Towards Mimicking the Fetal Liver Niche: The Influence of Elasticity and Oxygen Tension on Hematopoietic Stem/Progenitor Cells Cultured in 3D Fibrin Hydrogels.

Hematopoietic stem/progenitor cells (HSPCs) are responsible for the generation of blood cells throughout life. It is believed that, in addition to soluble cytokines and niche cells, biophysical cues like elasticity and oxygen tension are responsible for the orchestration of stem cell fate. Although several studies have examined the effects of bone marrow (BM) niche elasticity on HSPC behavior, no study has yet investigated the effects of the elasticity of other niche sites like the fetal liver (FL), where HSPCs expand more extensively. In this study, we evaluated the effect of matrix stiffness values similar to those of the FL on BM-derived HSPC expansion. We first characterized the elastic modulus of murine FL tissue at embryonic day E14.5. Fibrin hydrogels with similar stiffness values as the FL (soft hydrogels) were compared with stiffer fibrin hydrogels (hard hydrogels) and with suspension culture. We evaluated the expansion of total nucleated cells (TNCs), Lin-/cKit+ cells, HSPCs (Lin-/Sca+/cKit+ (LSK) cells), and hematopoietic stem cells (HSCs: LSK- Signaling Lymphocyte Activated Molecule (LSK-SLAM) cells) when cultured in 5% O2 (hypoxia) or in normoxia. After 10 days, there was a significant expansion of TNCs and LSK cells in all culture conditions at both levels of oxygen tension. LSK cells expanded more in suspension culture than in both fibrin hydrogels, whereas TNCs expanded more in suspension culture and in soft hydrogels than in hard hydrogels, particularly in normoxia. The number of LSK-SLAM cells was maintained in suspension culture and in the soft hydrogels but not in the hard hydrogels. Our results indicate that both suspension culture and fibrin hydrogels allow for the expansion of HSPCs and more differentiated progeny whereas stiff environments may compromise LSK-SLAM cell expansion. This suggests that further research using softer hydrogels with stiffness values closer to the FL niche is warranted.

Intercellular Transfer of Microvesicles from Young Mesenchymal Stromal Cells Rejuvenates Aged Murine Hematopoietic Stem Cells.

Donor age is one of the major concerns in bone marrow transplantation, as the aged hematopoietic stem cells (HSCs) fail to engraft efficiently. Here, using murine system, we show that a brief interaction of aged HSCs with young mesenchymal stromal cells (MSCs) rejuvenates them and restores their functionality via inter-cellular transfer of microvesicles (MVs) containing autophagy-related mRNAs. Importantly, we show that MSCs gain activated AKT signaling as a function of aging. Activated AKT reduces the levels of autophagy-related mRNAs in their MVs, and partitions miR-17 and miR-34a into their exosomes, which upon transfer into HSCs downregulate their autophagy-inducing mRNAs. Our data identify previously unknown mechanisms operative in the niche-mediated aging of HSCs. Inhibition of AKT in aged MSCs increases the levels of autophagy-related mRNAs in their MVs and reduces the levels of miR-17 and miR-34a in their exosomes. Interestingly, transplantation experiments showed that the rejuvenating power of these "rescued" MVs is even better than that of the young MVs. We demonstrate that such ex vivo rejuvenation of aged HSCs could expand donor cohort and improve transplantation efficacy. Stem Cells 2018;36:420-433.

APEX1 Nuclease and Redox Functions are Both Essential for Adult Mouse Hematopoietic Stem and Progenitor Cells.

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.

Mannose-binding dietary lectins induce adipogenic differentiation of the marrow-derived mesenchymal cells via an active insulin-like signaling mechanism.

We have recently demonstrated that the mannose-binding lectins, namely banana lectin (BL) and garlic lectin (GL), interacted with the insulin receptors on M210B4 cells--an established mesenchymal cell line of murine marrow origin--and initiate mitogen-activated protein kinase kinase (MEK)-dependent extracellular signal-regulated kinase (ERK) signaling in them. In this study, we show that this lectin-mediated active ERK signaling culminates into an adipogenic differentiation of these cells. Gene expression studies indicate that the effect takes place at the transcriptional level. Experiments carried out with pharmacological inhibitors show that MEK-dependent ERK and phosphatidylinositol 3-kinase-dependent AKT pathways are positive regulators of the lectin- and insulin-mediated adipogenic differentiation, while stress-activated kinase/c-jun N-terminal kinase pathway acts as a negative one. Since both lectins could efficiently substitute for insulin in the standard adipogenic induction medium, they may perhaps serve as molecular tools to study the mechanistic aspects of the adipogenic process that are independent of cell proliferation. Our study clearly demonstrates the ability of BL and GL to activate insulin-like signaling in the mesenchymal cells in vitro leading to their adipocytic differentiation. The dietary origin of these lectins underscores an urgent need to examine their in vivo effects on tissue homeostasis.

