Introduction Endometriosis describes the presence of endometrial glands outside of the uterus and can cause various symptoms such as chronic pelvic pain, hypermenorrhea and infertility. These complications pose an extreme burden on the patients, especially as up to date, the average time until diagnosis can consume several years and requires invasive laparoscopy. Objectives The aim of this study is to molecularly characterize endometrium and endometriosis using marker-independent Raman microspectroscopy to identify potential biomarkers and validate its diagnostic potential. Methods After histopathological characterization of tissue sections of human endometrium and endometriosis, Raman microspectroscopy was performed on the gland region. Multivariate analysis of the hyperspectral maps was used to localize major subcellular structures and further decipher their molecular composition. Samples from different anatomical regions and throughout all menstrual cycle phases were analyzed. Results Raman imaging enabled label-free visualization of tissue morphology and submolecular tissue characterization. Distinct differences between endometrium and endometriosis were found for collagen type I and nuclear signatures. Spectral deconvolution allowed identification of a Raman biomarker indicative of fibrotic changes in endometriosis samples. Additionally, a significant increase in epigenetic 5mC foci and an increased signal intensity relevant for methylations was detected in nuclei of endometriosis. Furthermore, a neural network-based classification of Raman data resulted in high accuracies in discriminating endometrial and peritoneal tissue from endometriosis. Conclusion The non-destructive approach by hyperspectral Raman imaging enabled for molecular sensitive characterization of endometriotic lesions which could not only be an asset in complementing histopathological tissue evaluation but combined with data-driven classification models support in situ tissue diagnosis. ### Competing Interest Statement The authors have declared no competing interest. Deutsche Forschungsgemeinschaft, GRK 2543/1, INST 2388/64-1, INST 2388/33-1 Bundesministerium für Bildung und Forschung, 01EJ2403B

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs1-3. Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast, restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness, such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses, whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover, sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion.

Hematopoietic stem cells (HSC) sustain life-long blood production and have the ability to recapitulate the hematopoietic system upon transplantation. This makes them therapeutically invaluable for the treatment of hematopoietic malignancies and genetic disorders. However, the regulatory processes governing HSC functional competence and engraftment ability are poorly understood and challenging to recapitulate ex vivo, making robust expansion of human HSCs difficult to achieve. We have identified a novel HSC regulatory protein, MYCT1 (MYC target 1), that is highly enriched in self-renewing human HSC but lost during differentiation and culture. Knockdown (KD) experiments in human fetal liver and cord blood revealed a critical requirement for MYCT1 in human HSPC ex vivo expansion and engraftment across HSC ontogeny. Single cell RNAseq of human HSPCs upon MYCT1 KD linked MYCT1 loss in HLF+ HSCs to a profound dysregulation of cellular programs essential for HSC stemness, including mitochondrial activity, proteostasis and splicing. Because MYCT1 becomes drastically downregulated during HSC ex vivo culture, we investigated if restoring MYCT1 expression in culture could improve HSC function. Indeed, maintaining MYCT1 levels during culture via lentiviral expression resulted in improved “stemness” transcriptional signatures, greater expansion of the most undifferentiated EPCR+ITGA3+ human HSPCs, and enhanced engraftment ability upon transplantation. Notably, we also uncovered that the novel albumin free culture conditions reported by Sakurai et al. 2023 to robustly expand human HSCs can also maintain higher levels of MYCT1 expression. This reinforces our findings that the loss of MYCT1 expression is a major contributor to the functional incompetence of human HSCs cultured ex vivo. Nonetheless, the MYCT1 regulated processes and why they are critical for HSC competence are not known. Immunofluorescence and IP-mass spectrometry revealed that MYCT1 localizes in the endosomal membrane and interacts with vesicle trafficking machinery and receptor signaling components essential for HSC function. MYCT1 KD in human HSCs and endothelial cells (EC) led to hyperactive endocytosis and caused exaggerated signaling responses to cytokines in the culture microenvironment. We found that ex vivo culture gradually increased endocytosis specifically in CD34+ EPCR+ HSPCs in parallel with the culture-induced downregulation of MYCT1 in these cells, but not in the more differentiated progeny. On the other hand, restoring MYCT1 expression during HSC culture dampened the excessive endocytosis. Furthermore, maintaining low endocytosis during culture, either by the endocytosis inhibitor chlorpromazine or by sorting HSPCs with lower endocytic internalization of fluorescent dextran, results in the enrichment of immunophenotypic LT-HSCs in culture. Our data show that the moderation of environmental sensing through MYCT1-controlled endocytosis is essential for preserving human HSC self-renewal and engraftment ability. As MYCT1 expression is downregulated in cultured human HSPC, our findings imply that loss of the molecular machinery required for proper sensing of microenvironment signals has far-reaching effects into essential cellular functions required for stemness, and is a key contributor to the functional incompetence of cultured human HSC.

