Reprogramming roadmap reveals route to human induced trophoblast stem cells.

The reprogramming of human somatic cells to primed or naive induced pluripotent stem cells recapitulates the stages of early embryonic development1-6. The molecular mechanism that underpins these reprogramming processes remains largely unexplored, which impedes our understanding and limits rational improvements to reprogramming protocols. Here, to address these issues, we reconstruct molecular reprogramming trajectories of human dermal fibroblasts using single-cell transcriptomics. This revealed that reprogramming into primed and naive pluripotency follows diverging and distinct trajectories. Moreover, genome-wide analyses of accessible chromatin showed key changes in the regulatory elements of core pluripotency genes, and orchestrated global changes in chromatin accessibility over time. Integrated analysis of these datasets revealed a role for transcription factors associated with the trophectoderm lineage, and the existence of a subpopulation of cells that enter a trophectoderm-like state during reprogramming. Furthermore, this trophectoderm-like state could be captured, which enabled the derivation of induced trophoblast stem cells. Induced trophoblast stem cells are molecularly and functionally similar to trophoblast stem cells derived from human blastocysts or first-trimester placentas7. Our results provide a high-resolution roadmap for the transcription-factor-mediated reprogramming of human somatic cells, indicate a role for the trophectoderm-lineage-specific regulatory program during this process, and facilitate the direct reprogramming of somatic cells into induced trophoblast stem cells.

The RNA-binding protein SRSF3 has an essential role in megakaryocyte maturation and platelet production.

RNA processing is increasingly recognized as a critical control point in the regulation of different hematopoietic lineages including megakaryocytes responsible for the production of platelets. Platelets are anucleate cytoplasts that contain a rich repertoire of RNAs encoding proteins with essential platelet functions derived from the parent megakaryocyte. It is largely unknown how RNA binding proteins contribute to the development and functions of megakaryocytes and platelets. We show that serine-arginine-rich splicing factor 3 (SRSF3) is essential for megakaryocyte maturation and generation of functional platelets. Megakaryocyte-specific deletion of Srsf3 in mice led to macrothrombocytopenia characterized by megakaryocyte maturation arrest, dramatically reduced platelet counts, and abnormally large functionally compromised platelets. SRSF3 deficient megakaryocytes failed to reprogram their transcriptome during maturation and to load platelets with RNAs required for normal platelet function. SRSF3 depletion led to nuclear accumulation of megakaryocyte mRNAs, demonstrating that SRSF3 deploys similar RNA regulatory mechanisms in megakaryocytes as in other cell types. Our study further suggests that SRSF3 plays a role in sorting cytoplasmic megakaryocyte RNAs into platelets and demonstrates how SRSF3-mediated RNA processing forms a central part of megakaryocyte gene regulation. Understanding SRSF3 functions in megakaryocytes and platelets provides key insights into normal thrombopoiesis and platelet pathologies as SRSF3 RNA targets in megakaryocytes are associated with platelet diseases.

3D-cardiomics: A spatial transcriptional atlas of the mammalian heart.

Understanding the spatial gene expression and regulation in the heart is key to uncovering its developmental and physiological processes, during homeostasis and disease. Numerous techniques exist to gain gene expression and regulation information in organs such as the heart, but few utilize intuitive true-to-life three-dimensional representations to analyze and visualise results. Here we combined transcriptomics with 3D-modelling to interrogate spatial gene expression in the mammalian heart. For this, we microdissected and sequenced transcriptome-wide 18 anatomical sections of the adult mouse heart. Our study has unveiled known and novel genes that display complex spatial expression in the heart sub-compartments. We have also created 3D-cardiomics, an interface for spatial transcriptome analysis and visualization that allows the easy exploration of these data in a 3D model of the heart. 3D-cardiomics is accessible from http://3d-cardiomics.erc.monash.edu/.

Smad4 promotes diabetic nephropathy by modulating glycolysis and OXPHOS.

