPSC-NS/PCs. Interestingly, we found unique subsets of scNS/PCs, which exhibited the transcriptome signature of mesenchymal lineages. Furthermore, these scNS/PCs expressed both neural (PSA-NCAM) and mesenchymal (CD73 and CD105) markers, and had an osteogenic differentiation capacity. Notably, eliminating CD73+ CD105+ cells from among parental hiPSC-NS/PCs ensured the quality of hiPSC-NS/PCs. Taken together, the existence of unexpected cell populations among NS/PCs may explain their tumorigenicity leading to potential safety issues of hiPSC-NS/PCs for future regenerative medicine.

CD73-Positive Cell Spheroid Transplantation Attenuates Colonic Atrophy.

The incidence of inflammatory bowel diseases (IBD) is increasing worldwide. Mesenchymal stem/stromal cells (MSCs) have immunomodulatory functions and are a promising source for cell transplantation therapy for IBD. However, owing to their heterogeneous nature, their therapeutic efficacy in colitis is controversial and depends on the delivery route and form of transplanted cells. Cluster of differentiation (CD) 73 is widely expressed in MSCs and used to obtain a homogeneous MSC population. Herein, we determined the optimal method for MSC transplantation using CD73+ cells in a colitis model. mRNA sequencing analysis showed that CD73+ cells exhibited a downregulation of inflammatory gene expression and an upregulation of extracellular matrix-related gene expression. Furthermore, three-dimensional CD73+ cell spheroids showed enhanced engraftment at the injured site through the enteral route, facilitated extracellular matrix remodeling, and downregulated inflammatory gene expression in fibroblasts, leading to the attenuation of colonic atrophy. Therefore, the interaction between intestinal fibroblasts and exogenous MSCs via tissue remodeling is one mechanism that can be exploited for colitis prevention. Our results highlight that the transplantation of homogeneous cell populations with well-characterized properties is beneficial for IBD treatment.

Evaluation of Human-Induced Pluripotent Stem Cells Derived from a Patient with Schwartz-Jampel Syndrome Revealed Distinct Hyperexcitability in the Skeletal Muscles.

Schwartz-Jampel syndrome (SJS) is an autosomal recessive disorder caused by loss-of-function mutations in heparan sulfate proteoglycan 2 (HSPG2), which encodes the core basement membrane protein perlecan. Myotonia is a major criterion for the diagnosis of SJS; however, its evaluation is based solely on physical examination and can be challenging in neonates and young children. Furthermore, the pathomechanism underlying SJS-related myotonia is not fully understood, and effective treatments for SJS are limited. Here, we established a cellular model of SJS using patient-derived human-induced pluripotent stem cells. This model exhibited hyper-responsiveness to acetylcholine as a result of abnormalities in the perlecan molecule, which were confirmed via comparison of their calcium imaging with calcium imaging of satellite cells derived from Hspg2-/--Tg mice, which exhibit myotonic symptoms similar to SJS symptoms. Therefore, our results confirm the utility of creating cellular models for investigating SJS and their application in evaluating myotonia in clinical cases, while also providing a useful tool for the future screening of SJS therapies.

Human induced pluripotent stem cell-derived salivary gland organoids model SARS-CoV-2 infection and replication.

Salivary glands act as virus reservoirs in various infectious diseases and have been reported to be targeted by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). However, the mechanisms underlying infection and replication in salivary glands are still enigmatic due to the lack of proper in vitro models. Here, we show that human induced salivary glands (hiSGs) generated from human induced pluripotent stem cells can be infected with SARS-CoV-2. The hiSGs exhibit properties similar to those of embryonic salivary glands and are a valuable tool for the functional analysis of genes during development. Orthotopically transplanted hiSGs can be engrafted at a recipient site in mice and show a mature phenotype. In addition, we confirm SARS-CoV-2 infection and replication in hiSGs. SARS-CoV-2 derived from saliva in asymptomatic individuals may participate in the spread of the virus. hiSGs may be a promising model for investigating the role of salivary glands as a virus reservoir.

Hematopoietic-Mesenchymal Signals Regulate the Properties of Mesenchymal Stem Cells.

