Karina Liuba

Identification of Flt3 + Lympho-Myeloid Stem Cells Lacking Erythro-Megakaryocytic Potential: A Revised Road Map for Adult Blood Lineage Commitment

All blood cell lineages derive from a common hematopoietic stem cell (HSC). The current model implicates that the first lineage commitment step of adult pluripotent HSCs results in a strict separation into common lymphoid and common myeloid precursors. We present evidence for a population of cells which, although sustaining a high proliferative and combined lympho-myeloid differentiation potential, have lost the ability to adopt erythroid and megakaryocyte lineage fates. Cells in the Lin-Sca-1+c-kit+ HSC compartment coexpressing high levels of the tyrosine kinase receptor Flt3 sustain granulocyte, monocyte, and B and T cell potentials but in contrast to Lin-Sca-1+c-kit+Flt3- HSCs fail to produce significant erythroid and megakaryocytic progeny. This distinct lineage restriction site is accompanied by downregulation of genes for regulators of erythroid and megakaryocyte development. In agreement with representing a lymphoid primed progenitor, Lin-Sca-1+c-kit+CD34+Flt3+ cells display upregulated IL-7 receptor gene expression. Based on these observations, we propose a revised road map for adult blood lineage development.

Kit Regulates Maintenance of Quiescent Hematopoietic Stem Cells

Hematopoietic stem cell (HSC) numbers are tightly regulated and maintained in postnatal hematopoiesis. Extensive studies have supported a role of the cytokine tyrosine kinase receptor Kit in sustaining cycling HSCs when competing with wild-type HSCs posttransplantation, but not in maintenance of quiescent HSCs in steady state adult bone marrow. In this study, we investigated HSC regulation in White Spotting 41 (KitW41/W41) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult KitW41/W41 mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin−Sca-1+Kithigh (LSK)CD34−Flt3− long-term HSCs by 12 wk of age, whereas LSKCD34+Flt3− short-term HSCs and LSKCD34+Flt3+ multipotent progenitors were less affected. Whereas homing and initial reconstitution of KitW41/W41 bone marrow cells in myeloablated recipients were close to normal, self-renewing KitW41/W41 HSCs were progressively depleted in not only competitive but also noncompetitive transplantation assays. Overexpression of the anti-apoptotic regulator BCL-2 partially rescued the posttransplantation KitW41/W41 HSC deficiency, suggesting that Kit might at least in the posttransplantation setting in part sustain HSC numbers by promoting HSC survival. Most notably, accelerated in vivo BrdU incorporation and cell cycle kinetics implicated a previously unrecognized role of Kit in maintaining quiescent HSCs in steady state adult hematopoiesis.

Myeloid and lymphoid contribution to non-haematopoietic lineages through irradiation-induced heterotypic cell fusion

Recent studies have suggested that regeneration of non-haematopoietic cell lineages can occur through heterotypic cell fusion with haematopoietic cells of the myeloid lineage. Here we show that lymphocytes also form heterotypic-fusion hybrids with cardiomyocytes, skeletal muscle, hepatocytes and Purkinje neurons. However, through lineage fate-mapping we demonstrate that such in vivo fusion of lymphoid and myeloid blood cells does not occur to an appreciable extent in steady-state adult tissues or during normal development. Rather, fusion of blood cells with different non-haematopoietic cell types is induced by organ-specific injuries or whole-body irradiation, which has been used in previous studies to condition recipients of bone marrow transplants. Our findings demonstrate that blood cells of the lymphoid and myeloid lineages contribute to various non-haematopoietic tissues by forming rare fusion hybrids, but almost exclusively in response to injuries or inflammation.

Polyclonal T-cell reconstitution of X-SCID recipients after in utero transplantation of lymphoid-primed multipotent progenitors

Although successful in utero hematopoietic cell transplantation (IUHCT) of X-linked severe combined immune deficiency (X-SCID) with enriched stem and progenitor cells was achieved more than a decade ago, it remains applied only in rare cases. Although this in part reflects that postnatal transplantations have overall given good results, there are no direct comparisons between IUHCT and postnatal transplantations of X-SCID. The proposed tolerance of the fetal immune system to foreign human leukocyte antigen early in gestation, a main rationale behind IUHCT, has recently been challenged by evidence for a considerable immune barrier against in utero transplanted allogeneic bone marrow cells. Consequently, there is need for further exploring the application of purified stem and progenitor cells to overcome this barrier also in IUHCT. Herein, we demonstrate in a congenic setting that recently identified lymphoid-primed multipotent progenitors are superior to hematopoietic stem cells in providing rapid lymphoid reconstitution after IUHCT of X-SCID recipients, and sustain in the long-term B cells, polyclonal T cells, as well as short-lived B-cell progenitors and thymic T-cell precursors. We further provide evidence for IUHCT of hematopoietic stem cells giving superior B- and T-cell reconstitution in fetal X-SCID recipients compared with neonatal and adolescent recipients.

