Human Cell Biology Research Articles

IER3IP1 mutations cause neonatal diabetes due to impaired proinsulin trafficking.

Immediate early response 3 interacting-protein 1 (IER3IP1) is an endoplasmic reticulum resident protein, highly expressed in pancreatic cells and the developing brain cortex. Homozygous mutations in IER3IP1 have been found in individuals with microcephaly and neonatal diabetes, yet the underlying mechanism causing beta cell failure remains unclear. Here, we utilized differentiation of genome edited-stem cells into pancreatic islet cells to elucidate the molecular basis of IER3IP1 neonatal diabetes. Using CRISPR-Cas9, we generated two distinct IER3IP1-mutant human embryonic stem cell lines: a homozygous knock-in model of a patient mutation (IER3IP1V21G), and a knockout model (IER3IP1-/-). While these mutant stem cell lines differentiated normally into definitive endoderm and pancreatic progenitors, we observed that IER3IP1-KO stem cell derived-islets (SC-islets) presented a significant decrease in beta cell numbers and elevated ER stress. Retention Using Selective Hooks (RUSH) assay revealed three-fold reduction in ER-to-Golgi trafficking of proinsulin in IER3IP1 mutant beta cells. Additionally, IER3IP1 mutant SC-islets implanted into immunocompromised mice displayed defective human insulin secretion, indicating the deleterious impact of IER3IP1 mutations on beta cell function. Our study provides valuable insights into the role of IER3IP1 in human beta cell biology and establishes a useful model to investigate ER-to-Golgi trafficking defects within beta cells.

What are the essential determinants of human papillomavirus carcinogenesis?

Human papillomavirus (HPV) infection is the leading viral cause of cancer. Over the past several decades, research on HPVs has provided remarkable insight into human cell biology and into the pathology of viral and non-viral cancers. The HPV E6 and E7 proteins engage host cellular proteins to establish an environment in infected cells that is conducive to virus replication. They rewire host cell signaling pathways to promote proliferation, inhibit differentiation, and limit cell death. The activity of the "high-risk" HPV E6 and E7 proteins is so potent that their dysregulated expression is sufficient to drive the initiation and maintenance of HPV-associated cancers. Consequently, intensive research efforts have aimed to identify the host cell targets of E6 and E7, in part with the idea that some or all of the virus-host interactions would be essential cancer drivers. These efforts have identified a large number of potential binding partners of each oncoprotein. However, over the same time period, parallel research has revealed that a relatively small number of genetic mutations drive carcinogenesis in most non-viral cancers. We therefore propose that a high-priority goal is to identify which of the many targets of E6 and E7 are critical drivers of HPV carcinogenesis. By identifying the cancer-driving targets of E6 and E7, it should be possible to better understand the distinct roles of other targets, perhaps in the viral life cycle, and to focus efforts to develop anti-cancer therapies on the subset of virus-host interactions for which therapeutic intervention would have the greatest impact.

Kinase-catalyzed biotinylation for discovery and validation of substrates to multispecificity kinases NME1 and NME2

Protein phosphorylation by kinases regulates mammalian cell functions, such as growth, division, and signal transduction. Among human kinases, NME1 and NME2 are associated with metastatic tumor suppression, but remain understudied due to the lack of tools to monitor their cellular substrates. In particular, NME1 and NME2 are multi-specificity kinases phosphorylating serine, threonine, histidine, and aspartic acid residues of substrate proteins, and the heat and acid sensitivity of phosphohistidine and phosphoaspartate complicates substrate discovery and validation. To provide new substrate monitoring tools, we established the γ-phosphate modified ATP analog, ATP-biotin, as a cosubstrate for phosphorylbiotinylation of NME1 and NME2 cellular substrates. Building upon this ATP-biotin compatibility, the Kinase-catalyzed Biotinylation with Inactivated Lysates for Discovery of Substrates (K-BILDS) method enabled validation of a known substrate and the discovery of seven NME1 and three NME2 substrates. Given the paucity of methods to study kinase substrates, ATP-biotin and the K-BILDS method are valuable tools to characterize the roles of NME1 and NME2 in human cell biology.

