Perspectives on systematic review protocol registration: a survey amongst stakeholders in the clinical research publication process.

BACKGROUND: As systematic reviews (SRs) inform healthcare decisions, it is key that they address relevant questions and use rigorous methodology. Registration of SR protocols helps researchers identify relevant topics for future reviews and aims to prevent bias and duplication of effort. However, most SRs protocols are currently not registered, despite its significance. To guide future recommendations to enhance preregistration of SRs, it is important to gain a comprehensive understanding of the perspectives within the research community. Therefore, this study aims to examine the experiences with and factors of influence (barriers and facilitators) on prospective SR registration amongst researchers, peer reviewers and journal editors. METHODS: Two different surveys were distributed to two groups: researchers and journal editors both identified from an existing sample of SRs. Researchers who indicated to have peer reviewed a SR were surveyed on their perspectives as peer reviewers as well. Survey design and analysis were informed by the Consolidated Framework for Implementation Research (CFIR). Shared and unique subthemes from the perspectives of researchers, peer reviewers and journal editors were identified and linked to the SR registration process (Innovation), to team, organisation (Inner setting) and (inter)national research community (Outer setting), and to characteristics of researchers, peer reviewers or journal editors (Individuals). RESULTS: The survey's response rates were 65/727 (9%) for researchers, of which 37 were peer reviewers, and 22/308 (7%) for journal editors. Most respondents (n = 76, 94%) were familiar with SR protocol registration and 81% of researchers had registered minimally one SR protocol. Shared SR registration process subthemes were the importance and advantages of SR protocol registration, as well as barriers such as a high administrative burden. Shared subthemes regarding the inner and outer setting centred on journal processes, external standards and time. Shared individual factors were knowledge, skills and awareness. CONCLUSIONS: The majority of the respondents were familiar with SR protocol registration and had a positive attitude towards it. This study identified suboptimal registration process, administrative burden and lack of mandatory SR protocol registration as barriers. By overcoming these barriers, SR protocol registration could contribute more effectively to the goals of open science. SYSTEMATIC REVIEW REGISTRATION: osf.io/gmv6z.

The score after 10 years of registration of systematic review protocols.

BACKGROUND: With the exponential growth of published systematic reviews (SR), there is a high potential for overlapping and redundant duplication of work. Prospective protocol registration gives the opportunity to assess the added value of a new study or review, thereby potentially reducing research waste and simultaneously increasing transparency and research quality. The PROSPERO database for SR protocol registration was launched 10 years ago. This study aims to assess the proportion SRs of intervention studies with a protocol registration (or publication) and explore associations of SR characteristics with protocol registration status. METHODS: PubMed was searched for SRs of human intervention studies published in January 2020 and January 2021. After random-stratified sampling and eligibility screening, data extraction on publication and journal characteristics, and protocol registration status, was performed. Both descriptive and multivariable comparative statistical analyses were performed. RESULTS: A total of 357 SRs (2020: n = 163; 2021: n = 194) were included from a random sample of 1267 publications. Of the published SRs, 38% had a protocol. SRs that reported using PRISMA as a reporting guideline had higher odds of having a protocol than publications that did not report PRISMA (OR 2.71; 95% CI: 1.21 to 6.09). SRs with a higher journal impact factor had higher odds of having a protocol (OR 1.12; 95% CI 1.04 to 1.25). Publications from Asia had a lower odds of having a protocol (OR 0.43; 95% CI 0.23 to 0.80, reference category = Europe). Of the 33 SRs published in journals that endorse PROSPERO, 45% did not have a protocol. Most SR protocols were registered in PROSPERO (n = 129; 96%). CONCLUSIONS: We found that 38% of recently published SRs of interventions reported a registered or published protocol. Protocol registration was significantly associated with a higher impact factor of the journal publishing the SR and a more frequent self-reported use of the PRISMA guidelines. In some parts of the world, SR protocols are more often registered or published than others. To guide strategies to increase the uptake of SR protocol registration, further research is needed to gain understanding of the benefits and informativeness of SRs protocols among different stakeholders. SYSTEMATIC REVIEW REGISTRATION: osf.io/9kj7r/.