A fully defined matrix to support a pluripotent stem cell derived multi-cell-liver steatohepatitis and fibrosis model.

Chronic liver injury, as observed in non-alcoholic steatohepatitis (NASH), progressive fibrosis, and cirrhosis, remains poorly treatable. Steatohepatitis causes hepatocyte loss in part by a direct lipotoxic insult, which is amplified by derangements in the non-parenchymal cellular (NPC) interactive network wherein hepatocytes reside, including, hepatic stellate cells, liver sinusoidal endothelial cells and liver macrophages. To create an in vitro culture model encompassing all these cells, that allows studying liver steatosis, inflammation and fibrosis caused by NASH, we here developed a fully defined hydrogel microenvironment, termed hepatocyte maturation (HepMat) gel, that supports maturation and maintenance of pluripotent stem cell (PSC) derived hepatocyte- and NPC-like cells for at least one month. The HepMat-based co-culture system modeled key molecular and functional features of TGFß-induced liver fibrosis and fatty-acid induced inflammation and fibrosis better than monocultures of its constituent cell populations. The novel co-culture system should open new avenues for studying mechanisms underlying liver steatosis, inflammation and fibrosis as well as for assessing drugs counteracting these effects.

Fetal hematopoietic stem cell homing is controlled by VEGF regulating the integrity and oxidative status of the stromal-vascular bone marrow niches.

Hematopoietic stem and progenitor cell (HSPC) engraftment after transplantation during anticancer treatment depends on support from the recipient bone marrow (BM) microenvironment. Here, by studying physiological homing of fetal HSPCs, we show the critical requirement of balanced local crosstalk within the skeletal niche for successful HSPC settlement in BM. Transgene-induced overproduction of vascular endothelial growth factor (VEGF) by osteoprogenitor cells elicits stromal and endothelial hyperactivation, profoundly impacting the stromal-vessel interface and vascular architecture. Concomitantly, HSPC homing and survival are drastically impaired. Transcriptome profiling, flow cytometry, and high-resolution imaging indicate alterations in perivascular and endothelial cell characteristics, vascular function and cellular metabolism, associated with increased oxidative stress within the VEGF-enriched BM environment. Thus, developmental HSPC homing to bone is controlled by local stromal-vascular integrity and the oxidative-metabolic status of the recipient milieu. Interestingly, irradiation of adult mice also induces stromal VEGF expression and similar osteo-angiogenic niche changes, underscoring that our findings may contribute targets for improving stem cell therapies.

Regulatory Dynamics of Tet1 and Oct4 Resolve Stages of Global DNA Demethylation and Transcriptomic Changes in Reprogramming.

Reprogramming somatic cells into induced pluripotent stem cells (iPSCs) involves the reactivation of endogenous pluripotency genes and global DNA demethylation, but temporal resolution of these events using existing markers is limited. Here, we generate murine transgenic lines harboring reporters for the 5-methylcytosine dioxygenase Tet1 and for Oct4. By monitoring dual reporter fluorescence during pluripotency entry, we identify a sequential order of Tet1 and Oct4 activation by proximal and distal regulatory elements. Full Tet1 activation marks an intermediate stage that accompanies predominantly repression of somatic genes, preceding full Oct4 activation, and distinguishes two waves of global DNA demethylation that target distinct genomic features but are uncoupled from transcriptional changes. Tet1 knockout shows that TET1 contributes to both waves of demethylation and activates germline regulatory genes in reprogramming intermediates but is dispensable for Oct4 reactivation. Our dual reporter system for time-resolving pluripotency entry thus refines the molecular roadmap of iPSC maturation.

Induction and Detection of Autophagy in Aged Hematopoietic Stem Cells by Exposing Them to Microvesicles Secreted by HSC-Supportive Mesenchymal Stromal Cells.