Diabetic nephropathy (DN) is the leading cause of end-stage kidney disease. TGF-ß1/Smad3 signalling plays a major pathological role in DN; however, the contribution of Smad4 has not been examined. Smad4 depletion in the kidney using anti-Smad4 locked nucleic acid halted progressive podocyte damage and glomerulosclerosis in mouse type 2 DN, suggesting a pathogenic role of Smad4 in podocytes. Smad4 is upregulated in human and mouse podocytes during DN. Conditional Smad4 deletion in podocytes protects mice from type 2 DN, independent of obesity. Mechanistically, hyperglycaemia induces Smad4 localization to mitochondria in podocytes, resulting in reduced glycolysis and oxidative phosphorylation and increased production of reactive oxygen species. This operates, in part, via direct binding of Smad4 to the glycolytic enzyme PKM2 and reducing the active tetrameric form of PKM2. In addition, Smad4 interacts with ATPIF1, causing a reduction in ATPIF1 degradation. In conclusion, we have discovered a mitochondrial mechanism by which Smad4 causes diabetic podocyte injury.

Comprehensive characterization of distinct states of human naive pluripotency generated by reprogramming.

Recent reports on the characteristics of naive human pluripotent stem cells (hPSCs) obtained using independent methods differ. Naive hPSCs have been mainly derived by conversion from primed hPSCs or by direct derivation from human embryos rather than by somatic cell reprogramming. To provide an unbiased molecular and functional reference, we derived genetically matched naive hPSCs by direct reprogramming of fibroblasts and by primed-to-naive conversion using different naive conditions (NHSM, RSeT, 5iLAF and t2iLGöY). Our results show that hPSCs obtained in these different conditions display a spectrum of naive characteristics. Furthermore, our characterization identifies KLF4 as sufficient for conversion of primed hPSCs into naive t2iLGöY hPSCs, underscoring the role that reprogramming factors can play for the derivation of bona fide naive hPSCs.

MOZ (KAT6A) is essential for the maintenance of classically defined adult hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are conventionally thought to be at the apex of a hierarchy that produces all mature cells of the blood. The quintessential property of these cells is their ability to reconstitute the entire hematopoietic system of hemoablated recipients. This characteristic has enabled HSCs to be used to replenish the hematopoietic system of patients after chemotherapy or radiotherapy. Here, we use deletion of the monocytic leukemia zinc finger gene (Moz/Kat6a/Myst3) to examine the effects of removing HSCs. Loss of MOZ in adult mice leads to the rapid loss of HSCs as defined by transplantation. This is accompanied by a reduction of the LSK-CD48-CD150+ and LSK-CD34-Flt3- populations in the bone marrow and a reduction in quiescent cells in G0 Surprisingly, the loss of classically defined HSCs did not affect mouse viability, and there was no recovery of the LSK-CD48-CD150+ and LSK-CD34-Flt3- populations 15 to 18 months after Moz deletion. Clonal analysis of myeloid progenitors, which produce short-lived granulocytes, demonstrate that these are derived from cells that had undergone recombination at the Moz locus up to 2 years earlier, suggesting that early progenitors have acquired extended self-renewal. Our results establish that there are essential differences in HSC requirement for steady-state blood cell production compared with the artificial situation of reconstitution after transplantation into a hemoablated host. A better understanding of steady-state hematopoiesis may facilitate the development of novel therapies engaging hematopoietic cell populations with previously unrecognized traits, as well as characterizing potential vulnerability to oncogenic transformation.

i-bodies, Human Single Domain Antibodies That Antagonize Chemokine Receptor CXCR4.