Reducing Inappropriate Proton Pump Inhibitors Use for Stress Ulcer Prophylaxis in Hospitalized Patients: Systematic Review of De-Implementation Studies.

BACKGROUND: A large proportion of proton pump inhibitor (PPI) prescriptions, including those for stress ulcer prophylaxis (SUP), are inappropriate. Our study purpose was to systematically review the effectiveness of de-implementation strategies aimed at reducing inappropriate PPI use for SUP in hospitalized, non-intensive care unit (non-ICU) patients. METHODS: We searched MEDLINE and Embase databases (from inception to January 2020). Two authors independently screened references, performed data extraction, and critical appraisal. Randomized trials and comparative observational studies were eligible for inclusion. Criteria developed by the Cochrane Effective Practice and Organisation of Care (EPOC) group were used for critical appraisal. Besides the primary outcome (inappropriate PPI prescription or use), secondary outcomes included (adverse) pharmaceutical effects and healthcare use. RESULTS: We included ten studies in this review. Most de-implementation strategies contained an educational component (meetings and/or materials), combined with either clinical guideline implementation (n = 5), audit feedback (n = 3), organizational culture (n = 4), or reminders (n = 1). One study evaluating the de-implementation strategy effectiveness showed a significant reduction (RR 0.14; 95% CI 0.03-0.55) of new inappropriate PPI prescriptions. Out of five studies evaluating the effectiveness of de-implementing inappropriate PPI use, four found a significant reduction (RR 0.21; 95% CI 0.18-0.26 to RR 0.76; 95% CI 0.68-0.86). No significant differences in the occurrence of pharmaceutical effects (n = 1) and in length of stay (n = 3) were observed. Adverse pharmaceutical effects were reported in two studies and five studies reported on PPI or total drug costs. No pooled effect estimates were calculated because of large statistical heterogeneity between studies. DISCUSSION: All identified studies reported mainly educational interventions in combination with one or multiple other intervention strategies and all interventions were targeted at providers. Most studies found a small to moderate reduction of (inappropriate) PPI prescriptions or use.

Antibiotic prophylaxis for prevention of postoperative wound infection in adults undergoing open elective inguinal or femoral hernia repair.