Understanding the immune system's foreign body response (FBR) is essential when developing and validating a biomaterial. Macrophage activation and proliferation are critical events in FBR that can determine the material's biocompatibility and fate in vivo. In this study, two different macro-encapsulation pouches intended for pancreatic islet transplantation were implanted into streptozotocin-induced diabetes rat models for 15 days. Post-explantation, the fibrotic capsules were analyzed by standard immunohistochemistry as well as non-invasive Raman microspectroscopy to determine the degree of FBR induced by both materials. The potential of Raman microspectroscopy to discern different processes of FBR was investigated and it was shown that Raman microspectroscopy is capable of targeting ECM components of the fibrotic capsule as well as pro and anti-inflammatory macrophage activation states, in a molecular-sensitive and marker-independent manner. In combination with multivariate analysis, spectral shifts reflecting conformational differences in Col I were identified and allowed to discriminate fibrotic and native interstitial connective tissue fibers. Moreover, spectral signatures retrieved from nuclei demonstrated changes in methylation states of nucleic acids in M1 and M2 phenotypes, relevant as indicator for fibrosis progression. This study could successfully implement Raman microspectroscopy as complementary tool to study in vivo immune-compatibility providing insightful information of FBR of biomaterials and medical devices, post-implantation.

Despite tremendous progress in deciphering breast cancer at the genomic level, the pronounced intra- and intertumoral heterogeneity remains a major obstacle to the advancement of novel and more effective treatment approaches. Frequent treatment failure and the development of treatment resistance highlight the need for patient-derived tumor models that reflect the individual tumors of breast cancer patients and allow a comprehensive analyses and parallel functional validation of individualized and therapeutically targetable vulnerabilities in protein signal transduction pathways. Here, we introduce the generation and application of breast cancer patient-derived 3D microtumors (BC-PDMs). Residual fresh tumor tissue specimens were collected from n = 102 patients diagnosed with breast cancer and subjected to BC-PDM isolation. BC-PDMs retained histopathological characteristics, and extracellular matrix (ECM) components together with key protein signaling pathway signatures of the corresponding primary tumor tissue. Accordingly, BC-PDMs reflect the inter- and intratumoral heterogeneity of breast cancer and its key signal transduction properties. DigiWest®-based protein expression profiling of identified treatment responder and non-responder BC-PDMs enabled the identification of potential resistance and sensitivity markers of individual drug treatments, including markers previously associated with treatment response and yet undescribed proteins. The combination of individualized drug testing with comprehensive protein profiling analyses of BC-PDMs may provide a valuable complement for personalized treatment stratification and response prediction for breast cancer.

