C-Myb(+) erythro-myeloid progenitor-derived fetal monocytes give rise to adult tissue-resident macrophages.

Although classified as hematopoietic cells, tissue-resident macrophages (MFs) arise from embryonic precursors that seed the tissues prior to birth to generate a self-renewing population, which is maintained independently of adult hematopoiesis. Here we reveal the identity of these embryonic precursors using an in utero MF-depletion strategy and fate-mapping of yolk sac (YS) and fetal liver (FL) hematopoiesis. We show that YS MFs are the main precursors of microglia, while most other MFs derive from fetal monocytes (MOs). Both YS MFs and fetal MOs arise from erythro-myeloid progenitors (EMPs) generated in the YS. In the YS, EMPs gave rise to MFs without monocytic intermediates, while EMP seeding the FL upon the establishment of blood circulation acquired c-Myb expression and gave rise to fetal MOs that then seeded embryonic tissues and differentiated into MFs. Thus, adult tissue-resident MFs established from hematopoietic stem cell-independent embryonic precursors arise from two distinct developmental programs.

Recapitulative haematopoietic development of human pluripotent stem cells in the absence of exogenous haematopoietic cytokines.

To improve the recapitulative quality of human pluripotent stem cell (hPSC) differentiation, we removed exogenous haematopoietic cytokines from the defined differentiation system. Here, we show that endogenous stimuli and VEGF are sufficient to induce robust hPSC-derived haematopoiesis, intensive generation of haematopoietic progenitors, maturation of blood cells and the emergence of definitive precursor cells including those that phenotypically identical to early human embryonic haematopoietic stem cells (HSCs). Moreover, the cytokine-free system produces significantly higher numbers of haematopoietic progenitors compared to the published protocols. The removal of cytokines revealed a broad developmental potential of the early blood cells, stabilized the hPSC-derived definitive precursors and led to spontaneous activation of inflammatory signalling. Our cytokine-free protocol is simple, efficient, reproducible and applicable for embryonic stem cells (ESCs) and induced PSCs. The spectrum of recapitulative features of the novel protocol makes the cytokine-free differentiation a preferred model for studying the early human haematopoietic development.

MYB bi-allelic targeting abrogates primitive clonogenic progenitors while the emergence of primitive blood cells is not affected.

MYB is a key regulator of definitive hematopoiesis and it is dispensable for the development of primitive hematopoietic cells in vertebrates. To delineate definitive versus primitive hematopoiesis during differentiation of human embryonic stem cells, we have introduced reporters into the MYB locus and inactivated the gene by bi-allelic targeting. To recapitulate the early developmental events more adequately, the mutant and wild type human embryonic stem cell lines were differentiated in defined culture conditions without the addition of hematopoietic cytokines. The differentiation of the reporter cell lines demonstrated that MYB is specifically expressed throughout emerging hematopoietic cell populations. Here we show that the disruption of the MYB gene leads to severe defects in the development and proliferation of primitive hematopoietic progenitors while the emergence of primitive blood cells is not affected. We also provide evidence that MYB is essential for neutrophil and T cell development and the upregulation of innate immunity genes during hematopoietic differentiation. Our results suggest that the endothelial origin of primitive blood cells is direct and does not include the intermediate step of primitive hematopoietic progenitors.

Development of an ovine model of occlusive arterial injury for the evaluation of endovascular interventions.

Endovascular intervention for occlusive arterial trauma is becoming more common in clinical practice. The aim of this study is to present an ovine model of extremity arterial injury for use in future endovascular translational research. Animals under general anesthesia had their left superficial femoral artery exposed, which was bluntly injured over a 2-cm section using a hemostat and injection of air. Occlusion was confirmed on angiography and the flow characteristics measured by ultrasonography. Of five animals enrolled, four occluding lesions were created successfully. Post injury, there was a significant reduction in the median (interquartile range) systolic velocity (cm/sec) on the left (injury) compared to the right (control) side (3.5 (0-16.5) vs. 29 (23.8-43.3); p < 0.001). The ovine superficial femoral artery can be used to consistently produce an occlusive lesion that is suitable for use in the future evaluation of endovascular trauma interventions.

Deconvoluting the ontogeny of hematopoietic stem cells.