CXCR4 is a G protein-coupled receptor with excellent potential as a therapeutic target for a range of clinical conditions, including stem cell mobilization, cancer prognosis and treatment, fibrosis therapy, and HIV infection. We report here the development of a fully human single-domain antibody-like scaffold termed an "i-body," the engineering of which produces an i-body library possessing a long complementarity determining region binding loop, and the isolation and characterization of a panel of i-bodies with activity against human CXCR4. The CXCR4-specific i-bodies show antagonistic activity in a range of in vitro and in vivo assays, including inhibition of HIV infection, cell migration, and leukocyte recruitment but, importantly, not the mobilization of hematopoietic stem cells. Epitope mapping of the three CXCR4 i-bodies AM3-114, AM4-272, and AM3-523 revealed binding deep in the binding pocket of the receptor.

Niche Extracellular Matrix Components and Their Influence on HSC.

Maintenance of hematopoietic stem cells (HSC) takes place in a highly specialized microenvironment within the bone marrow. Technological improvements, especially in the field of in vivo imaging, have helped unravel the complexity of the niche microenvironment and have completely changed the classical concept from what was previously believed to be a static supportive platform, to a dynamic microenvironment tightly regulating HSC homeostasis through the complex interplay between diverse cell types, secreted factors, extracellular matrix molecules, and the expression of different transmembrane receptors. To add to the complexity, non-protein based metabolites have also been recognized as a component of the bone marrow niche. The objective of this review is to discuss the current understanding on how the different extracellular matrix components of the niche regulate HSC fate, both during embryonic development and in adulthood. Special attention will be provided to the description of non-protein metabolites, such as lipids and metal ions, which contribute to the regulation of HSC behavior. J. Cell. Biochem. 118: 1984-1993, 2017. © 2017 Wiley Periodicals, Inc.

Progress in bio-manufacture of platelets for transfusion.

Blood transfusion services face an ever-increasing demand for donor platelets to meet clinical needs. Whilst strategies for increasing platelet storage life and improving the efficiency of donor platelet collection are important, in the longer term, platelets generated by bio-manufacturing processes will be required to meet demands. Production of sufficient numbers of in vitro-derived platelets for transfusion represents a significant bioengineering challenge. In this review, we highlight recent progress in this area of research and outline the main technical and biological obstacles that need to be met before this becomes feasible and economic. A critical consideration is assurance of the functional properties of these cells as compared to their fresh, donor collected, counterparts. We contend that platelet-like particles and in vitro-derived platelets that phenotypically resemble fresh platelets must deliver the same functions as these cells upon transfusion. We also note recent progress with immortalized megakaryocyte progenitor cell lines, molecular strategies for reducing expression of HLA Class I to generate universal donor platelets and the move to early clinical studies with in vitro-derived platelets.

The role of CD44 in fetal and adult hematopoietic stem cell regulation.

Throughout development, hematopoietic stem cells migrate to specific microenvironments, where their fate is, in part, extrinsically controlled. CD44 standard as a member of the cell adhesion molecule family is extensively expressed within adult bone marrow and has been previously reported to play important roles in adult hematopoietic regulation via CD44 standard-ligand interactions. In this manuscript, CD44 expression and function are further assessed and characterized on both fetal and adult hematopoietic stem cells. Using a CD44(-/-) mouse model, conserved functional roles of CD44 are revealed throughout development. CD44 is critical in the maintenance of hematopoietic stem and progenitor pools, as well as in hematopoietic stem cell migration. CD44 expression on hematopoietic stem cells as well as other hematopoietic cells within the bone marrow microenvironment is important in the homing and lodgment of adult hematopoietic stem cells isolated from the bone/bone marrow interface. CD44 is also involved in fetal hematopoietic stem cell migration out of the liver, via a process involving stromal cell-derived factor-1α. The absence of CD44 in neonatal bone marrow has no impact on the size of the long-term reconstituting hematopoietic stem cell pool, but results in an enhanced long-term engraftment potential of hematopoietic stem cells.

Brief Report: Factors Released by Megakaryocytes Thrombin Cleave Osteopontin to Negatively Regulate Hematopoietic Stem Cells.

