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Liver transplantation is currently the only curative therapy for patients with acute or chronic liver failure. However, a dramatic gap between the number of available liver grafts and the number of patients on the transplantation waiting list emphasizes the need for valid liver substitutes. Whole-organ engineering is an emerging field of tissue engineering and regenerative medicine. It aims to generate transplantable and functional organs to support patients on transplantation waiting lists until a graft becomes available. It comprises two base technologies developed in the last decade; (1) organ decellularization to generate a three-dimensional (3D) extracellular matrix scaffold of an organ, and (2) scaffold recellularization to repopulate both the parenchymal and vascular compartments of a decellularized organ. In this review article, recent advancements in both technologies, in relation to liver whole-organ engineering, are presented. We address the potential sources of hepatocytes and non-parenchymal liver cells for repopulation studies, and the role of stem-cell-derived liver progeny is discussed. In addition, different cell seeding strategies, possible graft modifications, and methods used to evaluate the functionality of recellularized liver grafts are outlined. Based on the knowledge gathered from recent transplantation studies, future directions are summarized. [ABSTRACT FROM AUTHOR]

The first definitive hematopoietic progenitors emerge through the process of endothelial-to-hematopoietic transition in vertebrate embryos. With molecular regulators for this process worked out, the role of metabolic pathways used remains unclear. Here, we performed nano-LC-MS/MS-based proteomic analysis and predicted a metabolic switch from a glycolytic to oxidative state upon hematopoietic transition. Mitochondrial activity, glucose uptake, and glycolytic flux analysis supported this hypothesis. Systemic inhibition of lactate dehydrogenase A (LDHA) increased oxygen consumption rate in the hemato-endothelial system and inhibited the emergence of intra-aortic hematopoietic clusters. These findings were corroborated using Tie2-Cre-mediated deletion of Ldha that showed similar effects on hematopoietic emergence. Conversely, stabilization of HIF-1a via inhibition of oxygen-sensing pathway led to decreased oxidative flux and promoted hematopoietic emergence in mid-gestation embryos. Thus, cell-intrinsic regulation of metabolic state overrides oxygenated microenvironment in the aorta to promote a glycolytic metabolic state that is crucial for hematopoietic emergence in mammalian embryos. [ABSTRACT FROM AUTHOR]

Decisions about what experimental therapies are advanced to clinical trials are based almost exclusively on findings in preclinical animal studies. Over the past 30 years, animal models have forecast the success of hundreds of neuroprotective pharmacological therapies for stroke, Alzheimer׳s disease, spinal cord injury, traumatic brain injury and amyotrophic lateral sclerosis. Yet almost without exception, all have failed. Rapid advances in stem cell technologies have raised new hopes that these neurological diseases may one day be treatable. Still, how can neuroregenerative therapies be translated into clinical realities if available animal models are such poor surrogates of human disease? To address this question we discuss human and rodent neurogenesis, evaluate mechanisms of action for cellular therapies and describe progress in translating neuroregeneration to date. We conclude that not only are appropriate animal models critical to the development of safe and effective therapies, but that the multiple mechanisms of stem cell-mediated therapies may be particularly well suited to the mechanistically diverse nature of central nervous system diseases in mice and man. [ABSTRACT FROM AUTHOR]

The interest in the use of stem cells as a source for therapy has increased dramatically over the last decades. Different stem cell types have been tested in both in vitro and in vivo models, because of their properties such as differentiation potential, trophic effects and immune modulatory properties. To further optimize the use of different stem cell types for the treatment of disease in a clinical setting, it is necessary to know more about the in vivo behavior of these cells following engraftment. Until now, the golden standard to preclinically evaluate cell therapy was histology, which is an invasive method as the animals need to be sacrificed. This hampers the generation of dynamic information and results in only one single point in time available for analysis per animal. For more information regarding cell migration, in situ persistence, viability, proliferation and differentiation, molecular imaging can be used for imaging cells after transplantation dynamically and longitudinally, in a noninvasive way. With this technology, it becomes possible to track cells within the same subjects over a long period of time. [ABSTRACT FROM AUTHOR]

Micro RNAs are a class of small non-coding RNAs involved in the transcriptional and post-transcriptional regulation of gene expression. The function of mi RNAs in liver disease including hepatocellular carcinoma ( HCC), hepatitis, and alcoholic liver disease, have been widely studied and extensively reviewed. Increasing evidence demonstrates that mi RNAs also play a critical role in normal liver development and in the fine-tuning of fundamental biological liver processes. In this review, we highlight the most recent findings on the role of mi RNAs in liver specification and differentiation, liver cell development, as well as in the many metabolic functions of the liver, including glucose, lipid, iron and drug metabolism. These findings demonstrate an important role of mi RNAs in normal liver development and function. Further researches will be needed to fully understand how mi RNAs regulate liver generation and metabolic function, which should then lead to greater insights in liver biology and perhaps open up the possibility to correct errors that cause liver diseases or metabolic disorders. [ABSTRACT FROM AUTHOR]

In this review, we discuss the migration and homing ability of mesenchymal stem cells (MSCs) and MSC-like cells and factors influencing this. We also discuss studies related to the mechanism of migration and homing and the approaches undertaken to enhance it. Finally, we describe the different methods available and frequently used to track and identify the injected cells in vivo. [ABSTRACT FROM AUTHOR]

The vasculature of the bone marrow remains poorly characterized, yet crucial to maintain hematopoiesis and retain stem cells in a quiescent state. A recent study by Itkin et al. (2016) in Nature reports how vascular barrier integrity and endothelial cell metabolism regulate hematopoietic stem cell quiescence and leukocyte trafficking. [ABSTRACT FROM AUTHOR]

The pharmaceutical industry is in high need of efficient and relevant in vitro liver models, which can be incorporated in their drug discovery pipelines to identify potential drugs and their toxicity profiles. Current liver models often rely on cancer cell lines or primary cells, which both have major limitations. However, the development of human induced pluripotent stem cells (hiPSCs) has created a new opportunity for liver disease modeling, drug discovery and liver toxicity research. hiPSCs can be differentiated to any cell of interest, which makes them good candidates for disease modeling and drug discovery. Moreover, hiPSCs, unlike primary cells, can be easily genome-edited, allowing the creation of reporter lines or isogenic controls for patient-derived hiPSCs. Unfortunately, even though liver progeny from hiPSCs has characteristics similar to their in vivo counterparts, the differentiation of iPSCs to fully mature progeny remains highly challenging and is a major obstacle for the full exploitation of these models by pharmaceutical industries. In this review, we discuss current liver-cell differentiation protocols and in vitro iPSC-based liver models that could be used for disease modeling and drug discovery. Furthermore, we will discuss the challenges that still need to be overcome to allow for the successful implementation of these models into pharmaceutical drug discovery platforms. [ABSTRACT FROM AUTHOR]