The ontogeny of human haematopoietic stem cells (HSCs) is poorly defined owing to the inability to identify HSCs as they emerge and mature at different haematopoietic sites1. Here we created a single-cell transcriptome map of human haematopoietic tissues from the first trimester to birth and found that the HSC signature RUNX1+HOXA9+MLLT3+MECOM+HLF+SPINK2+ distinguishes HSCs from progenitors throughout gestation. In addition to the aorta-gonad-mesonephros region, nascent HSCs populated the placenta and yolk sac before colonizing the liver at 6 weeks. A comparison of HSCs at different maturation stages revealed the establishment of HSC transcription factor machinery after the emergence of HSCs, whereas their surface phenotype evolved throughout development. The HSC transition to the liver marked a molecular shift evidenced by suppression of surface antigens reflecting nascent HSC identity, and acquisition of the HSC maturity markers CD133 (encoded by PROM1) and HLA-DR. HSC origin was tracked to ALDH1A1+KCNK17+ haemogenic endothelial cells, which arose from an IL33+ALDH1A1+ arterial endothelial subset termed pre-haemogenic endothelial cells. Using spatial transcriptomics and immunofluorescence, we visualized this process in ventrally located intra-aortic haematopoietic clusters. The in vivo map of human HSC ontogeny validated the generation of aorta-gonad-mesonephros-like definitive haematopoietic stem and progenitor cells from human pluripotent stem cells, and serves as a guide to improve their maturation to functional HSCs.

In article number 2002500 by Katja Schenke-Layland and co-workers, the basement membrane protein Nidogen-1 is shown to promote tissue survival and regeneration in ischemic conditions. Once thought as a simple linker protein, Nidogen-1 increases heart function and decreases scaring in a myocardial infarction model, rescues pancreatic beta-cell function in hypoxia, and modulates the immune system, demonstrating the multifaceted effect of Nidogen-1.

Water restriction is commonly used to motivate rodents to perform behavioral tasks; however, its effects on hydration and stress hormone levels are unknown. Here, we report daily body weight and bi-weekly packed red blood cell volume and corticosterone (CORT) in adult male rats across 80 days for three commonly used water restriction schedules. We also assessed renal adaptation to water restriction using postmortem histologic evaluation of renal medulla. A control group received ad libitum water. After one week of water restriction, rats on all restriction schedules resumed similar levels of growth relative to the control group. Normal hydration was observed, and water restriction did not drive renal adaptation. An intermittent restriction schedule was associated with an increase in CORT relative to the control group. However, intermittent restriction evokes a stress response which could affect behavioral and neurobiological results. Our results also suggest that stable motivation in behavioral tasks may only be achieved after one week of restriction.

Organoid models of early tissue development have been produced for the intestine, brain, kidney and other organs, but similar approaches for the heart have been lacking. Here we generate complex, highly structured, three-dimensional heart-forming organoids (HFOs) by embedding human pluripotent stem cell aggregates in Matrigel followed by directed cardiac differentiation via biphasic WNT pathway modulation with small molecules. HFOs are composed of a myocardial layer lined by endocardial-like cells and surrounded by septum-transversum-like anlagen; they further contain spatially and molecularly distinct anterior versus posterior foregut endoderm tissues and a vascular network. The architecture of HFOs closely resembles aspects of early native heart anlagen before heart tube formation, which is known to require an interplay with foregut endoderm development. We apply HFOs to study genetic defects in vitro by demonstrating that NKX2.5 -knockout HFOs show a phenotype reminiscent of cardiac malformations previously observed in transgenic mice.