Factor V (FV) and factor X (FX) activate and complex to form prothrombinase which subsequently cleaves prothrombin (PT), converting it to active thrombin. Thrombin cleaved osteopontin (tcOPN) contains a cryptic binding site for α4 ß1 and α9 ß1 integrins. We have previously shown that hematopoietic stem cells (HSC) bind to tcOPN via this site resulting in a decrease in their proliferation and differentiation. Therefore, tcOPN and the factors required for its generation are important components of the HSC niche. Herein we show mature megakaryocytes (MM, ≥8N) contain FV, FX, and PT mRNA and protein. Furthermore, we show 8N, 16N, 32N, and 64N MM all release the required factors to enable thrombin cleavage of OPN. Importantly, mice devoid of the myeloproliferative leukemia protein (Mpl), c-Mpl(-/-) mice, contain only approximately 10% of normal megakaryocyte numbers, showed significantly reduced FX and tcOPN protein levels in endosteal bone marrow (BM). In addition, WT hematopoietic progenitors and HSC showed reduced homing to the BM of c-Mpl(-/-) mice. This is the first report identifying MM as a key cellular component in the production of tcOPN in situ, allowing the BM microenvironment to self regulate HSC biology via tcOPN.

Validating Eaton's Hypothesis: Cubane as a Benzene Bioisostere.

Pharmaceutical and agrochemical discovery programs are under considerable pressure to meet increasing global demand and thus require constant innovation. Classical hydrocarbon scaffolds have long assisted in bringing new molecules to the market place, but an obvious omission is that of the Platonic solid cubane. Eaton, however, suggested that this molecule has the potential to act as a benzene bioisostere. Herein, we report the validation of Eaton's hypothesis with cubane derivatives of five molecules that are used clinically or as agrochemicals. Two cubane analogues showed increased bioactivity compared to their benzene counterparts whereas two further analogues displayed equal bioactivity, and the fifth one demonstrated only partial efficacy. Ramifications from this study are best realized by reflecting on the number of bioactive molecules that contain a benzene ring. Substitution with the cubane scaffold where possible could revitalize these systems, and thus expedite much needed lead candidate identification.

Design, synthesis and binding properties of a fluorescent α9ß1/α4ß1 integrin antagonist and its application as an in vivo probe for bone marrow haemopoietic stem cells.

The α9ß1 and α4ß1 integrin subtypes are expressed on bone marrow haemopoietic stem cells and have important roles in stem cell regulation and trafficking. Although the roles of α4ß1 integrin have been thoroughly investigated with respect to HSC function, the role of α9ß1 integrin remains poorly characterised. Small molecule fluorescent probes are useful tools for monitoring biological processes in vivo, to determine cell-associated protein localisation and activation, and to elucidate the mechanism of small molecule mediated protein interactions. Herein, we report the design, synthesis and integrin-dependent cell binding properties of a new fluorescent α9ß1 integrin antagonist (R-BC154), which was based on a series of N-phenylsulfonyl proline dipeptides and assembled using the Cu(I)-catalyzed azide alkyne cycloaddition (CuAAC) reaction. Using transfected human glioblastoma LN18 cells, we show that R-BC154 exhibits high nanomolar binding affinities to α9ß1 integrin with potent cross-reactivity against α4ß1 integrin under physiological mimicking conditions. On-rate and off-rate measurements revealed distinct differences in the binding kinetics between α9ß1 and α4ß1 integrins, which showed faster binding to α4ß1 integrin relative to α9ß1, but more prolonged binding to the latter. Finally, we show that R-BC154 was capable of binding rare populations of bone marrow haemopoietic stem and progenitor cells when administered to mice. Thus, R-BC154 represents a useful multi-purpose fluorescent integrin probe that can be used for (1) screening small molecule inhibitors of α9ß1 and α4ß1 integrins; (2) investigating the biochemical properties of α9ß1 and α4ß1 integrin binding and (3) investigating integrin expression and activation on defined cell phenotypes in vivo.

The activity of early-life gene regulatory elements is hijacked in aging through pervasive AP-1-linked chromatin opening.