TSLP-mediated fetal B lymphopoiesis?

To the editor: In the August 2003 issue of Nature Immunology1, as well as in later studies2, Voβhenrich et al. presented experimental data on the cytokine requirements of developing B cells, which led them to conclude that “TSLP [thymic stromal lymphopoietin] is the factor responsible for most of the fetal and perinatal B cell production that takes place when the IL-7–γc [interleukin 7–common γ-chain] signaling pathway is disrupted.”1 Although the data reported were technically sound and compatible with such a conclusion, the authors did not provide direct evidence to support (or exclude) the idea of a critical function for TSLP in IL-7-independent fetal B lymphopoiesis. The conclusions of Voβhenrich et al. were based on the demonstration that B lymphopoiesis was much more affected (tenfold more) in mice deficient in IL-7 receptor α-chain, essential for IL-7 as well as TSLP signaling, than in mice deficient in the common γ-chain (γc), required for IL-7 but not TSLP-mediated signaling3,4. However, these data could at best be considered strong indirect support for the idea of TSLP as the main cytokine driving IL-7-independent fetal B lymphopoiesis, as there could be other reasons for a difference in the phenotypes of γcdeficient mice and those deficient in the IL-7 receptor α-chain. Furthermore, Voβhenrich et al. used bone marrow of mice 4–12 weeks of age, not fetal liver, for their comparative in vivo analysis of B lymphopoiesis in these mice1,2. Instead, the extrapolation to the idea that TSLP is key to the fetal stages of B lymphopoiesis was based on the finding that fetal but not adult pro–B cells were responsive to TSLP in vitro1,2. In contrast, a lack of an important function for TSLP in adult B lymphopoiesis has been indicated by studies of TSLP receptor–deficient (Tpte2–/–) mice5,6. As fetal lymphopoiesis had not been examined in singly deficient Tslp–/– or Tpte2–/– mice, we investigated B lymphopoiesis in the livers of Tpte2–/– mice at embryonic day 17.5 but found no deficiency in Tpte2–/– fetuses at any stage of B cell development (Fig. 1a and Supplementary Fig. 1 online). Furthermore, when comparing B lymphopoiesis in the fetal livers of Il7–/– and Il7–/–Tpte2–/– mice, we obtained no evidence for substantial involvement of TSLP in IL-7independent regulation of fetal pro–B cells or pre–B cells, whereas we noted a slight additional reduction in the number of immature B cells in Il7–/–Tpte2–/– fetuses relative to that in Il7–/– fetuses (Fig. 1b and Supplementary Fig. 1). Thus, although Voβhenrich et al. provided compelling evidence that fetal pro–B cells are highly responsive to TSLP1, our studies of Tpte2–/– and Il7–/–Tpte2–/– fetuses fail to support their claim that TSLP is the most important cytokine promoting IL-7independent fetal B lymphopoiesis. Instead, although Voβhenrich et al. also concluded that “Flk-2 is involved, but TSLP is the main factor driving IL-7-independent fetal and perinatal lymphopoiesis,”1 we have done additional studies of mice deficient in the cytokine Flt3L (also called Flk-2 ligand) and IL-7 (Flt3l–/–Il7–/– mice) and of Flt3l–/–Tpte2–/– mice and have found that the reported complete loss of B-1 as well as B-2 B lymphopoiesis in Flt3l–/–Il7r–/– mice7 and Flk2–/–Il7r–/– mice1 is entirely due to the simultaneous loss of function of IL-7 and Flt3L (C.T.J. and S.E.W.J., unpublished observations). Collectively, our findings suggest that Flt3L rather than TSLP is the key regulator of IL-7-independent B lymphopoiesis and that intact TSLP function is insufficient to restore any detectable B lymphopoiesis in the absence of these two critical regulators of B cell progenitors.