Hematopoietic stem cells (HSCs) capable of sustaining life-long multilineage hematopoiesis are formed in the human embryo between 4 and 5 weeks of development. HSCs emerge from hemogenic endothelium (HE) in the aorta-gonad mesonephros (AGM) region in a process called endothelial-to-hematopoietic transition (EHT). EHT has been mainly studied in in vitro human pluripotent stem cell models, which do not fully recapitulate in vivo HSC specification. Thus, the precise molecular identity and source of the HSC-forming HE and the molecular programs governing EHT in human remain elusive. This is crucial, as the embryo and extraembryonic tissues also harbor HE that generates non-HSC hematopoietic progenitors. Here, we conducted single-cell RNA-sequencing on purified cells (CD34+/CD31+) from the AGM region of 4.5-5-week human embryos from elective pregnancy terminations. Trajectory inference analyses revealed a distinct cluster of nascent HSCs with a unique expression profile (RUNX1+HLF+HOXA+MLLT3+SPINK2+) and a closely associated putative arterial HE population with a molecular signature that distinguishes it from other arterial endothelium (ALDH1A1+COL23A1+AGTR2+DKK1+KCNK17+). We also identified a novel arterial population (IL33+SULF1+ALDH1A1+AGTR2+), herein termed pre-HE, that is largely non-proliferative and metabolically quiescent and precedes the HSC-forming HE. Pseudotime analyses showed that acquisition of hemogenic properties in endothelium is concomitant to the downregulation of Notch, TGFb and Wnt signaling, and to the induction of hematopoietic transcription factors and genes associated with autophagy, identifying key regulatory switches during EHT. Comparison of HSC-forming HE (4.5-5-week AGM) to earlier, non-HSC-forming HE (3-4-week AGM) informed that ALDH1A1, COL23A1, AGTR2 and DKK1 are HSC lineage-specific markers, while KCNK17 is a pan hemogenic marker also induced during progenitor development from HE. Altogether, these findings provide a high-resolution map of the cell types and molecular switches involved in human HSC formation from endothelium, and can ultimately enable the recapitulation of HSC development in vitro for therapeutic purposes. Hematopoietic stem cells (HSCs) capable of sustaining life-long multilineage hematopoiesis are formed in the human embryo between 4 and 5 weeks of development. HSCs emerge from hemogenic endothelium (HE) in the aorta-gonad mesonephros (AGM) region in a process called endothelial-to-hematopoietic transition (EHT). EHT has been mainly studied in in vitro human pluripotent stem cell models, which do not fully recapitulate in vivo HSC specification. Thus, the precise molecular identity and source of the HSC-forming HE and the molecular programs governing EHT in human remain elusive. This is crucial, as the embryo and extraembryonic tissues also harbor HE that generates non-HSC hematopoietic progenitors. Here, we conducted single-cell RNA-sequencing on purified cells (CD34+/CD31+) from the AGM region of 4.5-5-week human embryos from elective pregnancy terminations. Trajectory inference analyses revealed a distinct cluster of nascent HSCs with a unique expression profile (RUNX1+HLF+HOXA+MLLT3+SPINK2+) and a closely associated putative arterial HE population with a molecular signature that distinguishes it from other arterial endothelium (ALDH1A1+COL23A1+AGTR2+DKK1+KCNK17+). We also identified a novel arterial population (IL33+SULF1+ALDH1A1+AGTR2+), herein termed pre-HE, that is largely non-proliferative and metabolically quiescent and precedes the HSC-forming HE. Pseudotime analyses showed that acquisition of hemogenic properties in endothelium is concomitant to the downregulation of Notch, TGFb and Wnt signaling, and to the induction of hematopoietic transcription factors and genes associated with autophagy, identifying key regulatory switches during EHT. Comparison of HSC-forming HE (4.5-5-week AGM) to earlier, non-HSC-forming HE (3-4-week AGM) informed that ALDH1A1, COL23A1, AGTR2 and DKK1 are HSC lineage-specific markers, while KCNK17 is a pan hemogenic marker also induced during progenitor development from HE. Altogether, these findings provide a high-resolution map of the cell types and molecular switches involved in human HSC formation from endothelium, and can ultimately enable the recapitulation of HSC development in vitro for therapeutic purposes.

