Microfluidic devices for studying coagulation biology.

The ability to study the behavior of cells, proteins, and cell-cell or cell-protein interactions under dynamic forces such as shear stress under fluid flow, provides a more accurate understanding of the physiopathology of hemostasis. This review touches upon the traditional methods for studying blood coagulation and platelet aggregation and provides an overview on cellular and protein response to shear stress. We also elaborate on the biological aspects of how cells recognize mechanical forces and convert them into biochemical signals that can drive various signaling pathways. We give a detailed description of the various types of microfluidic devices that are employed to study the complex processes of platelet aggregation and blood coagulation under flow conditions as well as to investigate endothelial shear-response. We also highlight works mimicking artificial vessels as platforms to study the mechanisms of coagulation, and finish our review by describing anticipated clinical uses of microfluidics devices and their standardization.

Acceptability of prenatal diagnosis and prenatal treatment of haemophilia using cell and gene therapies within US haemophilia community.

BACKGROUND: The overall burden of disease in persons with haemophilia continues to be high despite the latest advancements in therapeutics. Clinical trials testing prenatal treatments for several genetic disorders are underway or are recruiting subjects, attesting to the much-needed change in paradigm of how patients with monogenic disorders can be treated. Here we investigate the overall attitude towards prenatal diagnosis, preferences on types of prenatal therapies for haemophilia, the level of 'acceptable' risk tolerated, and which social and moral pressures or disease personal experiences may predict willingness of individuals to consider foetal therapy in a future pregnancy. RESULTS: A multidisciplinary team designed the survey, and the study was carried out using REDCap, and publicized through the National Haemophilia Foundation. Subjects ≥18 years of age were eligible to participate in the study. We assessed participants' attitudes towards prenatal therapy and their level of 'acceptable' risk towards the procedure and therapy. The survey was completed by 67 adults, the majority females. Respondents were willing to undergo prenatal diagnosis, and their main concerns related to the well-being of the pregnant woman and the foetus regarding lasting therapeutic efficacy, side effects of the therapy, and procedural risks, but they were likely to accept a wide range of prenatal therapeutic options, particularly if the foetal therapy proved to be long-lasting and safe. CONCLUSIONS: These data demonstrate the willingness of persons with haemophilia, and the haemophilia community, to explore new treatment options beyond the currently offered approaches.

Soluble Fas affects erythropoiesis in vitro and acts as a potential predictor of erythropoiesis-stimulating agent therapy in patients with chronic kidney disease.

Serum soluble Fas (sFas) levels are associated with erythropoietin (Epo) hyporesponsiveness in patients with chronic kidney disease (CKD). Whether sFas could predict the need for erythropoiesis-stimulating agent (ESA) usage and its influence in erythropoiesis remain unclear. We evaluated the relation between sFas and ESA therapy in patients with CKD with anemia and its effect on erythropoiesis in vitro. First, we performed a retrospective cohort study with 77 anemic patients with nondialysis CKD. We performed in vitro experiments to investigate whether sFas could interfere with the behavior of hematopoietic stem cells (HSCs). HSCs were isolated from umbilical cord blood and incubated with recombinant sFas protein in a dose-dependent manner. Serum sFas positively correlated with Epo levels (r = 0.30, P = 0.001) but negatively with hemoglobin (r = -0.55, P < 0.001) and glomerular filtration rate (r = -0.58, P < 0.001) in patients with CKD at baseline. Elevated sFas serum levels (4,316 ± 897 vs. 2,776 ± 749, P < 0.001) with lower estimated glomerular filtration rate (26.2 ± 10.1 vs. 33.5 ± 14.3, P = 0.01) and reduced hemoglobin concentration (11.1 ± 0.9 vs. 12.5 ± 1.2, P < 0.001) were identified in patients who required ESA therapy compared with patients with non-ESA. Afterward, we detected that the sFas level was slight correlated with a necessity of ESA therapy in patients with nondialysis CKD and anemia. In vitro assays demonstrated that the erythroid progenitor cell frequency negatively correlated with sFas concentration (r = -0.72, P < 0.001). There was decreased erythroid colony formation in vitro when CD34+ HSCs were incubated with a higher concentration of sFas protein (1.56 ± 0.29, 4.33 ± 0.53, P < 0.001). Our findings suggest that sFas is a potential predictor for ESA therapy in patients with nondialysis CKD and that elevated sFas could affect erythropoiesis in vitro.

Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Selectively recruiting bone marrow (BM)-derived stem and progenitor cells to injury sites is a promising therapeutic approach. The coordinated action of soluble factors is thought to trigger the mobilization of stem cells from the BM and recruit them to lesions to contribute to tissue regeneration. Nevertheless, the temporal response profile of the major cellular players and soluble factors involved in priming the BM and recruiting BM-derived cells to promote regeneration is unknown. We show that injury alters the BM cellular composition, introducing population-specific fluctuations during tissue regeneration. We demonstrate that injury causes an immediate, transient response of mesenchymal stromal cells and endothelial cells followed by a nonoverlapping increase in hematopoietic stem and progenitor cells. Moreover, BM reaction is identical whether the injury is inflicted on skin and muscle or also involves a bone defect, but these 2 injury paradigms trigger distinct systemic cytokine responses. Together, our results indicate that the BM response to injury in the early stages of regeneration is independent of the tissue-of-injury based on the 2 models used, but the injured tissue dictates the systemic cytokine response.-Leitão, L., Alves, C. J., Alencastre, I. S., Sousa, D. M., Neto, E., Conceição, F., Leitão, C., Aguiar, P., Almeida-Porada, G., Lamghari, M. Bone marrow cell response after injury and during early stage of regeneration is independent of the tissue-of-injury in 2 injury models.

Deconstructed Microfluidic Bone Marrow On-A-Chip to Study Normal and Malignant Hemopoietic Cell-Niche Interactions.

Human hematopoietic niches are complex specialized microenvironments that maintain and regulate hematopoietic stem and progenitor cells (HSPC). Thus far, most of the studies performed investigating alterations of HSPC-niche dynamic interactions are conducted in animal models. Herein, organ microengineering with microfluidics is combined to develop a human bone marrow (BM)-on-a-chip with an integrated recirculating perfusion system that consolidates a variety of important parameters such as 3D architecture, cell-cell/cell-matrix interactions, and circulation, allowing a better mimicry of in vivo conditions. The complex BM environment is deconvoluted to 4 major distinct, but integrated, tissue-engineered 3D niche constructs housed within a single, closed, recirculating microfluidic device system, and equipped with cell tracking technology. It is shown that this technology successfully enables the identification and quantification of preferential interactions-homing and retention-of circulating normal and malignant HSPC with distinct niches.

Bone Marrow Endothelial Cells Influence Function and Phenotype of Hematopoietic Stem and Progenitor Cells after Mixed Neutron/Gamma Radiation.

The bone marrow (BM) microenvironment plays a crucial role in the maintenance and regeneration of hematopoietic stem (HSC) and progenitor cells (HSPC). In particular, the vascular niche is responsible for regulating HSC maintenance, differentiation, and migration of cells in and out of the BM. Damage to this niche upon exposure to ionizing radiation, whether accidental or as a result of therapy, can contribute to delays in HSC recovery and/or function. The ability of BM derived-endothelial cells (BMEC) to alter and/or protect HSPC after exposure to ionizing radiation was investigated. Our data show that exposure of BMEC to ionizing radiation resulted in alterations in Akt signaling, increased expression of PARP-1, IL6, and MCP-1, and decreased expression of MMP1 and MMP9. In addition, global analysis of gene expression of HSC and BMEC in response to mixed neutron/gamma field (MF) radiation identified 60 genes whose expression was altered after radiation in both cell types, suggesting that a subset of genes is commonly affected by this type of radiation. Focused gene analysis by RT-PCR revealed two categories of BMEC alterations: (a) a subset of genes whose expression was altered in response to radiation, with no additional effect observed during coculture with HSPC, and (b) a subset of genes upregulated in response to radiation, and altered when cocultured with HSPC. Coculture of BMEC with CD34+ HSPC induced HSPC proliferation, and improved BM function after MF radiation. Nonirradiated HSPC exhibited reduced CD34 expression over time, but when irradiated, they maintained higher CD34 expression. Nonirradiated HSPC cocultured with nonirradiated BMEC expressed lower levels of CD34 expression compared to nonirradiated alone. These data characterize the role of each cell type in response to MF radiation and demonstrate the interdependence of each cell's response to ionizing radiation. The identified genes modulated by radiation and coculture provide guidance for future experiments to test hypotheses concerning specific factors mediating the beneficial effects of BMEC on HSPC. This information will prove useful in the search for medical countermeasures to radiation-induced hematopoietic injury.