A study of macrodontia of the permanent maxillary central incisors among Delta State University students in Abraka, Nigeria

Macrodontia (or megadontia) is a rare dental anomaly characterized by excessive enlargement of all tooth structures, and in few cases may be associated with morphological anomalies. The goal of this study was to find out how common macrodontia of the maxillary central incisors was among Delta State University students in Abraka. The Research and Ethical Committee of the Department of Human Anatomy and Cell Biology, Delta State University, Abraka, gave approval for this study. A descriptive cross-sectional survey with a representative sample of 102 subjects was conducted (58 female and 44 male). The method used was a simple random sampling technique. The age and sex of each subject were obtained using a data form, and intra-oral measurements of the mesio-distal width of the maxillary right central incisors were taken. Results were arranged according to age and sex to ascertain the occurrence of sexual dimorphism; independent sample t test and chi-square test were used as inferential statistical tools. The prevalence of macrodontia was 35.3%; there was a significant sex difference, as p value was <0.05; there was no significant difference within the age groups, as p-value was > 0.05. The mean mesio-distal width was 8.54±0.25 mm in those who did not have macrodontia and 9.63±0.54 mm in those who did have macrodontia. Macrodontia of central incisors is dominant amid Delta State University learners in Abraka.

The lack of either IRF9, or STAT2, has surprisingly little effect on human natural killer cell development and function.

Analysis of genetically defined immunodeficient patients allows study of the effect of the absence of specific proteins on human immune function in real-world conditions. Here we have addressed the importance of type I interferon signalling for human NK cell development by studying the phenotype and function of circulating NK cells isolated from patients suffering primary immunodeficiency disease due to mutation of either the human interferon regulatory factor 9 (IRF9) or the signal transducer and activator of transcription 2 (STAT2) genes. IRF9, together with phosphorylated STAT1 and STAT2, form a heterotrimer called interferon stimulated gene factor 3 (ISGF3) which promotes the expression of hundreds of IFN-stimulated genes that mediate antiviral function triggered by exposure to type I interferons. IRF9- and STAT2-deficient patients are unable to respond efficiently to stimulation by type I interferons and so our experiments provide insights into the importance of type I interferon signalling and the consequences of its impairment on human NK cell biology. Surprisingly, the NK cells of these patients display essentially normal phenotype and function.

Single-cell RNA sequencing reveals peripheral blood leukocyte responses to spinal cord injury in mice with humanised immune systems

Next-generation humanised mouse models and single-cell RNA sequencing (scRNAseq) approaches enable in-depth studies into human immune cell biology. Here we used NSG-SGM3 mice engrafted with human umbilical cord haematopoietic stem cells to investigate how human immune cells respond to and/or are changed by traumatic spinal cord injury (SCI). We hypothesised that the use of such mice could help advance our understanding of spinal cord injury-induced immune depression syndrome (SCI-IDS), and also how human leukocytes change as they migrate from the circulation into the lesion site. Our scRNAseq experiments, supplemented by flow cytometry, demonstrate the existence of up to 11 human immune cell (sub-) types and/or states across the blood and injured spinal cord (7 days post-SCI) of humanised NSG-SGM3 mice. Further comparisons of human immune cell transcriptomes between naïve, sham-operated and SCI mice identified a total of 579 differentially expressed genes, 190 of which were ‘SCI-specific’ (that is, genes regulated only in response to SCI but not sham surgery). Gene ontology analysis showed a prominent downregulation of immune cell function under SCI conditions, including for T cell receptor signalling and antigen presentation, confirming the presence of SCI-IDS and the transcriptional signature of human leukocytes in association with this phenomenon. We also highlight the activating influence of the local spinal cord lesion microenvironment by comparing the transcriptomes of circulating versus infiltrated human immune cells; those isolated from the lesion site were enriched for genes relating to both immune cell activity and function (e.g., oxidative phosphorylation, T cell proliferation and antigen presentation). We lastly applied an integrated bioinformatics approach to determine where immune responses in humanised NSG-SGM3 mice appear congruent to the native responses of human SCI patients, and where they diverge. Collectively, our study provides a valuable resource and methodological framework for the use of these mice in translational research.

Cultures of Human Skin Mast Cells, an Attractive In Vitro Model for Studies of Human Mast Cell Biology.