Two different models describe the development of definitive hematopoiesis and hematopoietic stem cells (HSCs). In one of these, the visceral yolk sac serves as a starting point of relatively lengthy developmental process culminating in the fetal liver hematopoiesis. In another, the origin of adult hematopoiesis is split between the yolk sac and the dorsal aorta, which has a peculiar capacity to generate definitive HSCs. Despite a large amount of experimental data consistent with the latter view, it becomes increasingly unsustainable in the light of recent cell tracing studies. Moreover, analysis of the published studies supporting the aorta-centered version uncovers significant caveats in standard experimental approach and argumentation. As a result, the theory cannot offer feasible cellular mechanisms of the HSC emergence. This review summarizes key efforts to discern the developmental pathway of the adult-type HSCs and attempts to put forward a hypothesis on the inflammatory mechanisms of hematopoietic ontogenesis.

Early ontogenic origin of the hematopoietic stem cell lineage.

Several lines of evidence suggest that the adult hematopoietic system has multiple developmental origins, but the ontogenic relationship between nascent hematopoietic populations under this scheme is poorly understood. In an alternative theory, the earliest definitive blood precursors arise from a single anatomical location, which constitutes the cellular source for subsequent hematopoietic populations. To deconvolute hematopoietic ontogeny, we designed an embryo-rescue system in which the key hematopoietic factor Runx1 is reactivated in Runx1-null conceptuses at specific developmental stages. Using complementary in vivo and ex vivo approaches, we provide evidence that definitive hematopoiesis and adult-type hematopoietic stem cells originate predominantly in the nascent extraembryonic mesoderm. Our data also suggest that other anatomical sites that have been proposed to be sources of the definitive hematopoietic hierarchy are unlikely to play a substantial role in de novo blood generation.

Cell tracing shows the contribution of the yolk sac to adult haematopoiesis.

The first haematopoietic stem cells (HSCs) appear in the aorta-gonad-mesonephros (AGM) region, major vitelline and umbilical vessels, and placenta; however, whether they arise locally or from immigrant yolk sac precursor cells remains unclear. This issue is best addressed by measuring cell-lineage relationships rather than cell potentials. To undertake long-term in vivo tracing of yolk sac cells, we designed a non-invasive pulse-labelling system based on Cre/loxP recombination. Here we show that in Runx1(+/-) (runt-related transcription factor 1) heterozygous mice, yolk sac cells expressing Runx1 at embryonic day 7.5 develop into fetal lymphoid progenitors and adult HSCs. During mid-gestation the labelled (embryonic day 7.5) yolk sac cells colonize the umbilical cord, the AGM region and subsequently the embryonic liver. This raises the possibility that some HSCs associated with major embryonic vasculature are derived from yolk sac precursors. We observed virtually no contribution of the labelled cells towards the yolk sac vasculature, indicating early segregation of endothelial and haematopoietic lineages.

Genotypic Characterization of a Chinese Family with Osteogenesis Imperfecta and Generation of Disease-Specific Induced Pluripotent Stem Cells.

BACKGROUND: Osteogenesis imperfecta (OI) is a rare genetic disorder characterized by recurring bone fractures. Some OI patients have other clinical manifestations such as growth retardation, dental abnormalities, blue sclera, and hearing loss. The relationship between the phenotype and genotype of OI is indistinct, and there is no cure for OI. Therefore, an appropriate disease model is urgently needed to understand the pathophysiology of OI. Induced pluripotent stem cells (iPSCs) are capable of developing into three germ layers and have the same genetic background as the donor cells they were derived from; thus, they are an appropriate disease model. METHODS: Blood samples collected from the proband and her affected children and one unaffected child were used forgenotyping by whole genome sequencing. A patient-specific iPSC line and a healthy donor iPSC line were generated by reprogramming peripheral blood mononuclear cells with episomal plasmids containing seven transcription factors, namely, OCT4, SOX2, NANOG, LIN28, cMYC, KLF4, and SV40LT. RESULTS: The proband and her two affected children were homozygous for a mutation in collagen type I alpha 1 exon 10, c.725G>T, predicting a p.G242V substitution. A patient-specific iPSC line and a healthy donor iPSC line were generated and characterized in terms of their human embryonic stem cell-like morphology, expression of pluripotency markers, and the ability to differentiate into cells of three germ layers. CONCLUSIONS: Here, we report the phenotyping and iPSC disease modeling of an OI family. The detailed phenotyping of the OI family and establishment of iPSCs from an OI patient and healthy family member will provide a powerful tool to evaluate the pathophysiology of OI and develop targeted therapies.

