Hematopoietic stem and progenitor cells and potentials for application in fetal cell replacement therapy

Fetal cell replacement therapy or in utero hematopoietic cell transplantation (IUHCT) is proposed as a non-myeloablative alternative to bone marrow transplantation (BMT) for a number of inborn immunologic, hematologic and metabolic disorders. IUHCT represents the method through which variable amounts of natural or genetically modified hematopoietic cells can be transferred to the fetal recipient in hope of correcting the disorder and preventing postnatal permanent organ damage. Although proof-of-principle has been achieved by succesful correction of X linked severe combined immune deficiency (X-SCID), in the majority of target diseases treated with IUHCT engraftment was insufficient for clinical benefit. Thus, the therapeutical promise of IUHCT remains unfullfilled and many challenges stand. In the present thesis we investigate the optimal cell population for IUHCT by first identifying a novel commitment/differentiation step of hematopoietic stem cells (HSCs) in adult murine hematopoiesis and then evaluating the therapeutical potential of the lymphoid primed multipotent progenitors (LMPPs) for immune reconstitution in a model of fetal X-SCID transplantation. We find that LMPPs generate rapid and sustained lymphoid reconstitution with polyclonal T cells, but that HSCs are most likely required for long term engraftment. We also find that the fetal microenvironment is apparently more receptive to donor HSCs (but also LMPPs) as it allows higher levels of chimerism after IUHCT then after BMT in neonatal or adult age. In the last part we investigate in adult and fetal animal models the proposed plasticity of HSCs, a feature that holds promise for clinical BMT (or IUHCT) to non-hematopoietic disorders. We find that HSCs plasticity is a result of heterotypic cell fusion, probably induced by inflammation/injury in the target tissue. We also show that heterotypic cell fusion is not a physiological frequently occuring event during development and we demonstrate that not only myeloid, but also lymphoid cells are efficient fusogenic partners to non-hematopoietic tissues

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. (Blood. 2009;113:4790-4798

Tru-Cut Biopsy in Gynecological Cancer: Adequacy, Accuracy, Safety and Clinical Applicability

Purpose: Tru-cut biopsy is a minimally invasive technique used to obtain tissue samples for the diagnosis of tumors, especially in patients where primary surgery is not indicated. The aim of this study was to assess the adequacy, accuracy and safety of the tru-cut biopsy for diagnosis in gynecological cancer.Methods: A retrospective population-based review of 328 biopsies was conducted. The indications for tru-cut biopsies were diagnosis of primary tumors, metastases of gynecological and non-gynecological tumors, and suspected recurrences. A tissue sample was considered adequate when the quality/quality was sufficient to identify the subtype/origin of the tumor. Potential factors affecting adequacy were analyzed using logistic regressions analyses. Accuracy was defined as agreement between the diagnosis of the tru-cut biopsy and the postoperative histology. The therapy plan was registered, and the clinical applicability of the tru-cut biopsy was investigated. Complications within 30 days after the biopsy procedure were registered.Results: In total, 300 biopsies were identified as tru-cut biopsies. The overall adequacy was 86.3%, varying between 80.8% and 93.5%, respectively, when performed by a gynecological oncologist or a gynecologist with a subspecialty in ultrasound diagnosis. Sampling of a pelvic mass had a lower adequacy (81.6%) compared with sampling of the omentum (93.9%) or carcinomatosis (91.5%). The accuracy was 97.5%, and the complication rate was 1.3%.Conclusion: The tru-cut biopsy is a safe and reliable diagnostic method with a high accuracy and a good adequacy, depending on the site of the tissue sample, indications for the biopsy and the experience of the operator

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 (Kit(W41/W41)) mice, with a partial loss of function of Kit. Although the extensive fetal HSC expansion was Kit-independent, adult Kit(W41/W41) mice had an almost 2-fold reduction in long-term HSCs, reflecting a loss of roughly 10,000 Lin(-)Sca-1(+)Kit(high) (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 Kit(W41/W41) bone marrow cells in myeloablated recipients were close to normal, self-renewing Kit(W41/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 Kit(W41/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

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

The Cardiac State Diagram As A Novel Approach For The Evaluation Of Pre- And Post-Ejection Phases Of The Cardiac Cycle In Asphyxiated Fetal Lambs

The aim of this study was to investigate myocardial wall motion using echocardiography and color-coded tissue velocity imaging and to generate a cardiac state diagram for evaluation of the duration of the pre- and post-ejection phases in asphyxiated fetal lambs. Six near-term lambs were partly exteriorized and brought to cardiac arrest through asphyxia. Echocardiography measurements were recorded simultaneously with arterial blood sampling for lactate and blood gases. All fetal lambs exhibited prolongation of the pre- and post-ejection phases at the time when the most pronounced changes in lactate concentration and pH occurred. The mean change in duration of the pre- and post-ejection phases for all fetal lambs was 36 +/- 7 ms (p < 0.002) and 77 +/- 17 ms (p < 0.019), respectively, and the percentage change was 50% (p < 0.001) and 38% (p < 0.049), respectively. As asphyxia progressed in fetal lambs, the duration of the pre- and post-ejection phases increased. The cardiac state diagram has the potential to be a comprehensible tool for detecting fetal asphyxia. (E-mail: [email protected]) (C) 2013 World Federation for Ultrasound in Medicine & Biology

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