Studies of mast cell biology are dependent on relevant and validated in vitro models. Here, we present detailed information concerning the phenotype of both freshly isolated human skin mast cells (MCs) and of in vitro cultures of these cells that were obtained by analyzing their total transcriptome. Transcript levels of MC-related granule proteins and transcription factors were found to be remarkably stable over a 3-week culture period. Relatively modest changes were also seen for important cell surface receptors including the high-affinity receptor for IgE, FCER1A, the low-affinity receptor for IgG, FCGR2A, and the receptor for stem cell factor, KIT. FCGR2A was the only Fc receptor for IgG expressed by these cells. The IgE receptor increased by 2-5-fold and an approximately 10-fold reduction in the expression of FCGR2A was observed most likely due to the cytokines, SCF and IL-4, used for expanding the cells. Comparisons of the present transcriptome against previously reported transcriptomes of mouse peritoneal MCs and mouse bone marrow-derived MCs (BMMCs) revealed both similarities and major differences. Strikingly, cathepsin G was the most highly expressed granule protease in human skin MCs, in contrast to the almost total absence of this protease in both mouse MCs. Transcript levels for the majority of cell surface receptors were also very low compared to the granule proteases in both mouse and human MCs, with a difference of almost two orders of magnitude. An almost total absence of T-cell granzymes was observed in human skin MCs, indicating that granzymes have no or only a minor role in human MC biology. Ex vivo skin MCs expressed high levels of selective immediate early genes and transcripts of heat shock proteins. In validation experiments, we determined that this expression was an inherent property of the cells and not the result of the isolation process. Three to four weeks in culture results in an induction of cell growth-related genes accompanying their expansion by 6-10-fold, which increases the number of cells for in vitro experiments. Collectively, we show that cultured human skin MCs resemble their ex vivo equivalents in many respects and are a more relevant in vitro model compared to mouse BMMCs for studies of MC biology, in particular human MC biology.

Anthropometric Study of the External Ear Among Delta State University Students in Abraka, Nigeria

Context: The morphology of the ear auricle contributes to the important facial features, which influence the facial appearance of an individual. Aim: The aim of this inquiry was to present the anthropometric details of the ear auricle and lobe among Delta State University students in Abraka, Nigeria. Setting and Design: This enquiry adopted a descriptive cross-sectional study design and considered both male and female Delta State University Students in Abraka aged between 17 and 31 years. The sample size for this study was 384 (204 males and 180 females), and the simple random sampling technique was used. Data were obtained with the use of Vernier caliper (standard digital sliding Vernier caliper), a tape rule, data collection sheet, and pencil, which were used for measurement and recording. Ethical clearance/approval was secured from the Ethical Board of the Department of Human Anatomy and Cell Biology in the Delta State University in Abraka, Nigeria. The data obtained were analyzed with the Statistical Package for the Social Sciences (SPSS) version 20.0.0. Results: The left and right auricular width and height and lobular width and height were observed to be higher in males when compared with females. There was no significant gender difference in the right ear auricular height, left and right ear lobular height, and left and right ear lobular width (P > 0.05). However, there was a significant gender difference in the left auricular height and left auricular width (P < 0.05). Conclusion: This enquiry provides a template for designers and ergonomists in designing prostheses and manufacturing ear microphone for various age brackets as well as provides a guide for plastic surgeons in construction and determination of the exact auricular framework across various age brackets.

Immunology of Cytokine Storm Syndromes: Natural Killer Cells.

Natural killer (NK) cells are innate immune lymphocytes that rapidly produce cytokines upon activation and kill target cells. NK cells have been of particular interest in primary hemophagocytic lymphohistiocytosis (pHLH) since all of the genetic defects associated with this disorder cause diminished cytotoxic capacity of NK cells and T lymphocytes, and assays of NK cell killing are used clinically for the diagnosis of HLH. Herein, we review human NK cell biology and the significance of alterations in NK cell function in the diagnosis and pathogenesis of HLH.

Chatbot responses suggest that hypothetical biology questions are harder than realistic ones.