Indocyanine green loaded hyaluronan-derived nanoparticles for fluorescence-enhanced surgical imaging of pancreatic cancer.

Pancreatic ductal adenocarcinoma is highly lethal and surgical resection is the only potential curative treatment for the disease. In this study, hyaluronic acid derived nanoparticles with physico-chemically entrapped indocyanine green, termed NanoICG, were utilized for intraoperative near infrared fluorescence detection of pancreatic cancer. NanoICG was not cytotoxic to healthy pancreatic epithelial cells and did not induce chemotaxis or phagocytosis, it accumulated significantly within the pancreas in an orthotopic pancreatic ductal adenocarcinoma model, and demonstrated contrast-enhancement for pancreatic lesions relative to non-diseased portions of the pancreas. Fluorescence microscopy showed higher fluorescence intensity in pancreatic lesions and splenic metastases due to NanoICG compared to ICG alone. The in vivo safety profile of NanoICG, including, biochemical, hematological, and pathological analysis of NanoICG-treated healthy mice, indicates negligible toxicity. These results suggest that NanoICG is a promising contrast agent for intraoperative detection of pancreatic tumors.

The hematopoietic system in the context of regenerative medicine.

Hematopoietic stem cells (HSC) represent the prototype stem cell within the body. Since their discovery, HSC have been the focus of intensive research, and have proven invaluable clinically to restore hematopoiesis following inadvertent radiation exposure and following radio/chemotherapy to eliminate hematologic tumors. While they were originally discovered in the bone marrow, HSC can also be isolated from umbilical cord blood and can be "mobilized" peripheral blood, making them readily available in relatively large quantities. While their ability to repopulate the entire hematopoietic system would already guarantee HSC a valuable place in regenerative medicine, the finding that hematopoietic chimerism can induce immunological tolerance to solid organs and correct autoimmune diseases has dramatically broadened their clinical utility. The demonstration that these cells, through a variety of mechanisms, can also promote repair/regeneration of non-hematopoietic tissues as diverse as liver, heart, and brain has further increased their clinical value. The goal of this review is to provide the reader with a brief glimpse into the remarkable potential HSC possess, and to highlight their tremendous value as therapeutics in regenerative medicine.

Sheep CD34+ amniotic fluid cells have hematopoietic potential and engraft after autologous in utero transplantation.

Unmatched allogeneic in utero stem cell transplantation (IUSCT) produces poor engraftment unless the fetus has congenital immunodeficiency, probably because of maternal and fetal immune responses to injected cells. We studied the functional hematopoietic potential of transduced green fluorescent protein (GFP+) sheep amniotic fluid (AF) stem cells, before and after autologous IUSCT. CD34+ cells were selected from first trimester sheep AF, transduced overnight, and injected intravenously into NOD-SCID-gamma (NSG) mice. At 3 months, primary recipient bone marrow (BM) was injected into secondary NSG recipients. GFP+ cells were detected in the hematopoietic organs and peripheral blood of primary and secondary recipients at 3 months. Autologous IUSCT (transduced GFP+CD34+AF) was performed in fetal sheep. Six months postnatally, lamb BM was injected into secondary NSG recipients. GFP+ cells were detected in the peripheral blood of primary and secondary recipients. This confirms the hematopoietic potential of AF stem cells supporting the concept of autologous IUSCT to treat congenital hematopoietic disease.

Temporal definition of haematopoietic stem cell niches in a large animal model of in utero stem cell transplantation.