Perinatal adaptive response of the adrenal and carotid blood flow in sheep fetuses subjected to total cord occlusion

OBJECTIVE To investigate the perinatal adaptive response of the adrenal blood flow/adrenal fractional moving blood volume (AFMBV) and carotid blood flow (CBF), in sheep fetuses subjected to severe acute intrauterine hypoxia/asphyxia induced by total cord occlusion. METHODS Adrenal blood flow velocity, AFMBV and CBF were measured in 13 exteriorized fetal sheep; eight of them underwent total umbilical cord occlusion to induce severe acute hypoxia/asphyxia. Five lambs were used as sham controls. Middle adrenal artery pulsatility index (MAAPI) and mean velocity (MAAMV) were recorded with pulsed Doppler ultrasound. AFMBV was estimated using power Doppler ultrasound. CBF was recorded with a transonic flowmeter. In the neonatal period, after resuscitation all lambs were followed for a 4-hour period and AFMBV and CBF were recorded. Mean arterial blood pressure (MABP) and fetal heart rate were recorded continuously. Arterial cortisol levels were measured at the beginning and at the end of the fetal and neonatal periods. RESULTS Following the total cord occlusion, there was a significant reduction in the CBF, MABP, and heart rate and adrenal flow/AFMBV after 2, 4 and 5 min, respectively. Cortisol levels in the asphyctic lambs at the end of the cord occlusion were significantly lower than those in controls. After resuscitation, the asphyctic lambs showed increased AFMBV and cortisol levels, and reduced MABP as compared to control lambs. No differences were found in CBF, MAAPI and MAAMV. Thereafter, no differences were observed between the two groups in any of the studied parameters. At the end of the cord occlusion period, there was a significant correlation between AFMBV and MABP (r=0.69), between AFMBV and CBF (r =0.65) and between CBF and MABP (r=0.89). CONCLUSION During severe acute intrauterine hypoxia, the fetal lamb is able to maintain the blood flow to the brain and the adrenal gland for 3-5 min. Changes in the AFMBV and the CBF were highly correlated to the changes in MABP. Adrenal FMBV and cortisol levels were higher in lamb neonates exposed to severe intrauterine asphyxia.

Blood‐flow streams in the fetal inferior vena cava: experimental animal study using ultrasound contrast agent

During fetal life, oxygenated blood from the placenta and deoxygenated blood from the lower part of the body travel together in the inferior vena cava (IVC) before reaching the heart. Only a few reports have provided evidence that these two blood-flow streams do not mix, or mix only in a limited way, before entering the right atrium1,2. In this study we aimed to use an ultrasound contrast agent (UCA) as a tracer to characterize these two blood-flow streams in the fetal IVC in an experimental fetal lamb model. The study was performed in 13 pregnant ewes of mixed breed at 136 days of gestation (term = 140 days). Under isoflurane anesthesia and mechanical ventilation, a midline laparotomy and hysterotomy were performed. One catheter (diameter = 0.4 mm) was placed in the fetal tibial vein and advanced until it reached the IVC. Another catheter was placed in one of the umbilical veins and advanced until it reached the origin of the ductus venosus (DV). The uterus was closed and the catheters were flushed with saline and heparin solution. Umbilical vein blood samples of 0.3 mL were obtained for acid–base evaluation. The UCA Levovist (99.9% galactose and 0.1% palmitic acid; 400 mg/mL) was infused at a rate of 1 mL/min during periods of 30 seconds. Ultrasound recordings were performed with an HDI-5000 ultrasound system (Philips, Bothell, WA, USA) and a linear 12–5MHz transducer. Injections of UCA were first performed in the tibial vein; the ultrasound probe was located on the uterine wall close to the fetal thorax and orientated either for obtaining a longitudinal view with complete visualization of the IVC, or in cross-sectional planes either side of the junction with the DV. UCA was observed in the IVC 3–5 seconds after starting the injection. When injection of the UCA was stopped, a period of 5 min was allotted to allow microbubbles to clear from the circulation. Before each subsequent UCA injection, an ultrasound examination was performed to confirm that the UCA had been cleared from the circulatory system. For umbilical vein UCA injections, two planes were recorded: longitudinal (before entering the heart) and cross-sectional (cranial to the junction with the DV). All recordings from the tibial and umbilical veins were performed twice. The total experimental time was 60–65 min. For each recording, a sequence containing at least 10 consecutive images was analyzed. Each image was evaluated using Image J software (public domain: http://imagej.nih.gov/ij; National Institutes of Health, Bethesda, MD, USA). A mean of 20 images (10 per Ultrasound contrast agent in the inferior vena cava

Operator auditory perception and spectral quantification of umbilical artery Doppler ultrasound signals.