The biology education literature includes compelling assertions that unfamiliar problems are especially useful for revealing students' true understanding of biology. However, there is only limited evidence that such novel problems have different cognitive requirements than more familiar problems. Here, we sought additional evidence by using chatbots based on large language models as models of biology students. For human physiology and cell biology, we developed sets of realistic and hypothetical problems matched to the same lesson learning objectives (LLOs). Problems were considered hypothetical if (i) known biological entities (molecules and organs) were given atypical or counterfactual properties (redefinition) or (ii) fictitious biological entities were introduced (invention). Several chatbots scored significantly worse on hypothetical problems than on realistic problems, with scores declining by an average of 13%. Among hypothetical questions, redefinition questions appeared especially difficult, with many chatbots scoring as if guessing randomly. These results suggest that, for a given LLO, hypothetical problems may have different cognitive demands than realistic problems and may more accurately reveal students' ability to apply biology core concepts to diverse contexts. The Test Question Templates (TQT) framework, which explicitly connects LLOs with examples of assessment questions, can help educators generate problems that are challenging (due to their novelty), yet fair (due to their alignment with pre-specified LLOs). Finally, ChatGPT's rapid improvement toward expert-level answers suggests that future educators cannot reasonably expect to ignore or outwit chatbots but must do what we can to make assessments fair and equitable.

Effects of tumor necrosis factor-α and interleukin-1β on human retinal endothelial cells

Uveitis, or intraocular inflammation, is a potentially blinding condition that mostly affects the working-age population. The cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β, play a role in the pathogenesis of non-infectious uveitis and have been linked to the breakdown of the inner blood-retinal barrier, composed mainly of retinal endothelial cells, leading to macular oedema and vascular leakage. However, the effects of TNF-α and IL-1β on human retinal endothelial function are not fully understood. In this work, we investigated the impact of TNF-α and IL-1β on several aspects of human retinal endothelial cell biology. Through a real-time biosensor, the impact of TNF-α and IL-1β on formation of a retinal endothelial cell barrier was analyzed. Changes in junctional components were assessed via RT-qPCR and immunolabelling. Cell survival, necrosis and apoptosis were appraised via cell proliferation and flow cytometric studies. Tumor necrosis factor-α and IL-1β impaired the electrical resistance of the retinal endothelial cell barrier, while the addition of a potentially barrier-impairing cytokine, IL-6, did not enhance the effect of TNF-α and IL-1β. Level of the gene transcript encoding zonula occludens (ZO)-1 was diminished, while ZO-1 protein configuration was changed by TNF-α and IL-1β. Both cytokines affected human retinal endothelial cell proliferation and viability, while only TNF-α increased rates of necrosis. These results indicate that TNF-α and IL-1β are important drivers of retinal endothelial dysfunction in non-infectious uveitis, suggesting that targeting these cytokines is critical when treating complications of uveitis, such as macular oedema and vascular leakage.

DMRT1 regulates human germline commitment

Germline commitment following primordial germ cell (PGC) specification during early human development establishes an epigenetic programme and competence for gametogenesis. Here we follow the progression of nascent PGC-like cells derived from human embryonic stem cells in vitro. We show that switching from BMP signalling for PGC specification to Activin A and retinoic acid resulted in DMRT1 and CDH5 expression, the indicators of migratory PGCs in vivo. Moreover, the induction of DMRT1 and SOX17 in PGC-like cells promoted epigenetic resetting with striking global enrichment of 5-hydroxymethylcytosine and locus-specific loss of 5-methylcytosine at DMRT1 binding sites and the expression of DAZL representing DNA methylation-sensitive genes, a hallmark of the germline commitment programme. We provide insight into the unique role of DMRT1 in germline development for advances in human germ cell biology and in vitro gametogenesis.

The effects of propranolol on the biology and Notch signaling pathway of human umbilical vein endothelial cells.

Propranolol is the first choice for treating infantile hemangioma (IH). How propranolol works in IH remains unclear. Infantile hemangioma endothelial cells (HemECs) express Notch1, Jagged, Hey1, and other molecules in the Notch pathway, suggesting that Notch pathway-related molecules play an important role in affecting vascular endothelial cell proliferation. Whether propranolol can affect the Notch signaling pathway in IH treatment is unclear. We performed this study to observe the effect of propranolol on the expression of Notch signaling pathway molecules in human umbilical vein endothelial cells (HUVECs) and to explore the therapeutic mechanism of propranolol on IH. HUVECs cultured in vitro were exposed to 60, 120, 240, 360, or 480 µM propranolol. The morphological changes of the HUVECs were observed under an inverted microscope. HUVECs proliferation was detected with Cell Counting Kit-8 (CCK-8). The effects of propranolol on HUVECs apoptosis were detected by flow cytometry. The role of Notch in propranolol inhibition of HUVEC proliferation was analyzed with real-time polymerase chain reaction (PCR) and western blotting. Propranolol reduced HUVECs numbers and altered their morphology. The inhibitory effect of propranolol on cell proliferation was dependent on the reaction time and drug concentration. Propranolol upregulated Jagged1, Notch1, and Hey1 expression and downregulated delta-like ligand4 (DLL4) expression. Propranolol may play a role in IH treatment by increasing Jagged1 expression in endothelial cells, activating the Notch pathway and inducing the upregulation of the downstream target gene HEY1.