Peripheral vascular disease (PVD) is a growing medical problem in Western societies and presents itself mainly in two different clinical forms. Intermittent claudication is an early moderate manifestation, while patients with critical limb ischemia suffer from severe muscle tissue loss or ulcers and are at high risk for limb amputation. Unfortunately, many patients cannot be helped with currently available surgical or endovascular revascularization procedures because of the complex anatomy of the vascular occlusion and/or the presence of other risk factors. Noninvasive stem cell therapy has been proposed as an alternative for such patients. Although pioneering clinical experience with stem cell-related therapy seems promising, it is too early for general clinical use of this technique, since many questions remain unanswered. Indeed, while questions about safety, dose, and administration route/timing/frequency are the first ones to be addressed when designing a stem cell-based clinical approach, there is accumulating evidence from recent (pre-)clinical studies that other issues may also be at stake. For instance, the choice of stem cells to be used and its precise mechanism of action, the need/possibility for concurrent tissue regeneration in case of irreversible tissue loss, the differentiation degree and specific vascular identity of the transplanted cells, and the long-term survival of engrafted cells in the absence of a normal supportive tissue environment should be well considered. Here, rather than presenting a comprehensive and extensive overview on the current literature on stem/progenitor cells and revascularization, we highlight some of the outstanding issues emerging from the recent (pre-)clinical literature that may codetermine the successful application of stem cells in a wide range of PVD patients in the future. [ABSTRACT FROM AUTHOR]

Provides information about the possible etiology of Multipotent Adult Progenitor Cell (MAPC). Proliferation of the cell without obvious signs of senescence; Suggestion that MAPC are derived from a population of cells that either has active telomerase in vivo, or that is highly quiescent in vivo; Role played by smooth muscle cells (SMC) in vascular development and morphogenesis of blood vessels defined by a noncontractile, or synthetic SMC phenotype.

Pluripotent stem cells have the capacity to self renew and to differentiate to cells of the three somatic germ layers that comprise an organism. Embryonic stem cells are the most studied pluripotent stem cells. Pluripotent stem cells have also been derived from adult tissues. Both embryonic and adult stem cells represent valuable sources of cells for applications in cell therapy, drug screening and tissue engineering. While expanding stem cells in culture, it is critical to maintain their self-renewal and differentiation capacity. In generating particular cell types for specific applications, it is important to direct their differentiation to the desired lineage. Challenges in expansion of undifferentiated stem cells for clinical applications include the removal of feeder layers and non-defined components in the culture medium. Our limited basic knowledge on the requirements for maintaining pluripotency of adult pluripotent stem cells and the lack of appropriate markers associated with pluripotency hinders the progress toward their wide spread application. In vitro differentiation of stem cells usually produces a mixed population of different cell lineages with the desired cell type present only at a small proportion. Use of growth factors that promote differentiation, expansion or survival of specific cell types is key in controlling the differentiation towards specific cell lineages. A variety of bioreactors for cell cultivation exist and can be readily adapted for stem cell cultivation and differentiation. They provide a well-controlled environment for studying the process of stem cell propagation and differentiation. Their wide use will facilitate the development of processes for stem cell application. [Copyright &y& Elsevier]

Objective: We have previously shown that hematopoietic progenitor cells (HPCs) from umbilical cord blood (UCB) can be maintained in a cytokine-supplemented stroma-noncontact (SNC) system. Here, we tested if all-trans retinoic acid (ATRA), known to improve expansion of murine hematopoietic stem cells, would enhance human HPC maintenance in a SNC culture system. Methods: CD34+CD38-Lin- cells from UCB were cultured in transwells above AFT024 in the presence of Flt-3 ligand (FLT) and thrombopoietin (TPO), with or without ATRA. Total nucleated cells (TNC), colony-forming units (CFUs), long-term culture-initiating cells (LTC-ICs), myeloid-lymphoid initiating cells (ML-ICs) and SCID repopulating cells (SRCs) were evaluated 1 to 5 weeks after culture. Results: All-trans retinoic acid (1 μmol/L) reduced expansion of CD34+CD38-Lin- TNC and CFUs after 2 to 5 weeks of culture. However, it significantly increased LTC-IC expansion after 1 to 3 and, even more so, 5 weeks of culture. ATRA also increased recovery of more primitive ML-ICs and SRCs. Increased HPC recovery appeared dependent on the presence of stromal cells, as LTC-IC expansion was significantly reduced when ATRA was added to stroma-free cultures. Conclusion: All-trans retinoic acid increases expansion of early HPCs in a stromal cell–dependent fashion. [Copyright &y& Elsevier]

Objective: Assays that can evaluate the potential of individual human hematopoietic stem cells (HSC) are still lacking. We previously developed the myeloid-lymphoid initiating cell (ML-IC) assay that enumerates single CD34+ cells that generate long-term culture-initiating (LTC-IC) and NK-initiating (NK-IC) daughter cells, or single primitive progenitors with multilineage potential. When transplanted in vivo, umbilical cord blood (UCB) has greater repopulating ability than bone marrow (BM) or mobilized peripheral blood (MPB). Whether the greater in vivo repopulating ability is due to an increased frequency of HSC in UCB and generative potential of UCB, BM, and MPB CD34+ cells is not known. Materials and methods: Single UCB, BM, and MPB CD34+CD38-Lin- or CD34+CD38-CD33- cells were plated in ML-IC assay and after 2 to 4 weeks, progeny was evaluated for frequency and generative potential of ML-IC. We also tested whether the ML-IC assay could be used to define if increased numbers of primitive progenitors generated by different cytokines in expansion cultures are mediated by recruitment of quiescent cells or by increasing their generative potential. Results: The frequency of ML-IC in BM, UCB, and MPB was similar, but the generative potential of UCB ML-IC was significantly higher. Substitution of Flt3-L, SCF, and IL-7 with Flt3-L and thrombopoietin significantly increased the generative potential of ML-IC, whereas Flt3-L, SCF, and hyper-IL-6 increased both ML-IC frequency and generative potential. Conclusion: The ML-IC assay demonstrates that the greater repopulating ability of UCB is due to the higher generative ability of HSC in UCB. Furthermore, the ML-IC assay can discriminate between cytokine-mediated expansion of hematopoietic progenitors by enhancing generation of immature daughter cells or by recruiting otherwise quiescent cells. [Copyright &y& Elsevier]

Reports that progress in the understanding of the molecular pathophysiology of chronic myeloid leukemia (CML) has led to the development of targeted and effective therapies. Discussion on the molecular pathophysiology of CML; Therapies for CML; Principle underlying the antisense-based therapeutic approach to CML.