It is well known that the properties of hematopoietic stem/progenitor cells (HSCs), such as their self-renewal ability and multipotency, are maintained through interactions with mesenchymal stem/stromal cells (MSCs). MSCs are rare cells that are present in the bone marrow and are useful for clinical applications due to their functional ability. To obtain the necessary number of cells, MSCs must be cultured to expand, but this causes a remarkable decrease in stem cell properties, such as multipotency and proliferation ability. In this study, we show that the c-Mpl signal, which is related to the maintenance of hematopoietic stem cells, has an important effect on the proliferation and differentiation ability of MSCs. Utilizing a co-culture system comprising MSCs and HSCs, it is suggested that signaling from hematopoietic cells to MSCs supports cell proliferation. Interestingly, the enhanced proliferation ability of the HSCs was decreased in c-Mpl knock-out HSCs (c-Mpl-KO). In addition, the MSCs co-cultured with c-Mpl-KO HSCs had reduced MSC marker expression (PDGFRa and Sca-1) compared to the MSCs co-cultured with c-Mpl-wild-type HSCs. These results suggest that a hematopoietic-mesenchymal signal exists, and that the state of the HSCs is important for the stability of MSC properties.

Role of Snai2 and Notch signaling in salivary gland myoepithelial cell fate.

Myoepithelial (ME) cells in exocrine glands exhibit both epithelial and mesenchymal features, contributing to fluid secretion through contraction. However, the regulation mechanism of behind this unique phenotype in salivary glands remains unclear. We established a flow cytometry-based purification method using cell surface molecules, epithelial cell adhesion molecule (EpCAM) and alpha 6 integrin (CD49f), to characterize ME cells. EpCAM+CD49fhigh cells showed relatively high expression of ME cell-marker genes, such as alpha-smooth muscle actin (α-SMA). For lineage tracing and strict isolation, tdTomato+EpCAM+CD49fhigh-ME cells were obtained from myosin heavy chain 11 (Myh11) -CreERT2/tdTomato mice. Transcriptome analysis revealed that expression of genes involved in the epithelial-mesenchymal transition, including Snai2, were upregulated in the ME cell-enriched subset. Snai2 suppression in stable ME cells decreased α-SMA and increased Krt14 expression, suggesting that ME cell features may be controlled by the epithelial-mesenchymal balance regulated by Snai2. In contrast, ME cells showed reduced ME properties and expressed the ductal markers Krt18/19 under sphere culture conditions. Notch signaling was activated under sphere culture conditions; excessive activation of Notch signaling accelerated Krt18/19 expression, but reduced α-SMA and Snai2 expression, suggesting that the behavior of Snai2-expressing ME cells may be controlled by Notch signaling.

MDS cells impair osteolineage differentiation of MSCs via extracellular vesicles to suppress normal hematopoiesis.

Myelodysplastic syndrome (MDS) is a clonal disorder of hematopoietic stem cells (HSCs), characterized by ineffective hematopoiesis and frequent progression to leukemia. It has long remained unresolved how MDS cells, which are less proliferative, inhibit normal hematopoiesis and eventually dominate the bone marrow space. Despite several studies implicating mesenchymal stromal or stem cells (MSCs), a principal component of the HSC niche, in the inhibition of normal hematopoiesis, the molecular mechanisms underlying this process remain unclear. Here, we demonstrate that both human and mouse MDS cells perturb bone metabolism by suppressing the osteolineage differentiation of MSCs, which impairs the ability of MSCs to support normal HSCs. Enforced MSC differentiation rescues the suppressed normal hematopoiesis in both in vivo and in vitro MDS models. Intriguingly, the suppression effect is reversible and mediated by extracellular vesicles (EVs) derived from MDS cells. These findings shed light on the novel MDS EV-MSC axis in ineffective hematopoiesis.

Novel Mesenchymal Stem Cell Spheroids with Enhanced Stem Cell Characteristics and Bone Regeneration Ability.