A genome-wide genetic screen uncovers determinants of human pigmentation.

Skin color, one of the most diverse human traits, is determined by the quantity, type, and distribution of melanin. In this study, we leveraged the light-scattering properties of melanin to conduct a genome-wide screen for regulators of melanogenesis. We identified 169 functionally diverse genes that converge on melanosome biogenesis, endosomal transport, and gene regulation, of which 135 represented previously unknown associations with pigmentation. In agreement with their melanin-promoting function, the majority of screen hits were up-regulated in melanocytes from darkly pigmented individuals. We further unraveled functions of KLF6 as a transcription factor that regulates melanosome maturation and pigmentation in vivo, and of the endosomal trafficking protein COMMD3 in modulating melanosomal pH. Our study reveals a plethora of melanin-promoting genes, with broad implications for human variation, cell biology, and medicine.

Size-Specific Modulation of a Multienzyme Glucosome Assembly during the Cell Cycle.

Enzymes in glucose metabolism have been subjected to numerous studies, revealing the importance of their biological roles during the cell cycle. However, due to the lack of viable experimental strategies for measuring enzymatic activities particularly in living human cells, it has been challenging to address whether their enzymatic activities and thus anticipated glucose flux are directly associated with cell cycle progression. It has remained largely elusive how human cells regulate glucose metabolism at a subcellular level to meet the metabolic demands during the cell cycle. Meanwhile, we have characterized that rate-determining enzymes in glucose metabolism are spatially organized into three different sizes of multienzyme metabolic assemblies, termed glucosomes, to regulate the glucose flux between energy metabolism and building block biosynthesis. In this work, we first determined using cell synchronization and flow cytometric techniques that enhanced green fluorescent protein-tagged phosphofructokinase is adequate as an intracellular biomarker to evaluate the state of glucose metabolism during the cell cycle. We then applied fluorescence single-cell imaging strategies and discovered that the percentage of Hs578T cells showing small-sized glucosomes is drastically changed during the cell cycle, whereas the percentage of cells with medium-sized glucosomes is significantly elevated only in the G1 phase, but the percentage of cells showing large-sized glucosomes is barely or minimally altered along the cell cycle. Should we consider our previous localization-function studies that showed assembly size-dependent metabolic roles of glucosomes, this work strongly suggests that glucosome sizes are modulated during the cell cycle to regulate glucose flux between glycolysis and building block biosynthesis. Therefore, we propose the size-specific modulation of glucosomes as a behind-the-scenes mechanism that may explain functional association of glucose metabolism with the cell cycle and, thereby, their metabolic significance in human cell biology.

High-throughput screening identifies cell cycle-associated signaling cascades that regulate a multienzyme glucosome assembly in human cells.

We have previously demonstrated that human liver-type phosphofructokinase 1 (PFK1) recruits other rate-determining enzymes in glucose metabolism to organize multienzyme metabolic assemblies, termed glucosomes, in human cells. However, it has remained largely elusive how glucosomes are reversibly assembled and disassembled to functionally regulate glucose metabolism and thus contribute to human cell biology. We developed a high-content quantitative high-throughput screening (qHTS) assay to identify regulatory mechanisms that control PFK1-mediated glucosome assemblies from stably transfected HeLa Tet-On cells. Initial qHTS with a library of pharmacologically active compounds directed following efforts to kinase-inhibitor enriched collections. Consequently, three compounds that were known to inhibit cyclin-dependent kinase 2, ribosomal protein S6 kinase and Aurora kinase A, respectively, were identified and further validated under high-resolution fluorescence single-cell microscopy. Subsequent knockdown studies using small-hairpin RNAs further confirmed an active role of Aurora kinase A on the formation of PFK1 assemblies in HeLa cells. Importantly, all the identified protein kinases here have been investigated as key signaling nodes of one specific cascade that controls cell cycle progression in human cells. Collectively, our qHTS approaches unravel a cell cycle-associated signaling network that regulates the formation of PFK1-mediated glucosome assembly in human cells.