Explores the mechanisms underlying abnormal trafficking and expansion of malignant progenitors in chronic myelogenous leukemia (CML). Discussion on abnormal progenitor/microenvironment interactions in CML; Role of p210[supBCR/APL] in abnormal adhesion and adhesion-mediated regulation of CML progenitors; Molecular mechanisms underlying abnormal integrin in CML.

It has been known for decades that stem cells with limited differentiation potential are present in post-natal tissues of mammals, and adult stem cells are already used clinically. For instance, hematopoietic stem cells can reestablish the hematopoietic system following myeloablation, and stem cells are being used to regenerate corneal and skin tissue. But recent studies report that adult tissues might contain cells with pluripotent characteristics. These have evoked significant excitement, given the medical implications, but have also met with much skepticism. Indeed, most studies still await independent confirmation, there is a low frequency with which the apparent lineage switching occurs, and importantly such lineage switching defies established developmental biology and stem cell principles. Here, I critically review the published data indicating that postnatal stem cells persist that have greater differentiation potential than previously thought. [Copyright &y& Elsevier]

A role for extrarenal cells in the regeneration following acute renal failure. Background. Recovery of renal function following acute tubular necrosis (ATN) is dependent on the replacement of necrotic tubular cells with functional tubular epithelium. The source of these new tubular cells is thought to be resident renal tubular cells. The discovery of pluripotent bone marrow-derived stem cells has led to a reexamination of the cellular source and processes involved in the recovery from organ injury. Methods. To test the hypothesis in humans that extrarenal cells participate in the recovery following ATN, we examined the origin of tubular cells in male patients with resolving ATN who had received a kidney transplant from a female donor. Immunohistochmistry of kidney biopsies was performed to identify renal tubular epithelial cells (cytokeratin positive) and leukocytes (CD45 positive). Fluorescent in-situ hybridization was used to detect Y chromosome containing cells with DAPI serving as a nuclear stain. All staining was performed on the same section. Results. The Y chromosome was detected in approximately 40% of tubular cell nuclei in male kidneys (positive control) and in no nuclei of female kidneys (negative control). In male recipients of female kidneys who developed ATN, 1% of tubules contained Y chromosome cells defined by their morphology, positive staining for cytokeratin, and negative staining for CD45. When present, multiple cells in a positive tubule stained for the Y chromosome. No Y chromosome containing tubular cells were seen in similar sex mismatched transplants in male recipients who did not develop ATN, suggesting that recipient derived cells do not routinely repopulate the transplanted kidney. Conclusions. This proof-of-principle clinical observation demonstrates that extrarenal cells can participate in the regenerative response following ATN. These findings provide rationale for the cellular therapy of acute renal failure. [ABSTRACT FROM AUTHOR]

: ObjectiveThe study of hematopoietic stem cells (HSC) is limited by lack of specific markers for HSC. Rhodamine 123 (Rho) is one of the substrates of P-glycoprotein (Pgp), and the presence of active Pgp can be shown by the efflux of Rho. Rho can also be used to measure the mitochondrial transmembrane potential (energy state) of a cell. We reasoned that selection of hematopoietic progenitors using a combination of Rho efflux and phenotypic markers might be superior to use of phenotypic markers alone.: Materials and MethodsWe used the myeloid-lymphoid initiating cell (ML-IC) assay as functional measure of primitive progenitors. Umbilical cord blood CD34+CD33−CD38−, CD34+CD33−CD38−Rho−, and CD34+CD33−CD38−Rho−c-kit+ cells were sorted singly onto AFT024 feeders to assess their capacity to become ML-IC.: ResultsThe frequency of ML-IC in CD34+CD33−CD38−Rho− cells was significantly higher (15 ± 0.4%) than that in CD34+CD33−CD38− cells (6.2 ± 0.9%, p < 0.05). However, the frequency of long-term culture-initiating cells (LTC-IC) (17 ± 3% vs 12 ± 1.5%) and natural killer culture-initiating cells (NK-IC) (25 ± 3% vs 20 ± 4%) was similar in the two populations. Following the treatment of CD34+CD33−CD38−Rho− cells with verapamil, which blocks Pgp function, no increase in ML-IC was detected compared with CD34+CD33−CD38− cells (6 ± 0.7%), suggesting that differences in the energy state, which is reflected by Rho staining after verapamil treatment, cannot be used as a criterion to identify human HSC. Further selection of CD34+CD33−CD38−Rho− cells based on expression of c-kit significantly increased the frequency of ML-IC, LTC-IC and NK-IC by 1.75-, 1.3-, and 1.8-fold, respectively.: ConclusionCombining the function of Pgp and phenotypic features of hematopoietic progenitors enriches the frequency of cord blood ML-IC to greater than 25%. Use of such enriched populations will allow us to characterize the biological behavior of human HSC. [Copyright &y& Elsevier]

Discusses the engraftment capabilities of hematopoietic stem cell sources. HSC characteristics; Ontogenic differences in HSCs; Frequency of human HSCs in fluorescence-activated cell sort-selected CD34.

Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, Yamanaka S. Successful reprogramming of differentiated human somatic cells into a pluripotent state would allow creation of patient- and disease-specific stem cells. We previously reported generation of induced pluripotent stem (iPS) cells, capable of germline transmission, from mouse somatic cells by transduction of four defined transcription factors. Here, we demonstrate the generation of iPS cells from adult human dermal fibroblasts with the same four factors: Oct3/4, Sox2, Klf4, and c-Myc. Human iPS cells were similar to human embryonic stem (ES) cells in morphology, proliferation, surface antigens, gene expression, epigenetic status of pluripotent cell-specific genes, and telomerase activity. Furthermore, these cells could differentiate into cell types of the three germ layers in vitro and in teratomas. These findings demonstrate that iPS cells can be generated from adult human fibroblasts. [Abstract reproduced by permission of Cell 2007;131:861–872] [Copyright &y& Elsevier]

Terminally differentiated cells are commonly regarded as the most stable cell state in adult organisms, characterized by growth arrest while fulfilling their specialized functions. A better understanding of the mechanisms involved in promoting cell cycle exit will improve the ability to differentiate pluripotent cells into mature tissues for both pharmacological and therapeutic use. Here, it demonstrates that a hyperosmolar environment enforces a protective p53‐independent quiescent state in immature hepatoma cells and in pluripotent stem cell‐derived models of human hepatocytes and endothelial cells. Prolonged culture in hyperosmolar conditions stimulates changes in gene expression promoting functional cell maturation. Interestingly, hyperosmolar conditions do not only trigger growth arrest and cellular maturation but are also necessary to maintain this maturated state, as switching back to plasma osmolarity reverses the changes in expression of maturation and proliferative markers. Transcriptome analysis revealed sequential stages of osmolarity‐regulated growth arrest followed by cell maturation, mediated by activation of NF‐κВ, and repression of WNT signaling, respectively. This study reveals that a modulated increase in osmolarity serves as a biochemical signal to promote long‐term growth arrest and cellular maturation into different lineages, providing a practical method to generate differentiated hiPSCs that resemble their mature counterpart more closely. [ABSTRACT FROM AUTHOR]

Although human pluripotent stem cell (PSC)‐derived brain organoids have enabled researchers to gain insight into human brain development and disease, these organoids contain solely ectodermal cells and are not vascularized as occurs during brain development. Here it is created less complex and more homogenous large neural constructs starting from PSC‐derived neuroprogenitor cells (NPC), by fusing small NPC spheroids into so‐called concentroids. Such concentroids consisted of a pro‐angiogenic core, containing neuronal and outer radial glia cells, surrounded by an astroglia‐dense outer layer. Incorporating PSC‐derived endothelial cells (EC) around and/or in the concentroids promoted vascularization, accompanied by differential outgrowth and differentiation of neuronal and astroglia cells, as well as the development of ectodermal‐derived pericyte‐like mural cells co‐localizing with EC networks. Single nucleus transcriptomic analysis revealed an enhanced neural cell subtype maturation and diversity in EC‐containing concentroids, which better resemble the fetal human brain compared to classical organoids or NPC‐only concentroids. This PSC‐derived "vascularized" concentroid brain model will facilitate the study of neurovascular/blood‐brain barrier development, neural cell migration, and the development of effective in vitro vascularization strategies of brain mimics. [ABSTRACT FROM AUTHOR]

Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neurodegenerative disorder, characterized by selective loss of motor neurons (MNs). A number of causative genetic mutations underlie the disease, including mutations in the fused in sarcoma (FUS) gene, which can lead to both juvenile and late-onset ALS. Although ALS results from MN death, there is evidence that dysfunctional glial cells, including oligodendroglia, contribute to neurodegeneration. Here, we used human induced pluripotent stem cells (hiPSCs) with a R521H or a P525L mutation in FUS and their isogenic controls to generate oligodendrocyte progenitor cells (OPCs) by inducing SOX10 expression from a TET-On SOX10 cassette. Mutant and control iPSCs differentiated efficiently into OPCs. RNA sequencing identified a myelin sheath-related phenotype in mutant OPCs. Lipidomic studies demonstrated defects in myelin-related lipids, with a reduction of glycerophospholipids in mutant OPCs. Interestingly, FUSR521H OPCs displayed a decrease in the phosphatidylcholine/phosphatidylethanolamine ratio, known to be associated with maintaining membrane integrity. A proximity ligation assay further indicated that mitochondria-associated endoplasmic reticulum membranes (MAM) were diminished in both mutant FUS OPCs. Moreover, both mutant FUS OPCs displayed increased susceptibility to ER stress when exposed to thapsigargin, and exhibited impaired mitochondrial respiration and reduced Ca2+ signaling from ER Ca2+ stores. Taken together, these results demonstrate a pathological role of mutant FUS in OPCs, causing defects in lipid metabolism associated with MAM disruption manifested by impaired mitochondrial metabolism with increased susceptibility to ER stress and with suppressed physiological Ca2+ signaling. As such, further exploration of the role of oligodendrocyte dysfunction in the demise of MNs is crucial and will provide new insights into the complex cellular mechanisms underlying ALS. [ABSTRACT FROM AUTHOR]

This document is a correction notice for an article titled "Disruption of MAM integrity in mutant FUS oligodendroglial progenitors from hiPSCs" published in Acta Neuropathologica. The correction states that there was an error in Figure 5 of the article, where the incorrect dataset was used to compare FUSR521H and Isogenic 1 OPCs. The corrected figures are provided in the notice. The article provides information on dysregulated glycerophospholipid metabolism in mutant FUS iPSC-derived OPCs. It also includes joint-pathway analysis, volcano plots, and bar graphs to illustrate the dysregulated genes and metabolic pathways. The correction notice is licensed under a Creative Commons Attribution 4.0 International License. [Extracted from the article]

In this issue of Cancer Cell, report that endothelial cells (ECs) from murine prostate tumors unexpectedly differentiate into cells with characteristics of bone and cartilage and that human and murine prostate tumors contain “calcified” ECs. These observations open the possibility for targeted antiangiogenic therapy. [Copyright &y& Elsevier]

Discusses the role of thrombopoietin in optimizing hematopoietic stem cell (HSC) engraftment. Uses of HSC and progenitors; Role of progenitors in the initial hemato poietic recovery; Factors that govern the different steps required for reestablishing hematopoiesis.