BACKGROUND: Inguinal or femoral hernia is a tissue protrusion in the groin region and has a cumulative incidence of 27% in adult men and of 3% in adult women. As most hernias become symptomatic over time, groin hernia repair is one of the most frequently performed surgical procedures worldwide. This type of surgery is considered 'clean' surgery with wound infection rates expected to be lower than 5%. For clean surgical procedures, antibiotic prophylaxis is not generally recommended. However after the introduction of mesh-based hernia repair and the publication of studies that have high wound infection rates the debate as to whether antibiotic prophylaxis is required to prevent postoperative wound infections started again. OBJECTIVES: To determine the effectiveness of antibiotic prophylaxis in reducing postoperative (superficial and deep) wound infections in elective open inguinal and femoral hernia repair. SEARCH METHODS: We searched several electronic databases: Cochrane Registry of Studies Online, MEDLINE Ovid, Embase Ovid, Scopus and Science Citation Index (search performed on 12 November 2019). We also searched two trial registers and the reference list of included studies. SELECTION CRITERIA: We included randomised controlled trials comparing any type of antibiotic prophylaxis versus placebo or no treatment for preventing postoperative wound infections in adults undergoing inguinal or femoral open hernia repair surgery (tissue repair and mesh repair). DATA COLLECTION AND ANALYSIS: Two review authors independently selected studies, extracted data and assessed risk of bias. We separately analysed results for two different surgical methods (herniorrhaphy and hernioplasty). Several studies revealed infection rates that were higher than the expected 5% for clean surgery and we therefore divided studies into two subgroups: high infection risk environments (≥ 5% infection rate); and low infection risk environments (< 5% infection rate). We performed meta-analyses with random-effects models. We analysed three outcomes: superficial surgical site infections (SSSI); deep surgical site infections (DSSI); and all postoperative wound infections (SSSI + DSSI). MAIN RESULTS: In this review update we identified and included 10 new studies. In total, we included 27 studies with 8308 participants in this review. It is uncertain whether antibiotic prophylaxis as compared to placebo (or no treatment) prevents all types of postoperative wound infections after herniorrhaphy surgery (risk ratio (RR) 0.86, 95% confidence interval (CI) 0.56 to 1.33; 5 studies, 1865 participants; very low quality evidence). Subgroup analysis did not change these results. We could not perform meta-analyses for SSSI or DSSI as these outcomes were not reported separately. Twenty-two studies related to hernioplasty surgery (total of 6443 participants) and we analysed three outcomes: SSSI; DSSI; SSSI + DSSI. Within the low infection risk environment subgroup, antibiotic prophylaxis as compared to placebo probably makes little or no difference for the outcomes 'prevention of all wound infections' (RR 0.71, 95% CI 0.44 to 1.14; moderate-quality evidence) and 'prevention of SSSI' (RR 0.71, 95% CI 0.44 to 1.17, moderate-quality evidence). Within the high infection risk environment subgroup it is uncertain whether antibiotic prophylaxis reduces all types of wound infections (RR 0.58, 95% CI 0.43 to 0.77, very low quality evidence) or SSSI (RR 0.56, 95% CI 0.41 to 0.77, very low quality evidence). When combining participants from both subgroups, antibiotic prophylaxis as compared to placebo probably reduces the risk of all types of wound infections (RR 0.61, 95% CI 0.48 to 0.78) and SSSI (RR 0.60, 95% CI 0.46 to 0.78; moderate-quality evidence). Antibiotic prophylaxis as compared to placebo probably makes little or no difference in reducing the risk of postoperative DSSI (RR 0.65, 95% CI 0.26 to 1.65; moderate-quality evidence), both in a low infection risk environment (RR 0.67, 95% CI 0.11 to 4.13; moderate-quality evidence) and in the high infection risk environment (RR 0.64, 95% CI 0.22 to 1.89; low-quality evidence). AUTHORS' CONCLUSIONS: Evidence of very low quality shows that it is uncertain whether antibiotic prophylaxis reduces the risk of postoperative wound infections after herniorrhaphy surgery. Evidence of moderate quality shows that antibiotic prophylaxis probably makes little or no difference in preventing wound infections (i.e. all wound infections, SSSI or DSSI) after hernioplasty surgery in a low infection risk environment. Evidence of low quality shows that antibiotic prophylaxis in a high-risk environment may reduce the risk of all wound infections and SSSI, while evidence of very low quality shows that it is uncertain whether antibiotic prophylaxis reduces DSSI after hernioplasty surgery.

The duration of the third stage in relation to postpartum hemorrhage.

INTRODUCTION: In this study, we describe the distribution of placenta delivery and the incidence of postpartum hemorrhage in both spontaneous placental delivery and manual removal of the placenta. METHODS: A retrospective study was performed of 7603 singleton vaginal deliveries of a gestational age over 32 weeks, registered between September 2011 and 2016. We calculated the incidence of postpartum hemorrhage (≥1000 mL blood loss) per 10-minute duration of the third stage. The odds ratio for developing postpartum hemorrhage was assessed, adjusted for risk factors. The incidence of postpartum hemorrhage was compared between women that did and did not receive manual removal of placenta. RESULTS: The median duration of the third stage was 10 minutes (interquartile range 7-16 minutes). The median amount of blood loss was 300 mL (200-400 mL). The overall incidence of postpartum hemorrhage was 8.5%. With every additional 10 minutes of third-stage duration, the risk of developing postpartum hemorrhage significantly increased. In a third stage longer than 60 minutes, the incidence of postpartum hemorrhage was 21.2% without manual removal of the placenta and 70.3% with manual removal. CONCLUSIONS: The incidence of postpartum hemorrhage increases significantly from 10 to 19 minutes into the third stage. Women with the removal of the placenta had a significantly higher percentage of postpartum hemorrhage. The optimal timing for manual removal of the placenta should be investigated in a carefully designed randomized controlled trial to examine whether earlier manual removal of placenta lowers the incidence and limits the severity of postpartum hemorrhage.