Hepatocyte growth factor-Met signaling is required for Runx1 extinction and peptidergic differentiation in primary nociceptive neurons.

Nociceptors in peripheral ganglia display a remarkable functional heterogeneity. They can be divided into the following two major classes: peptidergic and nonpeptidergic neurons. Although RUNX1 has been shown to play a pivotal role in the specification of nonpeptidergic neurons, the mechanisms driving peptidergic differentiation remain elusive. Here, we show that hepatocyte growth factor (HGF)-Met signaling acts synergistically with nerve growth factor-tyrosine kinase receptor A to promote peptidergic identity in a subset of prospective nociceptors. We provide in vivo evidence that a population of peptidergic neurons, derived from the RUNX1 lineage, require Met activity for the proper extinction of Runx1 and optimal activation of CGRP (calcitonin gene-related peptide). Moreover, we show that RUNX1 in turn represses Met expression in nonpeptidergic neurons, revealing a bidirectional cross talk between Met and RUNX1. Together, our novel findings support a model in which peptidergic versus nonpeptidergic specification depends on a balance between HGF-Met signaling and Runx1 extinction/maintenance.

Ultrasound-guided versus blind vascular access followed by REBOA on board of a medical helicopter in a hemorrhagic ovine model.

BACKGROUND: The aim of this study is to evaluate the feasibility of en-route resuscitative endovascular balloon occlusion of the aorta (REBOA) on board of a helicopter. METHODS: Six sedated male sheep (weighing 42-54 kg) underwent a controlled hemorrhage until the systolic blood pressure (BP) dropped to <90 mmHg, and were placed into a low capacity Eurocopter AS-350 (France). During the 30-minutes normal flight, every animal underwent blind (left side) and ultrasound-guided (US) (right side) vascular access (VA) to the femoral artery followed by REBOA: the first catheter (Rescue balloon, Japan) - into Zone I, the second one (MIT, Russia) - Zone III. In case of blind VA failure, an alternate US-puncture was attempted. Six experienced flight anesthetists were enrolled into the study. Vascular access and REBOA catheter placement (confirmed by X-Ray later) success rate and timing were recorded. RESULTS: Among six blind punctures one was successful, 2/6 - were into the vein, 3/6 - completely failed and switched to US-punctures (making total number of US-punctures nine). Eight out of nine US-punctures were successful. However, correct wire insertion and sheath placement was performed in 1/6 animal in the 'blind' group and only in 6/9 animals in the 'US' group. It took a median of 65 seconds (range 5-260) for US-puncture and a median of 4 minutes to get the sheath in. Among the 9 VAs, there were 2 REBOA failures (1 ruptured balloon [MIT] and 1 mistaken vena cava placement primarily recognized by a sudden drop of BP and later confirmed by X-Ray). Five out of seven balloons were placed in a desired intra-aortic position: 4/5 in Zone I and 1/2 - in Zone III. A median time for a successful REBOA procedure was 5.0 (range 2.5-10.0) minutes (1 min after sheath placement). CONCLUSION: Our study demonstrates the potential feasibility of the en-route REBOA which can be performed within 5 minutes. Ultrasound-guidance is critically important to achieve en-route VA.

Establishment of an induced pluripotent stem cell line SPHi001-A from a systemic lupus erythematosus patient combined with preeclampsia and psoriasis.

Systemic lupus erythematosus (SLE) is a heterogeneous, autoimmune disease that can affect multiple organs and systems such as skin, joints, kidneys, hematologic system or central nervous system. Women of childbearing age are the predominate population affected by SLE. In this study, we generated an iPS cell line from a 30-year-old female who was pregnant with a gestational age of 27 weeks and diagnosed with severe preeclampsia, SLE and psoriasis. This patient-specific iPSC line will be useful to create the specific disease model of systemic lupus erythematosus to elucidate the pathological mechanisms and develop drug screening.