Autophagy is an important cellular process for maintenance of quality and functionality of cells. This happens through repair and renewal of cellular components like proteins and mitochondria. Reduction in autophagy process in aged hematopoietic stem cells (HSCs) leads to their compromised stemness and self-renewal capacity, and consequently, their applicability in various regenerative therapies also reduces. HSC functions are regulated by their microenvironment, known as "HSC niche," which comprises of mesenchymal stromal cells (MSCs), osteoblasts, endothelial cells, etc. In this niche, the MSCs are known to closely interact with the HSCs, and therefore, they can directly influence the stem cell fate. In our earlier studies, we have demonstrated that young MSCs or aged MSCs rejuvenated by treating them with LY294002, a PI3K inhibitor (rescued aged MSCs), rejuvenate aged HSCs via intercellular transfer of microvesicles (MVs) harboring autophagy-inducing mRNAs.Here, we describe the protocol for induction of autophagy in aged HSCs by incubating them with microvesicles (MVs) collected from young MSCs or rescued aged MSCs. We also describe the protocols for determination of autophagy levels in these HSCs.

Generation of hepatocyte- and endocrine pancreatic-like cells from human induced endodermal progenitor cells.

Multipotent Adult Progenitor Cells (MAPCs) are one potential stem cell source to generate functional hepatocytes or ß-cells. However, human MAPCs have less plasticity than pluripotent stem cells (PSCs), as their ability to generate endodermal cells is not robust. Here we studied the role of 14 transcription factors (TFs) in reprogramming MAPCs to induced endodermal progenitor cells (iENDO cells), defined as cells that can be long-term expanded and differentiated to both hepatocyte- and endocrine pancreatic-like cells. We demonstrated that 14 TF-iENDO cells can be expanded for at least 20 passages, differentiate spontaneously to hepatocyte-, endocrine pancreatic-, gut tube-like cells as well as endodermal tumor formation when grafted in immunodeficient mice. Furthermore, iENDO cells can be differentiated in vitro into hepatocyte- and endocrine pancreatic-like cells. However, the pluripotency TF OCT4, which is not silenced in iENDO cells, may contribute to the incomplete differentiation to mature cells in vitro and to endodermal tumor formation in vivo. Nevertheless, the studies presented here provide evidence that reprogramming of adult stem cells to an endodermal intermediate progenitor, which can be expanded and differentiate to multiple endodermal cell types, might be a valid alternative for the use of PSCs for creation of endodermal cell types.

Neuropilin-1 Is an Important Niche Component and Exerts Context-Dependent Effects on Hematopoietic Stem Cells.

Marrow adipocytes pose a significant problem in post-transplant regeneration of hematopoiesis owing to their negative effects on regeneration of hematopoiesis. However, the precise mechanism operative in this negative regulation is not clear. In this study, we show that marrow adipocytes express neuropilin-1 (NRP1) as a function of differentiation and inhibit regeneration of hematopoiesis by three principal mechanisms: one, by inducing apoptosis in hematopoietic stem/progenitor cells (HSPCs) through the death receptor-mediated pathway; two, by downregulating CXCR4 expression on the HSPCs through ligand-mediated internalization; and three, by secreting copious amounts of transforming growth factor ß1 (TGFß1), a known inhibitor of hematopoiesis. Silencing of NRP1 in these adipocytes rescued the apoptosis of cocultured HSPCs and boosted the CXCR4 surface expression on them, showing an active role of NRP1 in these processes. However, such silencing had no effect on TGFß1 secretion and consequent inhibition of hematopoiesis by them, showing that secretion of TGFß1 by adipocytes is independent of NRP1 expression by them. Surprisingly, mesenchymal stromal cells modified with NRP1 supported expansion of HSPCs having enhanced functionality, suggesting that NRP1 exerts a context-dependent effect on hematopoiesis. Our data demonstrate that NRP1 is an important niche component and exerts context-dependent effects on HSPCs. Based on these data, we speculate that antibody- or peptide-mediated blocking of NRP1-HSC interactions coupled with a pharmacological inhibition of TGFß1 signaling may help in combating the negative regulation of post-transplant regeneration of hematopoiesis in a more effective manner.

Irradiation-induced secretion of BMP4 by marrow cells causes marrow adipogenesis post-myelosuppression.