X-chromosome dosage compensation in female placental mammals is achieved by X-chromosome inactivation (XCI). Human pre-implantation embryos are an exception, in which dosage compensation occurs by X-chromosome dampening (XCD). Here, we examined whether XCD extends to human prenatal germ cells given their similarities to naive pluripotent cells. We found that female human primordial germ cells (hPGCs) display reduced X-linked gene expression before entering meiosis. Moreover, in hPGCs, both X chromosomes are active and express the long non-coding RNAs X active coating transcript (XACT) and X inactive specific transcript (XIST)-the master regulator of XCI-which are silenced after entry into meiosis. We find that XACT is a hPGC marker, describe XCD associated with XIST expression in hPGCs and suggest that XCD evolved in humans to regulate X-linked genes in pre-implantation embryos and PGCs. Furthermore, we found a unique mechanism of X-chromosome regulation in human primordial oocytes. Therefore, future studies of human germline development must consider the sexually dimorphic X-chromosome dosage compensation mechanisms in the prenatal germline.

Regenerative strategies such as stem cell-based therapies and tissue engineering applications are being developed with the aim of replacing, remodeling, regenerating, or supporting damaged tissues and organs. When aiming for ultimately clinical applications, in addition to, or instead of, a careful cell type selection, the design of appropriate 3D scaffolds is essential for the generation of bioinspired replacement tissues. Such scaffolds can be effective and efficient drug carrier systems that are composed of degradable or nondegradable biomaterials. Extracellular matrix (ECM) proteins have great potential to be utilized as scaffolds for bioengineering and regenerative medicine. The native ECM is a heterogeneous network of soluble and fibrous proteins, proteoglycans, and glycosaminoglycans, which is maintained by both covalent and noncovalent interactions. Recapitulating the composition, structure, and native function of the ECM is a highly challenging task for bioengineers and biochemists. This said, researchers have worked successfully over the past decades on many novel approaches either to produce or further manufacture ECM proteins and natural materials or to harness the knowledge obtained from studies focusing on the ECM. This has enabled them to mimic the native function of the ECM using ECM-like materials supporting cell attachment, growth, and proliferation as well as differentiation. This book chapter provides an overview of approaches currently used in the field of bioengineering.

Ischemia impacts multiple organ systems and is the major cause of morbidity and mortality in the developed world. Ischemia disrupts tissue homeostasis, driving cell death, and damages tissue structure integrity. Strategies to heal organs, like the infarcted heart, or to replace cells, as done in pancreatic islet β-cell transplantations, are often hindered by ischemic conditions. Here, it is discovered that the basement membrane glycoprotein nidogen-1 attenuates the apoptotic effect of hypoxia in cardiomyocytes and pancreatic β-cells via the αvβ3 integrin and beneficially modulates immune responses in vitro. It is shown that nidogen-1 significantly increases heart function and angiogenesis, while reducing fibrosis, in a mouse postmyocardial infarction model. These results demonstrate the protective and regenerative potential of nidogen-1 in ischemic conditions.

The developmental trajectory of human skeletal myogenesis and the transition between progenitor and stem cell states are unclear. We used single-cell RNA sequencing to profile human skeletal muscle tissues from embryonic, fetal, and postnatal stages. In silico, we identified myogenic as well as other cell types and constructed a "roadmap" of human skeletal muscle ontogeny across development. In a similar fashion, we also profiled the heterogeneous cell cultures generated from multiple human pluripotent stem cell (hPSC) myogenic differentiation protocols and mapped hPSC-derived myogenic progenitors to an embryonic-to-fetal transition period. We found differentially enriched biological processes and discovered co-regulated gene networks and transcription factors present at distinct myogenic stages. This work serves as a resource for advancing our knowledge of human myogenesis. It also provides a tool for a better understanding of hPSC-derived myogenic progenitors for translational applications in skeletal muscle-based regenerative medicine.