Objective An experienced sonographer can by listening to the Doppler audio signals perceive various timbres that distinguish different types of umbilical artery flow despite an unchanged pulsatility index (PI). Our aim was to develop an objective measure of the Doppler audio signals recorded from fetoplacental circulation in a sheep model. Methods Various degrees of pathological flow velocity waveforms in the umbilical artery, similar to those in human complicated pregnancies, were induced by microsphere embolization of the placental bed (embolization model, 7 lamb fetuses, 370 Doppler recordings) or by fetal hemodilution (anemia model, 4 lamb fetuses, 184 recordings). A subjective 11-step operator auditory scale (OAS) was related to conventional Doppler parameters, PI and time average mean velocity (TAM), and to sound frequency analysis of Doppler signals (sound frequency with the maximum energy content [MAXpeak] and frequency band at maximum level minus 15 dB [MAXpeak-15 dB] over several heart cycles). Results We found a negative correlation between the OAS and PI: median Rho −0.73 (range −0.35– −0.94) and −0.68 (range −0.57– −0.78) in the two lamb models, respectively. There was a positive correlation between OAS and TAM in both models: median Rho 0.80 (range 0.58–0.95) and 0.90 (range 0.78–0.95), respectively. A strong correlation was found between TAM and the results of sound spectrum analysis; in the embolization model the median r was 0.91 (range 0.88–0.97) for MAXpeak and 0.91 (range 0.82–0.98) for MAXpeak-15 dB. In the anemia model, the corresponding values were 0.92 (range 0.78–0.96) and 0.96 (range 0.89–0.98), respectively. Conclusion Audio-spectrum analysis reflects the subjective perception of Doppler sound signals in the umbilical artery and has a strong correlation to TAM-velocity. This information might be of importance for clinical management of complicated pregnancies as an addition to conventional Doppler parameters.

OP03.04: Distribution of central venous blood flow in the fetal lamb examined with contrast‐enhanced ultrasound

Objective: To investigate the intra-cardiac distribution of blood from central veins in a fetal lamb model using contrast-enhanced ultrasound imaging. Methods: Eighteen lambs at 135–138 days of gestation were studied using an acute intrauterine fetal lamb model. In 10 lambs contrast agent (CA) (Levovist 400 mg/ml) was injected through the tibial vein into the inferior vena cava (IVC), in 6 lambs into one of the umbilical veins (UV), and in 2 lambs through the right jugular vein into the superior vena cava (SVC). Continuous real-time ultrasound recordings were obtained of the fetal heart at the level of 4-chamber view and at the level of great vessels. Sequences of at least 50–60 images for each plane were analyzed off-line. Distribution of CA was estimated using the brightness intensity scale. Results: Almost 60% of the blood flow from the lower part of fetal body was directed to the left heart. About 40% of the UV blood reached the IVC through the ductus venosus, and 60% of the ductus venosus blood was directed to the left heart. Of the SVC blood flow, more than 90% was directed to the right atrium. Conclusion: Using CA we found that, in the near term fetal lamb, the left heart handles 60% of the oxygenated blood from ductus venosus, 60% of the blood from the lower part of fetal body and 10% of the SVC blood.

OP03.05: Blood flow in the inferior vena cava of fetal lamb visualized with contrast‐enhanced ultrasound

Objective: To investigate the intra-cardiac distribution of blood from central veins in a fetal lamb model using contrast-enhanced ultrasound imaging. Methods: Eighteen lambs at 135–138 days of gestation were studied using an acute intrauterine fetal lamb model. In 10 lambs contrast agent (CA) (Levovist 400 mg/ml) was injected through the tibial vein into the inferior vena cava (IVC), in 6 lambs into one of the umbilical veins (UV), and in 2 lambs through the right jugular vein into the superior vena cava (SVC). Continuous real-time ultrasound recordings were obtained of the fetal heart at the level of 4-chamber view and at the level of great vessels. Sequences of at least 50–60 images for each plane were analyzed off-line. Distribution of CA was estimated using the brightness intensity scale. Results: Almost 60% of the blood flow from the lower part of fetal body was directed to the left heart. About 40% of the UV blood reached the IVC through the ductus venosus, and 60% of the ductus venosus blood was directed to the left heart. Of the SVC blood flow, more than 90% was directed to the right atrium. Conclusion: Using CA we found that, in the near term fetal lamb, the left heart handles 60% of the oxygenated blood from ductus venosus, 60% of the blood from the lower part of fetal body and 10% of the SVC blood.