Pre-transplant myeloablation is associated with marrow adipogenesis, resulting in delayed engraftment of hematopoietic stem cells (HSCs). This is strongly undesirable, especially when the donor HSCs are fewer in numbers or have compromised functionality. The molecular mechanisms behind irradiation-induced marrow adipogenesis have not been extensively investigated. Here we show that bone marrow (BM) cells, especially T-cells and stromal cells, express and secrete copious amounts of BMP4 in response to irradiation, which causes the bone marrow stromal cells to commit to adipocyte lineage, thereby contributing to an increase in bone marrow adipogenesis. We further demonstrate that Simvastatin inhibits the BMP4-mediated adipogenic commitment of marrow stromal cells by inhibiting Ppar-γ expression. Importantly, Simvastatin does not prevent BMP4 secretion by the BM cells, and thus does not interfere with its salutary role in post-transplant hematopoietic regeneration. Our data identify previously unknown mechanisms operative in marrow adipogenesis post-myeloablation. They also reveal the molecular mechanisms behind the advantage of using Simvastatin as a niche-targeting agent to improve HSC engraftment.

Vasculogenic mimicry of HT1080 tumour cells in vivo: critical role of HIF-1α-neuropilin-1 axis.

HT1080 - a human fibrosarcoma-derived cell line - forms aggressive angiogenic tumours in immuno-compromised mice. In spite of its extensive use as a model of tumour angiogenesis, the molecular event(s) initiating the angiogenic program in these cells are not known. Since hypoxia stimulates tumour angiogenesis, we examined the hypoxia-induced events evoked in these cells. In contrast to cells grown under normoxic conditions, hypoxia-primed (1% O(2)) HT1080 cells formed robust tubules on growth factor-reduced matrigel and formed significantly larger tumours in xenograft models in a chetomin-sensitive manner, indicating the role of HIF-1α-mediated transcription in these processes. Immuno-histochemical analyses of tumours formed by GFP-expressing HT1080 cells clearly showed that the tumour cells themselves expressed various angiogenic markers including Neuropilin-1 (NRP-1) and formed functional vessels containing red blood cells, thereby unambiguously demonstrating the vasculogenic mimicry of HT1080 cells in vivo. Experiments performed with the HT1080 cells stably transfected with plasmid constructs expressing shNRP-1 or full-length NRP-1 clearly established that the HIF1α-mediated up-regulation of NRP-1 played a deterministic role in the process. Hypoxia-exposure resulted in an up-regulation of c-Myc and OCT3/4 and a down-regulation of KLF4 mRNAs, suggesting their involvement in the tumour formation and angiogenesis. However, silencing of NRP-1 alone, though not affecting proliferation in culture, was sufficient to abrogate the tumour formation completely; clearly establishing that the hypoxia-mediated HIF-1α-dependent up-regulation of NRP-1 is a critical molecular event involved in the vasculogenic mimicry and tumor formation by HT1080 cells in vivo.

Oral administration of insulin receptor-interacting lectins leads to an enhancement in the hematopoietic stem and progenitor cell pool of mice.

Lectins form an important constituent of our daily diet, and thus, it is essential that their effect(s) on various tissues be examined systematically in order to assess whether they are beneficial or detrimental to human health. We examined the effect of oral administration of two dietary lectins that were isolated from banana (BL) and garlic (GL)-two quite commonly consumed food items-on the hematopoiesis of mice. Balb/c mice were fed weekly with lectins and their marrow mononuclear cells (MNCs) were subjected to various hematopoietic stem/progenitor (HSPC)-specific phenotypic and functional assays. It was observed that the lectin-fed mice harbored a considerably increased HSPC pool in their marrow. Marrow-derived MNCs isolated from these lectin-fed mice gave rise to large-sized colony-forming unit-fibroblast (CFU-F) colonies indicating that the lectins had a salutary effect on the stromal compartment. The molecular mechanisms involved in the process were examined by using a stromal cell line model, M210B4. The lectins pulled down pro-insulin and insulin receptors in an immunoprecipitation experiment and activated extracellular signal-regulated kinase (ERK) signaling in the treated cells, in a manner comparable to insulin, both in terms of kinetics as well as extent. M210B4 cells incubated with BL, GL, or insulin showed reduced levels of reactive oxygen species, suggesting that perhaps the lectins protected the stem cell pool of mice by activating ERK signaling and reducing the oxidative stress in the niche. Our data suggest that these lectins may serve as micronutrients for therapeutic purposes in hematological deficiencies.