Bioactive glass obliteration of the mastoid significantly improves surgical outcome in non-cholesteatomatous chronic otitis media patients.

This retrospective follow-up study evaluates the efficacy and safety of bioactive glass (BAG) S53P4 when applied as filler material in mastoid obliteration surgery performed on non-cholesteatomatous chronic otitis media (NC-COM) patients with chronically discharging ears despite conservative therapy. 94 Patients (96 ears) were included. Patients underwent either intact canal wall (ICW) or canal wall down (CWD) mastoid surgery between 2005 and 2015. The intervention group comprised 23 patients (23 ears) who were treated with additional mastoid obliteration using BAG S53P4; the remaining 71 patients (73 ears) were considered controls. All patients underwent preoperative CT scanning of the mastoid. Primary functional outcome, as defined by control of suppuration, was assessed using Merchant's scale. Hearing results as measured by air-bone gap and the incidence of adverse events were assessed as secondary outcomes. Thirty-two ears (44%) in the control group (n = 73) achieved complete control of infection at the most recent postoperative clinic visit vs 17 (74%) in the S53P4 obliteration group (n = 23). Comparing these outcomes yielded an odds ratio (OR) of 3.6 (p = 0.012, 95% CI 1.3-10.3). Complete failure to manage infection significantly differed (p = 0.048) between the control group (11 ears; 15%) and the S53P4 obliteration group (0 ears). No adverse events were observed in either group. Pre- and postoperative ABG results did not differ significantly between groups. Obliteration of the mastoid cavity using BAG S53P4 along with mastoidectomy in patients with chronically discharging NC-COM significantly improves the achievement of a dry and safe ear as compared to mastoidectomy alone. Importantly, no adverse events were observed with S53P4 BAG obliteration.

Mastoid obliteration with S53P4 bioactive glass in cholesteatoma surgery.

CONCLUSION: Evaluation of the follow-up of 67 patients shows that S53P4 bioactive glass (BAG) granules are safe and effective as obliteration material in cholesteatoma surgery. OBJECTIVES: To investigate the safety and efficacy of mastoid obliteration using S53P4 BAG in cholesteatoma surgery. Clinical outcomes were infection control (Merchant's grading), cholesteatoma recidivism, and audiometric performance. METHODS: Retrospective follow-up study at the Diakonessenhuis, Utrecht, the Netherlands. Eighteen young (age <17 years) and 49 adult (age ≥17 years) patients treated for cholesteatoma underwent tympanomastoidectomy with mastoid obliteration using S53P4 BAG in the period 2012-2015. Outcome was monitored with clinical otoscopy, otorrhea incidence measurement (Merchant's grading), DW-MRI, and audiographic performance analyses (pure tone average and air bone gap). RESULTS: During the follow-up period (mean = 22 months; range = 12-54 months) cholesteatoma recidivism was observed in 6% of the patients (four ears), mostly in young patients (three ears). An acceptably dry ear (Merchant grade 0-1) was achieved in 96% of all cases. The remaining 4% of cases scored a Merchant grade 2. Overall, both air conduction thresholds and air bone gap were slightly lowered when comparing post-operative values to pre-operative values and significantly in the case of ossicular reconstruction. In none of the patients (0%) did post-operative wound infections occur.

Altered intracellular localization and mobility of SBDS protein upon mutation in Shwachman-Diamond syndrome.

Shwachman-Diamond Syndrome (SDS) is a rare inherited disease caused by mutations in the SBDS gene. Hematopoietic defects, exocrine pancreas dysfunction and short stature are the most prominent clinical features. To gain understanding of the molecular properties of the ubiquitously expressed SBDS protein, we examined its intracellular localization and mobility by live cell imaging techniques. We observed that SBDS full-length protein was localized in both the nucleus and cytoplasm, whereas patient-related truncated SBDS protein isoforms localize predominantly to the nucleus. Also the nucleo-cytoplasmic trafficking of these patient-related SBDS proteins was disturbed. Further studies with a series of SBDS mutant proteins revealed that three distinct motifs determine the intracellular mobility of SBDS protein. A sumoylation motif in the C-terminal domain, that is lacking in patient SBDS proteins, was found to play a pivotal role in intracellular motility. Our structure-function analyses provide new insight into localization and motility of the SBDS protein, and show that patient-related mutant proteins are altered in their molecular properties, which may contribute to the clinical features observed in SDS patients.