Over the past decades, the importance of the immune system in a broad scope of pathologies, has drawn attention towards tissue-resident macrophages, such as microglia in the brain. To enable the study of for instance microglia, it is crucial to recreate in vitro (and in vivo ) assays. However, very fast loss of tissue-specific features of primary tissue resident macrophages, including microglia, upon in vitro culture has complicated such studies. Moreover, limited knowledge of macrophage developmental pathways and the role of local ‘niche factors’, has hampered the generation of tissue-resident macrophages from pluripotent stem cells (PSC). Recent data on the ontogeny of tissue-resident macrophages, combined with bulk and single cell RNAseq studies have identified the distinct origins and gene profile of microglia compared to other myeloid cells. As a result, over the past years, protocols have been published to create hPSC-derived microglia-‘like’ cells, as these cells are considered potential new therapeutic targets for therapies to treat neurodegenerative diseases. In this review we will provide an overview of different approaches taken to generate human microglia in vitro, taking into account their origin, and resemblance to their in vivo counterpart. Finally, we will discuss cell-extrinsic (culture conditions) and intrinsic factors (transcriptional machinery and epigenetics) that we believe can improve future differentiation protocols of tissue-resident macrophages from stem cells. [ABSTRACT FROM AUTHOR]

In the progressive phase of multiple sclerosis (MS), the hampered differentiation capacity of oligodendrocyte precursor cells (OPCs) eventually results in remyelination failure. We have previously shown that DNA methylation of Id2/Id4 is highly involved in OPC differentiation and remyelination. In this study, we took an unbiased approach by determining genome-wide DNA methylation patterns within chronically demyelinated MS lesions and investigated how certain epigenetic signatures relate to OPC differentiation capacity. We compared genome-wide DNA methylation and transcriptional profiles between chronically demyelinated MS lesions and matched normal-appearing white matter (NAWM), making use of post-mortem brain tissue (n = 9/group). DNA methylation differences that inversely correlated with mRNA expression of their corresponding genes were validated for their cell-type specificity in laser-captured OPCs using pyrosequencing. The CRISPR–dCas9-DNMT3a/TET1 system was used to epigenetically edit human-iPSC-derived oligodendrocytes to assess the effect on cellular differentiation. Our data show hypermethylation of CpGs within genes that cluster in gene ontologies related to myelination and axon ensheathment. Cell type-specific validation indicates a region-dependent hypermethylation of MBP, encoding for myelin basic protein, in OPCs obtained from white matter lesions compared to NAWM-derived OPCs. By altering the DNA methylation state of specific CpGs within the promotor region of MBP, using epigenetic editing, we show that cellular differentiation and myelination can be bidirectionally manipulated using the CRISPR–dCas9-DNMT3a/TET1 system in vitro. Our data indicate that OPCs within chronically demyelinated MS lesions acquire an inhibitory phenotype, which translates into hypermethylation of crucial myelination-related genes. Altering the epigenetic status of MBP can restore the differentiation capacity of OPCs and possibly boost (re)myelination. [ABSTRACT FROM AUTHOR]

Self-renewal and differentiation of hematopoietic stem and progenitor cells (HSPCs) are carefully controlled by extrinsic and intrinsic factors, to ensure the lifelong process of hematopoiesis. Apurinic/apyrimidinic endonuclease 1 (APEX1) is a multifunctional protein implicated in DNA repair and transcriptional regulation. Although previous studies have emphasized the necessity of studying APEX1 in a lineage-specific context and its role in progenitor differentiation, no studies have assessed the role of APEX1, nor its two enzymatic domains, in supporting adult HSPC function. In this study, we demonstrated that complete loss of APEX1 from murine bone marrow HSPCs (induced by CRISPR/Cas9) caused severe hematopoietic failure following transplantation, as well as a HSPC expansion defect in culture conditions maintaining in vivo HSC functionality. Using specific inhibitors against either the nuclease or redox domains of APEX1 in combination with single cell transcriptomics (CITE-seq), we found that both APEX1 nuclease and redox domains are regulating mouse HSPCs, but through distinct underlying transcriptional changes. Inhibition of the APEX1 nuclease function resulted in loss of HSPCs accompanied by early activation of differentiation programs and enhanced lineage commitment. By contrast, inhibition of the APEX1 redox function significantly downregulated interferon-stimulated genes and regulons in expanding HSPCs and their progeny, resulting in dysfunctional megakaryocyte-biased HSPCs, as well as loss of monocytes and lymphoid progenitor cells. In conclusion, we demonstrate that APEX1 is a key regulator for adult regenerative hematopoiesis, and that the APEX1 nuclease and redox domains differently impact proliferating HSPCs. [ABSTRACT FROM AUTHOR]

Pathological abnormalities in the tau protein give rise to a variety of neurodegenerative diseases, conjointly termed tauopathies. Several tau mutations have been identified in the tau-encoding gene MAPT, affecting either the physical properties of tau or resulting in altered tau splicing. At early disease stages, mitochondrial dysfunction was highlighted with mutant tau compromising almost every aspect of mitochondrial function. Additionally, mitochondria have emerged as fundamental regulators of stem cell function. Here, we show that compared to the isogenic wild-type triple MAPT-mutant human-induced pluripotent stem cells, bearing the pathogenic N279K, P301L, and E10+16 mutations, exhibit deficits in mitochondrial bioenergetics and present altered parameters linked to the metabolic regulation of mitochondria. Moreover, we demonstrate that the triple tau mutations disturb the cellular redox homeostasis and modify the mitochondrial network morphology and distribution. This study provides the first characterization of disease-associated tau-mediated mitochondrial impairments in an advanced human cellular tau pathology model at early disease stages, ranging from mitochondrial bioenergetics to dynamics. Consequently, comprehending better the influence of dysfunctional mitochondria on the development and differentiation of stem cells and their contribution to disease progression may thus assist in the potential prevention and treatment of tau-related neurodegeneration. [ABSTRACT FROM AUTHOR]

Human cerebral organoids (COs) are self-organizing three-dimensional (3D) neural structures that provide a human-specific platform to study the cellular and molecular processes that underlie different neurological events. The first step of CO generation from human pluripotent stem cells (hPSCs) is neural induction, which is an in vitro simulation of neural ectoderm development. Several signaling pathways cooperate during neural ectoderm development and in vitro differentiation of hPSCs toward neural cell lineages is also affected by them. In this study, we considered some of the known sources of these variable signaling cues arising from cell culture media components and sought to modulate their effects by applying a comprehensive combination of small molecules and growth factors for CO generation. Histological analysis demonstrated that these COs recapitulate the neural progenitor zone and early cortical layer organization, containing different types of neuronal and glial cells which was in accordance with single-nucleus transcriptome profiling results. Moreover, patch clamp and intracellular Ca2+ dynamic studies demonstrated that the COs behave as a functional neural network. Thus, this method serves as a facile protocol for generating hPSC-derived COs that faithfully mimic the features of their in vivo counterparts in the developing human brain. [ABSTRACT FROM AUTHOR]