The fetal sheep model has served as a biologically relevant and translational model to study in utero haematopoietic stem cell transplantation (IUHSCT), yet little is known about the ontogeny of the bone marrow (BM) niches in this model. Because the BMmicroenvironment plays a critical role in the outcome of haematopoietic engraftment, we have established the correlation between the fetal-sheep and fetal-human BM niche ontogeny, so that studies addressing the role of niche development at the time of IUHSCT could be accurately performed. Immunofluorescence confocal microscopic analysis of sheep fetal bone from gestational days (gd) 25-68 showed that the BM microenvironment commences development with formation of the vascular niche between 25 and 36 gd in sheep; correlating with the events at 10-11 gestational weeks (gw) in humans. Subsequently, between 45 and 51 gd in sheep (c. 14 gw in humans), the osteoblastic/endosteal niche started developing, the presence of CD34(+)  CD45(+) cells were promptly detected, and their number increased with gestational age. IUHSCT, performed in sheep at 45 and 65 gd, showed significant haematopoietic engraftment only at the later time point, indicating that a fully functional BM microenvironment improved engraftment. These studies show that sheep niche ontogeny closely parallels human, validating this model for investigating niche influence/manipulation in IUHSCT engraftment.

EphB2 isolates a human marrow stromal cell subpopulation with enhanced ability to contribute to the resident intestinal cellular pool.

To identify human bone marrow stromal cell (BMSC) subsets with enhanced ability to engraft/contribute to the resident intestinal cellular pool, we transplanted clonally derived BMSCs into fetal sheep. Analysis at 75 d post-transplantation showed 2 of the 6 clones engrafting the intestine at 4- to 5-fold higher levels (5.03±0.089 and 5.04±0.15%, respectively) than the other clones (P<0.01), correlating with the percentage of donor-derived Musashi-1(+) (12.01-14.17 vs. 1.2-3.8%; P<0.01) or leucine-rich repeat-containing G-protein coupled receptor 5 (Lgr5)(+) cells within the intestinal stem cell (ISC) region. Phenotypic and transcriptome analysis determined that the clones with enhanced intestinal contribution expressed high levels of Ephrin type B receptor 2 (EphB2). Intestinal explants demonstrated proliferation of the engrafted cells and ability to generate crypt-like structures in vitro still expressing EphB2. Additional transplants based on BMSC EphB2 expression demonstrated that, at 7 d post-transplant, the EphB2(high) BMSCs engrafted in the ISC region at levels of 2.1 ± 0.2%, while control EphB2(low) BMSCs engrafted at 0.3 ± 0.1% (P<0.01). Therefore we identified a marker for isolating and culturing an expandable subpopulation of BMSCs with enhanced intestinal homing and contribution to the ISC region.

Simulated Microgravity Impairs Human NK Cell Cytotoxic Activity Against Space Radiation-Relevant Leukemic Cells.

Natural killer (NK) cells are important effectors of the innate immune system. Unlike T cells, NK cells do not require antigen-priming, making them an important first-line of defense against malignant cells. Because of the potential for increased cancer risk as a result of astronaut exposure to space radiation, we performed studies to determine whether conditions of microgravity present during spaceflight affects the body's natural defenses against leukemogenesis. Human NK cells were cultured for 48 hours under normal gravity and simulated microgravity (sµG), and cytotoxicity against K-562 (CML) and MOLT-4 (T-ALL) cell lines was measured using standard methodology or under continuous conditions of sµG. Even this brief exposure to sµG markedly reduced NK cytotoxicity against both leukemic cells using standard assay procedures, and these deleterious effects were even more pronounced in continuous sµG. RNA-seq performed on NK cells from two healthy donors provided insight into the mechanism(s) by which sµG reduced cytotoxicity. Given our prior report that human HSC exposed to simulated space radiation gave rise to T-ALL in vivo , the reduced cytotoxicity against MOLT-4 is striking and raises the possibility that µG may add to astronaut risk of leukemogenesis during prolonged missions beyond LEO.

Evaluating sheep hemoglobins with MD simulations as an animal model for sickle cell disease.