Mesenchymal stem cells (MSCs) exhibit self-renewal, multi-lineage differentiation potential and immunomodulatory properties, and are promising candidates for cellular therapy of various tissues. Despite the effective function of MSCs, the gradual loss of stem cell characteristics that occurs with repeated passages may significantly limit their therapeutic potential. A novel 3D shaking method was previously established to generate MSC spheroids in growth medium (GM-spheroids) and successfully maintain the multipotency of expanded MSCs, yet the expression of MSC-related genes was still low. In this study, we used a neurosphere culture technique to optimize the shaking culture method using human bone marrow-derived MSCs (BM-MSCs). MSC spheroids generated in neurosphere medium (NM-spheroids) maintained high expression of MSC-related genes during 3 weeks of prolonged shaking culture. Moreover, NM-spheroids generated from expanded MSCs showed high viability, upregulation of MSC-related and immune-related genes, and recovery of differentiation potential in vitro. Expanded adherent MSCs, GM-spheroids, and NM-spheroids were transplanted into a rat femur bone defect model to investigate their therapeutic potential in bone repair. Adherent MSCs and GM-spheroids showed delayed bone healing. In contrast, NM-spheroids showed high transplantation efficiency and enhanced bone regeneration. These data suggest that NM-spheroids generated using modified neurosphere culture conditions under continuous shaking recovered their stem cell characteristics in vitro and enhanced bone regeneration in vivo. Therefore, NM-spheroids should have great clinical potential for bone and tissue regenerative therapies as a stem cell-based biomaterial therapy.

Regulation of activated microglia and macrophages by systemically administered DNA/RNA heteroduplex oligonucleotides.

Microglial activation followed by recruitment of blood-borne macrophages into the central nervous system (CNS) aggravates neuroinflammation. Specifically, in multiple sclerosis (MS) as well as in experimental autoimmune encephalomyelitis (EAE), a rodent model of MS, activated microglia and macrophages (Mg/Mφ) promote proinflammatory responses and expand demyelination in the CNS. However, a potent therapeutic approach through the systemic route for regulating their functions has not yet been developed. Here, we demonstrate that a systemically injected DNA/RNA heteroduplex oligonucleotide (HDO), composed of an antisense oligonucleotide (ASO) and its complementary RNA, conjugated to cholesterol (Chol-HDO) distributed more efficiently to demyelinating lesions of the spinal cord in EAE mice with significant gene silencing than the parent ASO. Importantly, systemic administration of Cd40-targeting Chol-HDO improved clinical signs of EAE with significant downregulation of Cd40 in Mg/Mφ. Furthermore, we successfully identify that macrophage scavenger receptor 1 (MSR1) is responsible for the uptake of Chol-HDO by Mg/Mφ of EAE mice. Overall, our findings demonstrate the therapeutic potency of systemically administered Chol-HDO to regulate activated Mg/Mφ in neuroinflammation.

DNA/RNA heteroduplex oligonucleotide technology for regulating lymphocytes in vivo.

Manipulating lymphocyte functions with gene silencing approaches is promising for treating autoimmunity, inflammation, and cancer. Although oligonucleotide therapy has been proven to be successful in treating several conditions, efficient in vivo delivery of oligonucleotide to lymphocyte populations remains a challenge. Here, we demonstrate that intravenous injection of a heteroduplex oligonucleotide (HDO), comprised of an antisense oligonucleotide (ASO) and its complementary RNA conjugated to α-tocopherol, silences lymphocyte endogenous gene expression with higher potency, efficacy, and longer retention time than ASOs. Importantly, reduction of Itga4 by HDO ameliorates symptoms in both adoptive transfer and active experimental autoimmune encephalomyelitis models. Our findings reveal the advantages of HDO with enhanced gene knockdown effect and different delivery mechanisms compared with ASO. Thus, regulation of lymphocyte functions by HDO is a potential therapeutic option for immune-mediated diseases.

Isolation and Characterization of Tissue Resident CD29-Positive Progenitor Cells in Livestock to Generate a Three-Dimensional Meat Bud.

The current process of meat production using livestock has significant effects on the global environment, including high emissions of greenhouse gases. In recent years, cultured meat has attracted attention as a way to acquire animal proteins. However, the lack of markers that isolate proliferating cells from bovine tissues and the complex structure of the meat make it difficult to culture meat in a dish. In this study, we screened 246 cell-surface antibodies by fluorescence-activated cell sorting for their capacity to form colonies and their suitability to construct spheroid "meat buds". CD29+ cells (Ha2/5 clone) have a high potency to form colonies and efficiently proliferate on fibronectin-coated dishes. Furthermore, the meat buds created from CD29+ cells could differentiate into muscle and adipose cells in a three-dimensional structure. The meat buds embedded in the collagen gel proliferated in the matrix and formed large aggregates. Approximately 10 trillion cells can theoretically be obtained from 100 g of bovine tissue by culturing and amplifying them using these methods. The CD29+ cell characteristics of bovine tissue provide insights into the production of meat alternatives in vitro.