Loss-of-function mutations in the PRKN, PINK1 and PARK7 genes (encoding parkin, PINK1 and DJ-1, respectively) cause autosomal recessive forms of Parkinson's disease. PINK1 and parkin jointly mediate selective autophagy of damaged mitochondria (mitophagy), but the mechanisms by which loss of DJ-1 induces Parkinson's disease, are not well understood. Here, we investigated PINK1/parkin-mediated mitophagy in cultured human fibroblasts and iPSC-derived neurons with homozygous PARK7 mutations. We found that DJ-1 is essential for PINK1/parkin-mediated mitophagy. Loss of DJ-1 did not interfere with PINK1 or parkin activation after mitochondrial depolarization, but blocked mitophagy further downstream by inhibiting recruitment of the selective autophagy receptor optineurin to depolarized mitochondria. By contrast, starvation-induced, non-selective autophagy was not affected by loss of DJ-1. In wild-type fibroblasts and iPSC-derived dopaminergic neurons, endogenous DJ-1 translocated to depolarized mitochondria in close proximity with optineurin. DJ-1 translocation to depolarized mitochondria was dependent on PINK1 and parkin and did not require oxidation of cysteine residue 106 of DJ-1. Overexpression of DJ-1 did not rescue the mitophagy defect of PINK1- or parkin-deficient cells. These findings position DJ-1 downstream of PINK1 and parkin in the same pathway and suggest that disruption of PINK1/parkin/DJ-1-mediated mitophagy is a common pathogenic mechanism in autosomal recessive Parkinson's disease. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cell (iPSC) technology enabled the production of pluripotent stem cell lines from somatic cells from a range of known genetic backgrounds. Their ability to differentiate and generate a wide variety of cell types has resulted in their use for various biomedical applications, including toxicity testing. Many of these iPSC lines are now registered in databases and stored in biobanks such as the European Bank for induced pluripotent Stem Cells (EBiSC), which can streamline the quality control and distribution of these individual lines. To generate the quantities of cells for banking and applications like high-throughput toxicity screening, scalable and robust methods need to be developed to enable the large-scale production of iPSCs. 3D suspension culture platforms are increasingly being used by stem cell researchers, owing to a higher cell output in a smaller footprint, as well as simpler scaling by increasing culture volume. Here we describe our strategies for successful scalable production of iPSCs using a benchtop bioreactor and incubator for 3D suspension cultures, while maintaining quality attributes expected of high-quality iPSC lines. Additionally, to meet the increasing demand for "ready-to-use" cell types, we report recent work to establish robust, scalable differentiation protocols to cardiac, neural, and hepatic fate to enable EBiSC to increase available research tools. • Flexible 3D protocols in one bioreactor system for undifferentiated cell expansion. • Subsequent differentiation to other cell types in the identical bioreactor system. • Virtually unlimited quantities of differentiated cell types for toxicity screening. • Differentiated cell products from healthy and diseased iPSC lines. [ABSTRACT FROM AUTHOR]

In cystic fibrosis (CF), genotype-phenotype correlation is complicated by the large number of CFTR variants, the influence of modifier genes, environmental effects, and the existence of complex alleles. We document the importance of complex alleles, in particular the F508C variant present in cis with the S1251N disease-causing variant, by detailed analysis of a patient with CF, with the [S1251N;F508]/G542X genotype and a very mild phenotype, contrasting it to that of four subjects with the [S1251N;F508C]/F508del genotype and classical CF presentation. Genetic differences were identified by Sanger sequencing and CFTR function was quantified using rectal organoids in rectal organoid morphology analysis (ROMA) and forskolin-induced swelling (FIS) assays. CFTR variants were further characterised in CF bronchial epithelial (CFBE) cell lines. The impact of involved amino acid changes in the CFTR 3D protein structure was evaluated. Organoids of the patient [S1251N;F508] with mild CF phenotype confirmed the CF diagnosis but showed higher residual CFTR function compared to the four others [S1251N;F508C]. CFBE cell lines showed a decrease in [S1251N;F508C]-CFTR function but not in processing when compared to [S1251N;F508]-CFTR. Analysis of the 3D CFTR structure suggested an additive deleterious effect of the combined presence of S1251N and F508C with respect to NBD1-2 dimerisation. In vitro and in silico data show that the presence of F508C in cis with S1251N decreases CFTR function without affecting processing. Complex CFTR alleles play a role in clinical phenotype and their identification is relevant in the context of personalised medicine for each patient with CF. [ABSTRACT FROM AUTHOR]

Introduction: Stem cell and tissue engineering (SC&TE) research remain controversial. Polemics are potential hurdles for raising public funds for research and clinical implementation. In view of future applications of SC&TE in perinatal conditions, we aimed to measure the background knowledge, perceptions or beliefs on SC&TE research among clinicians and academic researchers with perinatal applications on the department's research agenda. Material and Methods: We polled three professional categories: general obstetrician gynecologists, perinatologists and basic or translational researchers in development and regeneration. The survey included questions on demographics, work environment, educational background, general knowledge, expectations, opinions and ethical reflections of the respondent about SC&TE. Results: The response rate was 39 %. Respondents were mainly female (54 %) and under 40 years (63 %). The general background knowledge about SC&TE is low. Respondents confirm that remaining controversies still arise from the confusion that stem cell research coincides with embryo manipulation. Clinicians assume that stem cell research has reached the level of clinical implementation, and accept the risks associated of purposely harvesting fetal amniotic cells. Researchers in contrast are more cautious about both implementation and risks. Conclusion: Professionals in the field of perinatology may benefit of a better background knowledge and information on current SC & TE research. Though clinicians may be less aware of the current state of knowledge, they are open to clinical implementation, whereas dedicated researchers remain cautious. In view of the clinical introduction of SC & TE, purposed designed informative action should be taken and safety studies executed, hence avoid sustaining needless polemics. [ABSTRACT FROM AUTHOR]