TALEN-mediated biallelic inactivation of MYB in human embryonic stem cell lines WAe001-A-45 and WAe001-A-46.

MYB/c-MYB is a proto-oncogene encoding a helix-turn-helix transcription factor that plays a critical role in controlling proliferation and multilineage differentiation of hematopoietic progenitor and stem cells. Deregulation of MYB expression is associated with several types of leukemias and lymphomas. In an attempt to explore the role of the gene in the early human hematopoiesis, we have achieved bi-allelic targeting of MYB in human embryonic stem cells (hESCs) by TALEN-mediated homologous recombination. Furthermore, the gene targeting introduced eYFP Venus reporter gene into the MYB locus to delineate the expression pattern of MYB. The resulting two cell lines, WAe001-A-45 and WAe001-A-46, passed the standard assays for human pluripotent stem cells. Hematopoietic differentiation of these cell lines provides a model to study the role of MYB in human hematopoietic development.

Battlefield Extracorporeal Cardiopulmonary Resuscitation for Out-of-Hospital Cardiac Arrest: A Feasibility Study During Military Exercises.

PURPOSE: To evaluate the feasibility of prehospital extracorporeal cardiopulmonary resuscitation (E-CPR) in the military exercise setting. METHODS: Three 40kg Sus scrofa (wild swine) underwent controlled 35% blood loss and administration of potassium chloride to achieve cardiac arrest (CA). During CPR, initiated 1 minute after CA, the animals were transported to Role 1. Femoral vessels were cannulated, followed by E-CPR using a portable perfusion device. Crystalloid and blood transfusions were initiated, followed by tactical evacuation to Role 2 and 4-hour observation. RESULTS: All animals developed sustained asystole. Chest compressions supported effective but gradually deteriorating blood circulation. Two animals underwent successful E-CPR, with restoration of perfusion pressure to 80mmHg (70-90mmHg) 25 and 23 minutes after the induction of CA. After transportation to Role 2, one animal developed abdominal compartment syndrome as a result of extensive (9L) fluid replacement. The other animal received a lower volume of crystalloids (4L), and no complications occurred. In the third animal, multiple attempts to cannulate arteries were unsuccessful because of spasm and hypotension. Open aortic cannulation enabled the circuit to commence. No return of spontaneous circulation was ultimately achieved in either of the remaining animals. CONCLUSION: Our study demonstrates both the potential feasibility of battlefield E-CPR and the evolving capability in the care of severey injured combat casualties.

Generation of RUNX1-null reporter human embryonic stem cell line GIBHe008-A.

RUNX1/AML1/CBFA2 (runt-related transcription factor 1/acute myeloid leukemia 1 protein/core-binding factor subunit alpha-2), is a transcription factor that plays a critical role in the development of normal hematopoiesis. RUNX1 is also essential for the development of immune cells and sensory neurons. Chromosomal translocations involving the gene have been associated with several types of leukemia. To investigate the role of RUNX1 in human hematopoietic development we generated RUNX1-null human embryonic stem cell reporter line GIBHe008-A by TALEN mediated homologous recombination. This cell line GIBHe008-A was subjected to detailed characterization by standard assays for human pluripotent stem cells. It provides an ideal model to study the role of RUNX1 in the hESC-derived developmental models.

PEGylated doped- and undoped-TiO2 nanoparticles for photodynamic Therapy of cancers.

Titanium dioxide has been widely known for its phototoxicity in the environmental context, but little is known for its use in the photodynamic therapy of cancers. Previous studides have shown the hazardous effects of undoped-titanium dioxide nanoparticles (undoped-TiO2 NPs) in the ecosystem; however, it remains to explore the effect of polyethylene glycol (PEG) conjugation and doping of metal and non-metal on the photodynamic activity of TiO2. Here we report the synthesis, characterizations, and applications of doped- and undoped-TiO2 NPs stabilized by PEG in the photodynamic therapy of cancers. Our results demonstrate that in vitro PEG-NPs significantly reduced the survival of human cervical cancer cells (HeLa) upon solar and ultraviolet (UV) radiations. We found that doping of the metal (cobalt) and non-metal (nitrogen) onto TiO2 nanocrystals enhanced the photoactivation of doped-TiO2 NPs in the visible/near infrared (Vis/NIR) region, but these nanocrystals were revealed by cytotoxicity assays to be less potent in killing cancer cells compared to PEGylated undoped-TiO2. The significant photodynamic effect was shown by PEGylated undoped-TiO2 synthesized through the sol-gel method with 75% killing of HeLa cells at 5.5 µg/mL concentrations in exposure to UV or sunlight radiations. In vitro cytotoxicity was measured by Sulforhodamine B (SRB) and 3-(4, 5-Dimethylthiazol-2-Yl)-2,5-Diphenyltetrazolium Bromide (MTT) assays after irradiations with IR, UV, and sunlight for 15-30 minutes (min). All the synthesized NPs were characterized by XRD, AFM, SEM, EDX and DRS chemical analysis. Taken together, our data demonstrate that water-soluble PEGylated TiO2 NPs maybe a good candidate for the photodynamic therapy of cervical cancer cells. Our data propose that the use of PEG surfactant can enhance the potency of already available photochemical therpeutic agents.