Sickle cell disease (SCD) affects millions worldwide, yet there are few therapeutic options. To develop effective treatments, preclinical models that recapitulate human physiology and SCD pathophysiology are needed. SCD arises from a single Glu-to-Val substitution at position 6 in the ß subunit of hemoglobin (Hb), promoting Hb polymerization and subsequent disease. Sheep share important physiological and developmental characteristics with humans, including the same developmental pattern of fetal to adult Hb switching. Herein, we investigated whether introducing the SCD mutation into the sheep ß-globin locus would recapitulate SCD's complex pathophysiology by generating high quality SWISS-MODEL sheep Hb structures and performing MD simulations of normal/sickle human (huHbA/huHbS) and sheep (shHbB/shHbS) Hb, establishing how accurately shHbS mimics huHbS behavior. shHbS, like huHbS, remained stable with low RMSD, while huHbA and shHbB had higher and fluctuating RMSD. shHbB and shHbS also behaved identically to huHbA and huHbS with respect to ß2-Glu6 and ß1-Asp73 (ß1-Asn72 in sheep) solvent interactions. These data demonstrate that introducing the single SCD-causing Glu-to-Val substitution into sheep ß-globin causes alterations consistent with the Hb polymerization that drives RBC sickling, supporting the development of a SCD sheep model to pave the way for alternative cures for this debilitating, globally impactful disease.

Mesenchymal stem cells contribute to endogenous FVIII:c production.

Besides the liver, it has been difficult to identify which organ(s) and/or cellular component(s) contribute significantly to the production of human FVIII:c (FVIII). Thus far, only endothelial cells have been shown to constitute a robust extrahepatic source of FVIII, possibly explaining both the diverse presence of FVIII mRNA in the body, and the observed increase in FVIII levels during liver failure. Here, we investigate whether human mesenchymal stem cells (MSC), ubiquitously present in different organs, could also contribute to FVIII production. MSC isolated from human lung, liver, brain, and bone marrow expressed FVIII message as determined by quantitative-RT-PCR. Using an antibody specific for FVIII, confocal microscopy, and umbilical cord-derived endothelial cells (HUVEC) as a negative control, we demonstrated that, in MSC, FVIII protein was not stored in granules; rather, it localized to the perinuclear region. Furthermore, functional FVIII was detected in MSC supernatants and cell lysates by aPTT and chromogenic assays. These results demonstrate that MSC can contribute at low levels to the functional FVIII pool, and advance the understanding of the physiology of FVIII production and secretion.

Distinct contribution of human cord blood-derived endothelial colony forming cells to liver and gut in a fetal sheep model.

UNLABELLED: Although the vasculogenic potential of circulating and cord blood (CB)-derived endothelial colony-forming cells (ECFC) has been demonstrated in vitro and in vivo, little is known about the inherent biologic ability of these cells to home to different organs and contribute to tissue-specific cell populations. Here we used a fetal sheep model of in utero transplantation to investigate and compare the intrinsic ability of human CB-derived ECFC to migrate to the liver and to the intestine, and to define ECFC's intrinsic ability to integrate and contribute to the cytoarchitecture of these same organs. ECFCs were transplanted by an intraperitoneal or intrahepatic route (IH) into fetal sheep at concentrations ranging from 1.1-2.6 × 10(6) cells/fetus. Recipients were evaluated at 85 days posttransplant for donor (human) cells using flow cytometry and confocal microscopy. We found that, regardless of the route of injection, and despite the IH delivery of ECFC, the overall liver engraftment was low, but a significant percentage of cells were located in the perivascular regions and retained the expression of hallmark endothelial makers. By contrast, ECFC migrated preferentially to the intestinal crypt region and contributed significantly to the myofibroblast population. Furthermore, ECFC expressing CD133 and CD117 lodged in areas where endogenous cells expressed those same phenotypes. CONCLUSION: ECFC inherently constitute a potential source of cells for the treatment of intestinal diseases, but strategies to increase the numbers of ECFC persisting within the hepatic parenchyma are needed in order to enhance ECFC therapeutic potential for this organ.