SBDS expression and localization at the mitotic spindle in human myeloid progenitors.

BACKGROUND: Shwachman-Diamond Syndrome (SDS) is a hereditary disease caused by mutations in the SBDS gene. SDS is clinically characterized by pancreatic insufficiency, skeletal abnormalities and bone marrow dysfunction. The hematologic abnormalities include neutropenia, neutrophil chemotaxis defects, and an increased risk of developing Acute Myeloid Leukemia (AML). Although several studies have suggested that SBDS as a protein plays a role in ribosome processing/maturation, its impact on human neutrophil development and function remains to be clarified. METHODOLOGY/PRINCIPAL FINDINGS: We observed that SBDS RNA and protein are expressed in the human myeloid leukemia PLB-985 cell line and in human hematopoietic progenitor cells by quantitative RT-PCR and Western blot analysis. SBDS expression is downregulated during neutrophil differentiation. Additionally, we observed that the differentiation and proliferation capacity of SDS-patient bone marrow hematopoietic progenitor cells in a liquid differentiation system was reduced as compared to control cultures. Immunofluorescence analysis showed that SBDS co-localizes with the mitotic spindle and in vitro binding studies reveal a direct interaction of SBDS with microtubules. In interphase cells a perinuclear enrichment of SBDS protein which co-localized with the microtubule organizing center (MTOC) was observed. Also, we observed that transiently expressed SDS patient-derived SBDS-K62 or SBDS-C84 mutant proteins could co-localize with the MTOC and mitotic spindle. CONCLUSIONS/SIGNIFICANCE: SBDS co-localizes with the mitotic spindle, suggesting a role for SBDS in the cell division process, which corresponds to the decreased proliferation capacity of SDS-patient bone marrow CD34(+) hematopoietic progenitor cells in our culture system and also to the neutropenia in SDS patients. A role in chromosome missegregation has not been clarified, since similar spatial and time-dependent localization is observed when patient-derived SBDS mutant proteins are studied. Thus, the increased risk of myeloid malignancy in SDS remains unexplained.

Ventral embryonic tissues and Hedgehog proteins induce early AGM hematopoietic stem cell development.

Hematopoiesis is initiated in several distinct tissues in the mouse conceptus. The aorta-gonad-mesonephros (AGM) region is of particular interest, as it autonomously generates the first adult type hematopoietic stem cells (HSCs). The ventral position of hematopoietic clusters closely associated with the aorta of most vertebrate embryos suggests a polarity in the specification of AGM HSCs. Since positional information plays an important role in the embryonic development of several tissue systems, we tested whether AGM HSC induction is influenced by the surrounding dorsal and ventral tissues. Our explant culture results at early and late embryonic day 10 show that ventral tissues induce and increase AGM HSC activity, whereas dorsal tissues decrease it. Chimeric explant cultures with genetically distinguishable AGM and ventral tissues show that the increase in HSC activity is not from ventral tissue-derived HSCs, precursors or primordial germ cells (as was previously suggested). Rather, it is due to instructive signaling from ventral tissues. Furthermore, we identify Hedgehog protein(s) as an HSC inducing signal.

Shwachman-Diamond syndrome neutrophils have altered chemoattractant-induced F-actin polymerization and polarization characteristics.