IL-12 drives the differentiation of human T follicular regulatory cells

Abstract T follicular regulatory (Tfr) cells can counteract the B cell-helper activity of T follicular helper (Tfh) cells and hinder the production of antibodies against self-antigens or allergens. A mechanistic understanding of the cues initiating the differentiation of T regulatory (Treg) cells into Tfr cells, which is instrumental for the therapeutic manipulation of diseases associated with a Tfr cell imbalance, is still missing. Despite their opposed roles, Tfr and Tfh cell differentiation appears to be controlled by partially overlapping signals, including TCR stimulation and costimulatory molecules. However, it is still unknown if cytokines that have been shown to control the biology of human Tfh cells, including IL-12 and activin A, can influence the differentiation of Tfr cells. To address this question, we evaluated the impact of these two cytokines on the in vitro differentiation of Tfr cells. Herein, we report a role for IL-12 in driving, on activated Treg cells, the induction of molecules that belong to the Tfr cell program, including CXCR5, PD-1, BCL6 and ICOS meanwhile preserving Tfr regulatory function. Conversely, activin A presented a largely negligible effect on the in vitro differentiation of Tfr-like cells and only modestly strengthened the IL-12-driven differentiation of Tfr-like cells. Importantly, patients with inborn errors of immunity in IL12RB1 gene presented with a severe decrease in circulating Tfr cells and produced higher levels of anti-actin autoantibodies in vivo. Overall, this study unveils IL-12 as an inducer of Tfr cell differentiation in vivo and provides a novel approach for the in vitro generation of human Tfr-like cells. AI123738

Network expansion of genetic associations defines a pleiotropy map of human cell biology

Interacting proteins tend to have similar functions, influencing the same organismal traits. Interaction networks can be used to expand the list of candidate trait-associated genes from genome-wide association studies. Here, we performed network-based expansion of trait-associated genes for 1,002 human traits showing that this recovers known disease genes or drug targets. The similarity of network expansion scores identifies groups of traits likely to share an underlying genetic and biological process. We identified 73 pleiotropic gene modules linked to multiple traits, enriched in genes involved in processes such as protein ubiquitination and RNA processing. In contrast to gene deletion studies, pleiotropy as defined here captures specifically multicellular-related processes. We show examples of modules linked to human diseases enriched in genes with known pathogenic variants that can be used to map targets of approved drugs for repurposing. Finally, we illustrate the use of network expansion scores to study genes at inflammatory bowel disease genome-wide association study loci, and implicate inflammatory bowel disease-relevant genes with strong functional and genetic support.

Cardiovascular Organoids/3D Models Review Series: an Introduction.

Cardiovascular Organoids/3D Models Review Series: an Introduction.

Modeling human liver organ development and diseases with pluripotent stem cell-derived organoids.

The discoveries of human pluripotent stem cells (PSCs) including embryonic stem cells and induced pluripotent stem cells (iPSCs) has led to dramatic advances in our understanding of basic human developmental and cell biology and has also been applied to research aimed at drug discovery and development of disease treatments. Research using human PSCs has been largely dominated by studies using two-dimensional cultures. In the past decade, however, ex vivo tissue "organoids," which have a complex and functional three-dimensional structure similar to human organs, have been created from PSCs and are now being used in various fields. Organoids created from PSCs are composed of multiple cell types and are valuable models with which it is better to reproduce the complex structures of living organs and study organogenesis through niche reproduction and pathological modeling through cell-cell interactions. Organoids derived from iPSCs, which inherit the genetic background of the donor, are helpful for disease modeling, elucidation of pathophysiology, and drug screening. Moreover, it is anticipated that iPSC-derived organoids will contribute significantly to regenerative medicine by providing treatment alternatives to organ transplantation with which the risk of immune rejection is low. This review summarizes how PSC-derived organoids are used in developmental biology, disease modeling, drug discovery, and regenerative medicine. Highlighted is the liver, an organ that play crucial roles in metabolic regulation and is composed of diverse cell types.