A mechanistic connection between aging and development is largely unexplored. Through profiling age-related chromatin and transcriptional changes across 22 murine cell types, analyzed alongside previous mouse and human organismal maturation datasets, we uncovered a transcription factor binding site (TFBS) signature common to both processes. Early-life candidate cis-regulatory elements (cCREs), progressively losing accessibility during maturation and aging, are enriched for cell-type identity TFBSs. Conversely, cCREs gaining accessibility throughout life have a lower abundance of cell identity TFBSs but elevated activator protein 1 (AP-1) levels. We implicate TF redistribution toward these AP-1 TFBS-rich cCREs, in synergy with mild downregulation of cell identity TFs, as driving early-life cCRE accessibility loss and altering developmental and metabolic gene expression. Such remodeling can be triggered by elevating AP-1 or depleting repressive H3K27me3. We propose that AP-1-linked chromatin opening drives organismal maturation by disrupting cell identity TFBS-rich cCREs, thereby reprogramming transcriptome and cell function, a mechanism hijacked in aging through ongoing chromatin opening.

The relationship between bone, hemopoietic stem cells, and vasculature.

A large body of evidence suggests hemopoietic stem cells (HSCs) exist in an endosteal niche close to bone, whereas others suggest that the HSC niche is intimately associated with vasculature. In this study, we show that transplanted hemopoietic stem and progenitor cells (HSPCs) home preferentially to the trabecular-rich metaphysis of the femurs in nonablated mice at all time points from 15 minutes to 15 hours after transplantation. Within this region, they exist in an endosteal niche in close association with blood vessels. The preferential homing of HSPCs to the metaphysis occurs rapidly after transplantation, suggesting that blood vessels within this region may express a unique repertoire of endothelial adhesive molecules. One candidate is hyaluronan (HA), which is highly expressed on the blood vessel endothelium in the metaphysis. Analysis of the early stages of homing and the spatial dis-tribution of transplanted HSPCs at the single-cell level in mice devoid of Has3-synthesized HA, provides evidence for a previously undescribed role for HA expressed on endothelial cells in directing the homing of HSPCs to the metaphysis.

Bone, microenvironment and hematopoiesis.

PURPOSE OF REVIEW: Hematopoietic stem cells (HSCs) mature to form all blood cells and the study into how HSC fate decisions are made has exploded in recent years. In an effort to fully understand the function and organization of HSCs and hematopoietic progenitor cells (HPCs), many groups have identified the microenvironment in which they reside as playing a key role. This review highlights the findings within the last 18 months on the cells and molecules shown to be important within the bone marrow HSC niche for HSC regulation. RECENT FINDINGS: Previous research has heavily concentrated on the role of osteoblasts, mesenchymal stem cells (MSCs), reticular stromal cells, endothelial cells and nerve cells. More recently, research has not only expanded on the role of these cells, but has also shown that mature hematopoietic cells such as macrophages and megakaryocytes are also important in the maintenance of hematopoiesis within the HSC niche. SUMMARY: Identifying and understanding the roles of all cells comprising the HSC niche coupled with the development of better 3D imaging and 3D in-vitro mimicking of the HSC niche will increase our understanding of where HSCs reside and how they are regulated. Research will lead to better manipulation of HSCs for mobilization, homing and hematopoietic reconstitution following injury or disease.

Late fetal hematopoietic failure results from ZBTB11 deficiency despite abundant HSC specification.