Somites form during embryonic development and give rise to unique cell and tissue types, such as skeletal muscles and bones and cartilage of the vertebrae. Using somitogenesis-stage human embryos, we performed transcriptomic profiling of human presomitic mesoderm as well as nascent and developed somites. In addition to conserved pathways such as WNT-β-catenin, we also identified BMP and transforming growth factor β (TGF-β) signaling as major regulators unique to human somitogenesis. This information enabled us to develop an efficient protocol to derive somite cells in vitro from human pluripotent stem cells (hPSCs). Importantly, the in-vitro-differentiating cells progressively expressed markers of the distinct developmental stages that are known to occur during in vivo somitogenesis. Furthermore, when subjected to lineage-specific differentiation conditions, the hPSC-derived somite cells were multipotent in generating somite derivatives, including skeletal myocytes, osteocytes, and chondrocytes. This work improves our understanding of human somitogenesis and may enhance our ability to treat diseases affecting somite derivatives.

The human cerebral cortex possesses distinct structural and functional features that are not found in the lower species traditionally used to model brain development and disease. Accordingly, considerable attention has been placed on the development of methods to direct pluripotent stem cells to form human brain-like structures termed organoids. However, many organoid differentiation protocols are inefficient and display marked variability in their ability to recapitulate the three-dimensional architecture and course of neurogenesis in the developing human brain. Here, we describe optimized organoid culture methods that efficiently and reliably produce cortical and basal ganglia structures similar to those in the human fetal brain in vivo. Neurons within the organoids are functional and exhibit network-like activities. We further demonstrate the utility of this organoid system for modeling the teratogenic effects of Zika virus on the developing brain and identifying more susceptibility receptors and therapeutic compounds that can mitigate its destructive actions.

The ability to generate hematopoietic stem cells from human pluripotent cells would enable many biomedical applications. We find that hematopoietic CD34(+) cells in spin embryoid bodies derived from human embryonic stem cells (hESCs) lack HOXA expression compared with repopulation-competent human cord blood CD34(+) cells, indicating incorrect mesoderm patterning. Using reporter hESC lines to track the endothelial (SOX17) to hematopoietic (RUNX1C) transition that occurs in development, we show that simultaneous modulation of WNT and ACTIVIN signaling yields CD34(+) hematopoietic cells with HOXA expression that more closely resembles that of cord blood. The cultures generate a network of aorta-like SOX17(+) vessels from which RUNX1C(+) blood cells emerge, similar to hematopoiesis in the aorta-gonad-mesonephros (AGM). Nascent CD34(+) hematopoietic cells and corresponding cells sorted from human AGM show similar expression of cell surface receptors, signaling molecules and transcription factors. Our findings provide an approach to mimic in vitro a key early stage in human hematopoiesis for the generation of AGM-derived hematopoietic lineages from hESCs.

Elucidation of mechanisms in semilunar valve development may enable the development of new therapies. Here, we found differences in proliferation-associated genes and genes repressed by vascular endothelial growth factor between human semilunar valves from first and second trimester valve leaflets. The proliferation of valve interstitial cells and ventricular valve endothelial cells (VECs) and cellular density declined from the first to the second trimester. Cytoplasmic expression of nuclear factor of activated T-cells cytoplasmic 1 (NFATc-1) in VECs (4 weeks), and later cells in the leaflet/annulus junction mesenchyme expressing inactive NFATc-1 (5.5-9 weeks) were detected, indicative of EndMT in valvulogenesis. At this leaflet/annulus junction CD44+ cells clustered during elongation (11 weeks), extending toward the tip along the fibrosal layer in second trimester leaflets. Differing patterns of maturation in the fibrosa and ventricularis were detected via increased fibrosal periostin content, which tracked the presence of the CD44+ cells in the second trimester. We revealed that spatiotemporal NFATc-1 expression actively regulates EndMT during human valvulogenesis, as early as 4 weeks. Additionally, CD44+ cells play a role in leaflet maturation toward the trilaminar structure, possibly via migration of VECs undergoing EndMT, which subsequently ascend from the leaflet/annulus junction.