Cellular Heterogeneity of Mesenchymal Stem/Stromal Cells in the Bone Marrow.

Mesenchymal stem/stromal cells (MSCs) are present in various body tissues and help in maintaining homeostasis. The stemness of MSCs has been evaluated in vitro. In addition, analyses of cell surface antigens and gene expression patterns have shown that MSCs comprise a heterogeneous population, and the diverse and complex nature of MSCs makes it difficult to identify the specific roles in diseases. There is a lack of understanding regarding the classification of MSC properties. In this review, we explore the characteristics of heterogeneous MSC populations based on their markers and gene expression profiles. We integrated the contents of previously reported single-cell analysis data to better understand the properties of mesenchymal cell populations. In addition, the cell populations involved in the development of myeloproliferative neoplasms (MPNs) are outlined. Owing to the diversity of terms used to describe MSCs, we used the text mining technology to extract topics from MSC research articles. Recent advances in technology could improve our understanding of the diversity of MSCs and help us evaluate cell populations.

Soluble JAM-C Ectodomain Serves as the Niche for Adipose-Derived Stromal/Stem Cells.

Junctional adhesion molecules (JAMs) are expressed in diverse types of stem and progenitor cells, but their physiological significance has yet to be established. Here, we report that JAMs exhibit a novel mode of interaction and biological activity in adipose-derived stromal/stem cells (ADSCs). Among the JAM family members, JAM-B and JAM-C were concentrated along the cell membranes of mouse ADSCs. JAM-C but not JAM-B was broadly distributed in the interstitial spaces of mouse adipose tissue. Interestingly, the JAM-C ectodomain was cleaved and secreted as a soluble form (sJAM-C) in vitro and in vivo, leading to deposition in the fat interstitial tissue. When ADSCs were grown in culture plates coated with sJAM-C, cell adhesion, cell proliferation and the expression of five mesenchymal stem cell markers, Cd44, Cd105, Cd140a, Cd166 and Sca-1, were significantly elevated. Moreover, immunoprecipitation assay showed that sJAM-C formed a complex with JAM-B. Using CRISPR/Cas9-based genome editing, we also demonstrated that sJAM-C was coupled with JAM-B to stimulate ADSC adhesion and maintenance. Together, these findings provide insight into the unique function of sJAM-C in ADSCs. We propose that JAMs contribute not only to cell-cell adhesion, but also to cell-matrix adhesion, by excising their ectodomain and functioning as a niche-like microenvironment for stem and progenitor cells.

FZD5 regulates cellular senescence in human mesenchymal stem/stromal cells.

Human mesenchymal stem/stromal cells (hMSCs) have garnered enormous interest as a potential resource for cell-based therapies. However, the molecular mechanisms regulating senescence in hMSCs remain unclear. To elucidate these mechanisms, we performed gene expression profiling to compare clonal immature MSCs exhibiting multipotency with less potent MSCs. We found that the transcription factor Frizzled 5 (FZD5) is expressed specifically in immature hMSCs. The FZD5 cell surface antigen was also highly expressed in the primary MSC fraction (LNGFR+ THY-1+ ) and cultured MSCs. Treatment of cells with the FZD5 ligand WNT5A promoted their proliferation. Upon FZD5 knockdown, hMSCs exhibited markedly attenuated proliferation and differentiation ability. The observed increase in the levels of senescence markers suggested that FZD5 knockdown promotes cellular senescence by regulating the noncanonical Wnt pathway. Conversely, FZD5 overexpression delayed cell cycle arrest during the continued culture of hMSCs. These results indicated that the intrinsic activation of FZD5 plays an essential role in negatively regulating senescence in hMSCs and suggested that controlling FZD5 signaling offers the potential to regulate hMSC quality and improve the efficacy of cell-replacement therapies using hMSCs.