Defining degree of aortic occlusion for partial-REBOA: A computed tomography study on large animals.

INTRODUCTION: Partial resuscitative endovascular balloon occlusion of the aorta (P-REBOA) is a modified REBOA technique designed to help ameliorate ischemia-reperfusion injury. The balloon is partially deflated, allowing a proportion of aortic flow distal to the balloon. The aim of this study is to use an ovine model of haemorrhagic shock to correlate the degree of occlusion to several hemodynamic indices. MATERIALS AND METHODS: Six sheep weighing 35-46 kg underwent a controlled venous haemorrhage inside a CT scanner until the systolic arterial pressure (AP) dropped to <90 mmHg. A balloon positioned in an aortic zone I was incrementally filled with 1 mL of saline, with serial measurement of the proximal (carotid artery) and distal (femoral artery) mean APs (MAP) and intra-balloon pressure (IBP), along with CT imaging, following each inflation, until full occlusion was achieved. RESULTS: A diameter of the aorta at zone I was 16.0 (15.7-17.2) mm, with a cross-sectional area of 212 (194-233) mm2. Median volume of saline injected into the balloon until total occlusion was 7.0 (6.3-8.5) mL. During gradual balloon inflation, proximal MAP increased and distal MAP decreased proportionate to the degree of occlusion, in a linear fashion (proximal: r2 = 0.85, p < 0.001; distal: r2 = 0.95, p < 0.001). The femoral/carotid (F/C) pressure gradient also demonstrated a linear trend (r2 = 0.90, p < 0.001). The relationship between percentage occlusion and IBP was sigmoid. MAP values became significantly different at 40-49% occlusion and more (p < 0.01). Furthermore, a drop in the distal pulse pressure from 7.0 (5.5-16.5) to 2.0 (1.5-5.0) mmHg was observed at 80% occlusion. All animals had femoral pulse pressure <5 mmHg at 80% of occlusion and more, which also coincided with the observed loss of pulsatility of the femoral wave-form. CONCLUSION: Serial CT angiography at an ovine model of haemorrhagic shock demonstrates a correlation between the femoral MAP, F/C pressure gradient and degree of zone I P-REBOA during the staged partial aortic occlusion. These parameters should be considered potential parameters to define the degree of P-REBOA during animal research and clinical practice.

Field and en route resuscitative endovascular occlusion of the aorta: A feasible military reality?