In vertebrates, lifelong supply of all the blood cell types are maintained by the hematopoietic stem cells (HSCs). During development, these HSCs emerge in the aorta-gonad-mesonephros (AGM) from specialized embryonic vascular cells called hemogenic endothelium through a transdifferentiation process, called endothelial-to-hematopoietic transition (EHT). A complex interplay of key transcription factors and signalling pathways coordinates the whole process. Evidence showed that cellular phenotype and function can be driven according to the changes in cellular metabolism. Metabolic programs, which are stage specific, allow stem cells to adapt their function in different microenvironments. In adults, it is believed that the hypoxic bone marrow microenvironment regulates HSC function and protect them from oxidative stress. It is not clear whether hypoxic response and change in metabolic status regulate the generation and expansion of HSCs from the vasculature of midgestation embryo. Our proteomic studies predicted a metabolic switch from glycolytic to oxidative state during hematopoietic transition. Metabolic flux analysis, Mitochondrial activity and glucose uptake assays supported this hypothesis. Using pharmacologic and genetic approaches in mouse model, we targeted metabolic and oxygen- sensing enzymes in order to modulate the metabolic status of the hemato-endothelial system. Inhibition or deletion of Lactate Dehydrogenase A (LDHA) inhibited hematopoietic emergence. On the contrary, stabilization of HIF-1 α via inhibition of oxygen sensing pathway promoted intra-aortic hematopoietic cluster formation. Thus our study showed that, irrespective of the oxygenated microenvironment in the aorta, cell-intrinsic regulation of metabolic state promotes glycolytic metabolic state that is crucial for hematopoietic emergence in mammalian embryos. [ABSTRACT FROM AUTHOR]

The generation of tissue and organs requires close interaction with vasculature from the earliest moments of embryonic development. Tissue-specific organoids derived from pluripotent stem cells allow for the in vitro recapitulation of elements of embryonic development. However, they are not intrinsically vascularized, which poses a major challenge for their sustained growth, and for understanding the role of vasculature in fate specification and morphogenesis. Current organoid vascularization strategies do not recapitulate the temporal synchronization and spatial orientation needed to ensure in vivo-like early codevelopment. Here, we developed a human pluripotent stem cell (hPSC)-based approach to generate organoids which interact with vascular cells in a spatially determined manner. The spatial interaction between organoid and vasculature is enabled by the use of a custom designed 3D printed microfluidic chip which allows for a sequential and developmentally matched co-culture system. We show that on-chip hPSC-derived pericytes and endothelial cells sprout and self-assemble into organized vascular networks, and use cerebral organoids as a model system to explore interactions with this de novo generated vasculature. Upon co-development, vascular cells physically interact with the cerebral organoid and form an integrated neurovascular organoid on chip. This 3D printing-based platform is designed to be compatible with any organoid system and is an easy and highly cost-effective way to vascularize organoids. The use of this platform, readily performed in any lab, could open new avenues for understanding and manipulating the co-development of tissue-specific organoids with vasculature. [ABSTRACT FROM AUTHOR]

• Modelling platforms facilitate identification of therapeutic and prophylactic approaches in COVID-19. • Genome based strategies and computational platforms have introduced many drug candidates for COVID-19. • Conventional platforms still suffer from multiple limitations in modeling COVID-19. • Organoids and microfluidic devices encouraged recapitulation of virus-body interaction and drug discovery. Proposing efficient prophylactic and therapeutic strategies for coronavirus 2019 (COVID-19) requires precise knowledge of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pathogenesis. An array of platforms, including organoids and microfluidic devices, have provided a basis for studies of SARS-CoV-2. Here, we summarize available models as well as novel drug screening approaches, from simple to more advanced platforms. Notably, organoids and microfluidic devices offer promising perspectives for the clinical translation of basic science, such as screening therapeutics candidates. Overall, modifying these advanced micro and macro 3D platforms for disease modeling and combining them with recent advances in drug screening has significant potential for the discovery of novel potent drugs against COVID-19. [ABSTRACT FROM AUTHOR]

Abstract: Human hepatocytes, suitable for treatment of patients with liver failure, for the creation of bioartificial (BAL) devices, or for studies for toxicity and metabolization studies in the pharmaceutical industry, are in short supply due to the lack of donor organs. Therefore, methods that allow ex vivo expansion of hepatocytes with mature function are being pursued. One cell source, believed to be a possible inexhaustible source of hepatocytes, is pluripotent stem cells (PSCs). However, directed differentiation of PSCs to cells with features of adult hepatocytes is not yet possible. Differentiated progeny remains mixed and PSC progeny does not have a number of the functional features of mature hepatocytes. In this review article, we will address tools being developed that allow for the identification of mature hepatocytes, in a non-invasive manner; to perform lineage tracing of PSC progeny; and novel culture systems being created for the in vitro differentiation of PSCs to hepatocyte like cells, and for the maintenance of primary liver derived hepatocytes or PSC-derived hepatic progeny in culture. As conventional two-dimensional (2D) static culture conditions poorly recapitulate the in vivo cellular environment, we will discuss bioreactor systems for liver tissue engineering, both macro-scale and micro-scale culture systems. [Copyright &y& Elsevier]

Abstract: The liver is one of the few organs that possess a high capacity to regenerate after liver failure or liver damage. The parenchymal cells of the liver, hepatocytes, contribute to the majority of the regeneration process. Thus, hepatocyte transplantation presents an alternative method to treating liver damage. However, shortage of hepatocytes and difficulties in maintaining primary hepatocytes still remain key obstacles that researchers must overcome before hepatocyte transplantation can be used in clinical practice. The unique properties of pluripotent stem cells (PSCs) and induced pluripotent stem cells (iPSCs) have provided an alternative approach to generating enough functional hepatocytes for cellular therapy. In this review, we will present a brief overview on the current state of hepatocyte differentiation from PSCs and iPSCs. Studies of liver regenerative processes using different cell sources (adult liver stem cells, hepatoblasts, hepatic progenitor cells, etc.) will be described in detail as well as how this knowledge can be applied towards optimizing culture conditions for the maintenance and differentiation of these cells towards hepatocytes. As the outlook of stem cell-derived therapy begins to look more plausible, researchers will need to address the challenges we must overcome in order to translate stem cell research to clinical applications. [Copyright &y& Elsevier]