Shwachman-Diamond syndrome is a hereditary disorder characterized by pancreatic insufficiency and bone marrow failure. Most Shwachman-Diamond syndrome patients have mutations in the SBDS gene located at chromosome 7 and suffer from recurrent infections, due to neutropenia in combination with impaired neutrophil chemotaxis. Currently, the role of the actin cytoskeleton in Shwachman-Diamond syndrome neutrophils has not been investigated. Therefore, we performed immunofluorescence for SBDS and F-actin on human neutrophilic cells. Additionally, we examined in control neutrophils and cells from genetically defined Shwachman-Diamond syndrome patients F-actin polymerization and cytoskeletal polarization characteristics upon chemoattractant stimulation. These studies showed that SBDS and F-actin co-localize in neutrophilic cells and that F-actin polymerization and depolymerization characteristics are altered in Shwachman-Diamond syndrome neutrophils as compared to control neutrophils in response to both fMLP and C5a. Moreover, F-actin cytoskeletal polarization is delayed in Shwachman-Diamond syndrome neutrophils. Thus, Shwachman-Diamond syndrome neutrophils have aberrant chemoattractant-induced F-actin properties which might contribute to the impaired neutrophil chemotaxis.

Interleukin-1 regulates hematopoietic progenitor and stem cells in the midgestation mouse fetal liver.

BACKGROUND: Hematopoietic progenitors are generated in the yolk sac and aorta-gonad-mesonephros region during early mouse development. At embryonic day 10.5 the first hematopoietic stem cells emerge in the aorta-gonad-mesonephros. Subsequently, hematopoietic stem cells and progenitors are found in the fetal liver. The fetal liver is a potent hematopoietic site, playing an important role in the expansion and differentiation of hematopoietic progenitors and hematopoietic stem cells. However, little is known concerning the regulation of fetal liver hematopoietic stem cells. In particular, the role of cytokines such as interleukin-1 in the regulation of hematopoietic stem cells in the embryo has been largely unexplored. Recently, we observed that the adult pro-inflammatory cytokine interleukin-1 is involved in regulating aorta-gonad-mesonephros hematopoietic progenitor and hematopoietic stem cell activity. Therefore, we set out to investigate whether interleukin-1 also plays a role in regulating fetal liver progenitor cells and hematopoietic stem cells. DESIGN AND METHODS: We examined the interleukin-1 ligand and receptor expression pattern in the fetal liver. The effects of interleukin-1 on hematopoietic progenitor cells and hematopoietic stem cells were studied by FACS and transplantation analyses of fetal liver explants, and in vivo effects on hematopoietic stem cell and progenitors were studied in Il1r1(-/-) embryos. RESULTS: We show that fetal liver hematopoietic progenitor cells express the IL-1RI and that interleukin-1 increases fetal liver hematopoiesis, progenitor cell activity and promotes hematopoietic cell survival. Moreover, we show that in Il1r1(-/-) embryos, hematopoietic stem cell activity is impaired and myeloid progenitor activity is increased. CONCLUSIONS: The IL-1 ligand and receptor are expressed in the midgestation liver and act in the physiological regulation of fetal liver hematopoietic progenitor cells and hematopoietic stem cells.

Interleukin-1-mediated hematopoietic cell regulation in the aorta-gonad-mesonephros region of the mouse embryo.

Hematopoiesis during development is a dynamic process, with many factors involved in the emergence and regulation of hematopoietic stem cells (HSCs) and progenitor cells. Whereas previous studies have focused on developmental signaling and transcription factors in embryonic hematopoiesis, the role of well-known adult hematopoietic cytokines in the embryonic hematopoietic system has been largely unexplored. The cytokine interleukin-1 (IL-1), best known for its proinflammatory properties, has radioprotective effects on adult bone marrow HSCs, induces HSC mobilization, and increases HSC proliferation and/or differentiation. Here we examine IL-1 and its possible role in regulating hematopoiesis in the midgestation mouse embryo. We show that IL-1, IL-1 receptors (IL-1Rs), and signaling mediators are expressed in the aorta-gonad-mesonephros (AGM) region during the time when HSCs emerge in this site. IL-1 signaling is functional in the AGM, and the IL-1RI is expressed ventrally in the aortic subregion by some hematopoietic, endothelial, and mesenchymal cells. In vivo analyses of IL-1RI-deficient embryos show an increased myeloid differentiation, concomitant with a slight decrease in AGM HSC activity. Our results suggest that IL-1 is an important homeostatic regulator at the earliest time of HSC development, acting to limit the differentiation of some HSCs along the myeloid lineage.