Hematopoiesis produces diverse blood cell lineages to meet the basal needs and sudden demands of injury or infection. A rapid response to such challenges requires the expansion of specific lineages and a prompt return to balanced steady-state levels, necessitating tightly coordinated regulation. Previously we identified a requirement for the zinc finger and broad complex, tramtrak, bric-a-brac domain-containing 11 (ZBTB11) transcription factor in definitive hematopoiesis using a forward genetic screen for zebrafish myeloid mutants. To understand its relevance to mammalian systems, we extended these studies to mice. When Zbtb11 was deleted in the hematopoietic compartment, embryos died at embryonic day (E) 18.5 with hematopoietic failure. Zbtb11 hematopoietic knockout (Zbtb11hKO) hematopoietic stem cells (HSCs) were overabundantly specified from E14.5 to E17.5 compared with those in controls. Overspecification was accompanied by loss of stemness, inability to differentiate into committed progenitors and mature lineages in the fetal liver, failure to seed fetal bone marrow, and total hematopoietic failure. The Zbtb11hKO HSCs did not proliferate in vitro and were constrained in cell cycle progression, demonstrating the cell-intrinsic role of Zbtb11 in proliferation and cell cycle regulation in mammalian HSCs. Single-cell RNA sequencing analysis identified that Zbtb11-deficient HSCs were underrepresented in an erythroid-primed subpopulation and showed downregulation of oxidative phosphorylation pathways and dysregulation of genes associated with the hematopoietic niche. We identified a cell-intrinsic requirement for Zbtb11-mediated gene regulatory networks in sustaining a pool of maturation-capable HSCs and progenitor cells.

Stabilization and improved functionality of three-dimensional perfusable microvascular networks in microfluidic devices under macromolecular crowding.

BACKGROUND: There is great interest to engineer in vitro models that allow the study of complex biological processes of the microvasculature with high spatiotemporal resolution. Microfluidic systems are currently used to engineer microvasculature in vitro, which consists of perfusable microvascular networks (MVNs). These are formed through spontaneous vasculogenesis and exhibit the closest resemblance to physiological microvasculature. Unfortunately, under standard culture conditions and in the absence of co-culture with auxiliary cells as well as protease inhibitors, pure MVNs suffer from a short-lived stability. METHODS: Herein, we introduce a strategy for stabilization of MVNs through macromolecular crowding (MMC) based on a previously established mixture of Ficoll macromolecules. The biophysical principle of MMC is based on macromolecules occupying space, thus increasing the effective concentration of other components and thereby accelerating various biological processes, such as extracellular matrix deposition. We thus hypothesized that MMC will promote the accumulation of vascular ECM (basement membrane) components and lead to a stabilization of MVN with improved functionality. RESULTS: MMC promoted the enrichment of cellular junctions and basement membrane components, while reducing cellular contractility. The resulting advantageous balance of adhesive forces over cellular tension resulted in a significant stabilization of MVNs over time, as well as improved vascular barrier function, closely resembling that of in vivo microvasculature. CONCLUSION: Application of MMC to MVNs in microfluidic devices provides a reliable, flexible and versatile approach to stabilize engineered microvessels under simulated physiological conditions.

High ploidy large cytoplasmic megakaryocytes are hematopoietic stem cells regulators and essential for platelet production.

Megakaryocytes (MK) generate platelets. Recently, we and others, have reported MK also regulate hematopoietic stem cells (HSC). Here we show high ploidy large cytoplasmic megakaryocytes (LCM) are critical negative regulators of HSC and critical for platelet formation. Using a mouse knockout model (Pf4-Srsf3Δ/Δ) with normal MK numbers, but essentially devoid of LCM, we demonstrate a pronounced increase in BM HSC concurrent with endogenous mobilization and extramedullary hematopoiesis. Severe thrombocytopenia is observed in animals with diminished LCM, although there is no change in MK ploidy distribution, uncoupling endoreduplication and platelet production. When HSC isolated from a microenvironment essentially devoid of LCM reconstitute hematopoiesis in lethally irradiated mice, the absence of LCM increases HSC in BM, blood and spleen, and the recapitulation of thrombocytopenia. In contrast, following a competitive transplant using minimal numbers of WT HSC together with HSC from a microenvironment with diminished LCM, sufficient WT HSC-generated LCM regulates a normal HSC pool and prevents thrombocytopenia. Importantly, LCM are conserved in humans.