Zinc finger protein of the cerebellum (Zic)3, a member of Gli family of transcription factors (TFs), is essential for maintaining pluripotency of embryonic stem cells (ESCs) and has been reported to activate TF Nanog in an Oct4/Sox2-independent manner. Previously, we showed that Zic3 (Z), in combination with the Yamanka factors OCT4, SOX2, and KLF4 (OSK), induces neural progenitor-like cells from human fibroblasts. However, a similar combination of TFs (OSKZ) transduced in mouse embryonic fibroblasts resulted in enhanced induced pluripotent stem cells (iPSCs) formation compared with OSK alone, but not neuroprogenitors. OSKZ-derived iPSCs are indistinguishable from mESCs in colony morphology, expression of alkaline phosphatase and pluripotency genes, and embryoid body and teratoma formation. Zic3 activates the transcription of Nanog, a key pluripotency regulator, as evidenced by a luciferase promoter assay. During the course of iPSC derivation, Zic3-mediated enhanced expression of Nanog and Tbx3, gene known to enhance iPSCs derivation, is observed. Not only does Zic3 enhance the reprogramming efficiency, but also reactivation of the endogenous Zic3 protein is essential for the generation of iPSCs, as knockdown of Zic3 during the iPSC generation with OSKM significantly reduced the number of colonies. Together, our result uncovers an important role of Zic3 in generating mouse iPSCs. [ABSTRACT FROM AUTHOR]

Somatic, also termed adult, stem cells are highly attractive biomedical cell candidates because of their extensive replication potential and functional multilineage differentiation capacity. They can be used for drug and toxicity screenings in preclinical studies, as in vitro model to study differentiation or for regenerative medicine to aid in the repair of tissues or replace tissues that are lost upon disease, injury or ageing. Multipotent adult progenitor cells (MAPCs) and mesenchymal stem cells (MSCs) are two types of adult stem cells derived from bone marrow that are currently being used clinically for tissue regeneration and for their immunomodulatory and trophic effects. This review will give an overview of the phenotypic and functional differences between human MAPCs and MSCs, with a strong emphasis on their immunological characteristics. Finally, we will discuss the clinical studies in which MSCs and MAPCs are already used. [ABSTRACT FROM AUTHOR]

Overlaying differential changes in gene expression on protein interaction networks has proven to be a useful approach to interpreting the cell's dynamic response to a changing environment. Despite successes in finding active subnetworks in the context of a single species, the idea of overlaying lists of differentially expressed genes on networks has not yet been extended to support the analysis of multiple species' interaction networks. To address this problem, we designed a scalable, cross-species network search algorithm, neXus (Network - cross(X)-species - Search), that discovers conserved, active subnetworks based on parallel differential expression studies in multiple species. Our approach leverages functional linkage networks, which provide more comprehensive coverage of functional relationships than physical interaction networks by combining heterogeneous types of genomic data. We applied our cross-species approach to identify conserved modules that are differentially active in stem cells relative to differentiated cells based on parallel gene expression studies and functional linkage networks from mouse and human. We find hundreds of conserved active subnetworks enriched for stem cell-associated functions such as cell cycle, DNA repair, and chromatin modification processes. Using a variation of this approach, we also find a number of species-specific networks, which likely reflect mechanisms of stem cell function that have diverged between mouse and human. We assess the statistical significance of the subnetworks by comparing them with subnetworks discovered on random permutations of the differential expression data. We also describe several case examples that illustrate the utility of comparative analysis of active subnetworks. [ABSTRACT FROM AUTHOR]

Abstract: Because of their ability to self-renew and differentiate, adult stem cells are the in vivo source for replacing cells lost on a daily basis in high turnover tissues during the life of an organism. Adult stem cells however, do suffer the effects of aging resulting in decreased ability to self-renew and properly differentiate. Aging is a complex process and identification of the mechanisms underlying the aging of (stem) cell population(s) requires that relatively homogenous and well characterized populations can be isolated. Evaluation of the effect of aging on one such adult stem cell population, namely the hematopoietic stem cell (HSC), which can be purified to near homogeneity, has demonstrate that they do suffer cell intrinsic age associated changes. The cells that support HSC, namely marrow stromal cells, or mesenchymal stem cells (MSC), may similarly be affected by aging, although the inability to purify these cells to homogeneity precludes definitive assessment. As HSC and MSC are being used in cell-based therapies clinically, improved insight in the effect of aging on these two stem cell populations will probably impact the selection of sources for these stem cells. [Copyright &y& Elsevier]

Objective: We investigated the regulatory mechanism of primitive hematopoiesis in zebrafish (Danio rerio) embryos with particular reference to the role of a death receptor (zDR) gene, based on a morpholino (MO) knockdown approach. Methods: MOs targeting the zDR and chordin (Chd) were injected into naturally spawned embryos at one- to four-cell stage. A random sequence (RS) MO was used as a control. Effects on hemoglobin formation (Hb), apoptosis, and lineage-specific gene expression were examined. Embryos injected with zDR, Chd, and RS-MOs were denoted zDRmo, zChdmo, and zRSmo, respectively. Those coinjected with Chd+zDR-MOs and Chd+RS-MOs were abbreviated zChd+DRmo and zChd+RSmo. Results: zDR mRNA expression was restricted to the intermediate cell mass of wild-type (WT) and zChdmo embryos. At 48 hours postfertilization, zDRmo embryos showed increased Hb compared with WT or zRSmo embryos (2.36 × 10−2 ± 1.13 × 10−3 vs 1.85 × 10−2 ± 5.60 × 10−4 vs 1.79 × 10−2 ± 1.31 × 10−3 U, p < 0.05). zChd+DRmo embryos also showed increased Hb compared with zChdmo or zChd+RSmo embryos (4.60 × 10−2 ± 2.79 × 10−3 vs 3.17 × 10−2 ± 1.07 × 10−3 vs 3.05 × 10−2 ± 1.25 × 10−3 U, p < 0.05). zDR-MO reduced apoptosis, as shown by reduced terminal transferase-mediated dUTP nick end-labeling staining in zChd+DRmo compared with zChd+RSmo embryos and caspase-3 activity in zDRmo vs zRSmo (0.525 ± 0.094 vs 0.953 ± 0.113 U, p < 0.05), and zChd+DRmo vs zChd+RSmo embryos (0.247 ± 0.121 vs 1.180 ± 0.082, p < 0.05). zChd+DRmo embryos showed upregulation of erythroid-specific embryonic hemoglobin gene expression but not that of a myeloid-specific myeloperoxidase gene. Conclusion: Knockdown of zDR in zebrafish embryos decreased apoptosis and increased Hb, suggesting that zDR may regulate primitive hematopoiesis during development. [Copyright &y& Elsevier]