BACKGROUND: Severe noncompressible torso hemorrhage remains a leading cause of potentially preventable death in modern military conflicts. Resuscitative endovascular occlusion of the aorta (REBOA) has demonstrated potential as an effective adjunct to the treatment of noncompressible torso hemorrhage in the civilian early hospital and even prehospital settings-but the application of this technology for military prehospital use has not been well described. We aimed to assess the feasibility of both field and en route prehospital REBOA in the military exercise setting, simulating a modern armed conflict. METHODS: Two adult male Sus Scrofa underwent simulated junctional combat injury in the context of a planned military training exercise. Both underwent zone I REBOA in conjunction with standard tactical combat casualty care interventions-one during point of injury care and the other during en route flight care. Animals were sequentially evacuated to two separate forward surgical teams by rotary wing platform where the balloon position was confirmed by chest x-ray. Animals then underwent different damage control thoracic and abdominal procedures before euthanasia. RESULTS: The first swine underwent immediate successful REBOA at the point of injury 7 minutes and 30 seconds after the injury. It required 6 minutes total from initiation of procedure to effective aortic occlusion. Total occlusion time was 60 minutes. In the second animal, the REBOA placement procedure was initiated immediately after take off (17 minutes and 40 seconds after the injury). Although the movements and vibration of flight were not significant impediments, we only succeeded to put a 6-French (Fr) sheath into a femoral artery during the 14 minutes flight due to lighting and visualization challenges. After the sheath had been upsized in the forward surgical team, the REBOA catheter was primarily placed in zone I followed by its replacement to zone III. Both animals survived to study completion and the termination of training. No complications were observed in either animal. CONCLUSION: Our study demonstrates the potential feasibility of REBOA for use during tactical field and en route (flight) care of combat casualties. Further study is needed to determine the optimal training and utilization protocols required to facilitate the effective incorporation of REBOA into military prehospital care capabilities.

Contributions of the surgeon Nikolai Korotkov (1874-1920) to the management of extremity vascular injury.

The Russian military surgeon Nikolai Korotkov is known worldwide, mainly among internists and cardiovascular specialists, as the discoverer of the auscultatory method of measuring arterial blood pressure in 1905. This article reveals him as one of the first military vascular surgeons to carefully investigate, analyze, and register cases of vascular injury during his voluntarily trips to the Russian Far East in 1900 to 1901 and the Russo-Japanese War of 1904 to 1905. Examining 44 patients with extremity arterial and arterial-venous pseudoaneurysms following war-related injury, he routinely performed a measure termed the "arterial pressure index" using "Korotkov sounds." This pioneering approach to assessing extremity perfusion was the precursor to the modern-day ankle-brachial and injured extremity indices, and it initiated the quantitative assessment of the compensatory ability of the vascular system to restore circulation following axial artery ligation. Because of high thrombosis rates following direct vessel repair during his day, he proposed use of pharmacologic substances such as digitalis and amyl nitrite to improve extremity perfusion. As evidence of his innovative nature, Korotkov even proposed the use of "oxygenated nutrient solutions" in the future to improve extremity circulation. More than 100 years after his work, as continuous wave Doppler ultrasound, contrast angiography, and computed tomography are ubiquitous as diagnostic tools, the practice of surgery would be well served to recall Korotkov's foundational work and the rule of thumb for any physician: examine the patient.

The military surgical legacy of Vladimir Oppel (1872-1932).

Vladimir A. Oppel (1872-1932) was a forefather of military trauma systems. As a surgeon in the Russian Army in World War I, Oppel experienced the challenges and inefficiencies associated with caring for large numbers of combat wounded, the inefficiencies he observed leading to unacceptable morbidity and mortality. As a consequence, Oppel envisioned a coordinated sequence of surgical care on the battlefield and developed the concept of "targeted evacuation." In his work, Oppel was among the first to propose the "right operation for the right patient at the right location at the right time." Central to Oppel's precepts were (1) the forward positioning of surgical care close to the point of injury, (2) the development of a reserve of proficient and deployable military surgeons, and (3) the provision of specialized surgery to optimize survival and reduce morbidity. Oppel's teachings were validated during World War II in the performance of the Soviet casualty evacuation system and in all modern wars modern since. Today, nearly 100 years after the work of Vladimir Oppel, the benefits of a coordinated or "targeted" trauma system, working to optimize survival after trauma, are well recognized around the world.

Return to the hematopoietic stem cell origin.

Studying embryonic hematopoiesis is complicated by diversity of its locations in the constantly changing anatomy and by the mobility of blood cell precursors. Embryonic hematopoietic progenitors are identified in traditional in vivo and in vitro cell potential assays. Profound epigenetic plasticity of mammalian embryonic cells combined with significant inductive capacity of the potential assays suggest that our understanding of hematopoietic ontogenesis is substantially distorted. Non-invasive in vivo cell tracing methodology offers a better insight into complex processes of blood cell specification. In contrast to the widely accepted view based on the cell potential assays, the genetic tracing approach identified the yolk sac as the source of adult hematopoietic stem cell lineage. Realistic knowledge of the blood origin is critical for safe and efficient recapitulation of hematopoietic development in culture.