Bcl-2 expression and apoptosis in the regulation of hematopoietic stem cells.

Apoptosis or programmed cell death plays a pivotal role in regulating tissue homeostasis in the adult and in tissue remodeling during embryogenesis. As in other tissues, apoptosis plays an important role within the hematopoietic system in removing aged and non-functional cells. It plays a particularly important role in regulating the cells of the immune system. The signals and molecules regulating apoptosis in these immune cells have been intensely investigated over the years, providing great insight into the mechanisms involved. In contrast, much less is known about the regulation and role of apoptosis in the cells that produce differentiated hematopoietic cells, namely the hematopoietic stem cells (HSCs). It is appreciated that HSCs are under tight regulatory control, as either excessive proliferation or apoptosis will result in too many or too few hematopoietic cells (for example, leukemia or anemia). Apoptosis thus plays an essential role in maintaining the appropriate balance of HSC and mature blood cells and in protecting the HSC pool for life-long hematopoiesis. This review summarizes the current knowledge concerning apoptosis and its role in the physiology of the hematopoietic system, especially within the HSC compartment.

The role of apoptosis in the development of AGM hematopoietic stem cells revealed by Bcl-2 overexpression.

Apoptosis is an essential process in embryonic tissue remodeling and adult tissue homeostasis. Within the adult hematopoietic system, it allows for tight regulation of hematopoietic cell subsets. Previously, it was shown that B-cell leukemia 2 (Bcl-2) overexpression in the adult increases the viability and activity of hematopoietic cells under normal and/or stressful conditions. However, a role for apoptosis in the embryonic hematopoietic system has not yet been established. Since the first hematopoietic stem cells (HSCs) are generated within the aortagonad-mesonephros (AGM; an actively remodeling tissue) region beginning at embryonic day 10.5, we examined this tissue for expression of apoptosis-related genes and ongoing apoptosis. Here, we show expression of several proapoptotic and antiapoptotic genes in the AGM. We also generated transgenic mice overexpressing Bcl-2 under the control of the transcriptional regulatory elements of the HSC marker stem cell antigen-1 (Sca-1), to test for the role of cell survival in the regulation of AGM HSCs. We provide evidence for increased numbers and viability of Sca-1(+) cells in the AGM and subdissected midgestation aortas, the site where HSCs are localized. Most important, our in vivo transplantation data show that Bcl-2 overexpression increases AGM and fetal liver HSC activity, strongly suggesting that apoptosis plays a role in HSC development.

Identification of 2 novel genes developmentally regulated in the mouse aorta-gonad-mesonephros region.

The first adult-repopulating hematopoietic stem cells (HSCs) emerge in the mouse aorta-gonad-mesonephros (AGM) region at embryonic day 10.5 prior to their appearance in the yolk sac and fetal liver. Although several genes are implicated in the regulation of HSCs, there are gaps in our understanding of the processes taking place in the AGM at the time of HSC emergence. To identify genes involved in AGM HSC emergence, we performed differential display reverse transcriptase-polymerase chain reaction (DD RT-PCR). Differentially expressed genes included beta-catenin and homologs of human TM9SF2 and TAB2. We characterized the expression pattern of Wnt/beta-catenin signaling, mTM9SF2, and mTAB2 in the embryo and adult. Interestingly, the expression of mouse TAB2 (mTAB2) in the E11 dorsal aorta endothelium suggests a role for mTAB2 in HSC emergence and/or regulation. The identification of differentially expressed genes in the AGM region should yield further insights into the development of this tissue and into the emergence and regulation of HSCs.

Embryonal subregion-derived stromal cell lines from novel temperature-sensitive SV40 T antigen transgenic mice support hematopoiesis.

Throughout life, the hematopoietic system requires a supportive microenvironment that allows for the maintenance and differentiation of hematopoietic stem cells (HSC). To understand the cellular interactions and molecules that provide these functions, investigators have previously established stromal cell lines from the late gestational stage and adult murine hematopoietic microenvironments. However, the stromal cell microenvironment that supports the emergence, expansion and maintenance of HSCs during mid-gestational stages has been largely unexplored. Since several tissues within the mouse embryo are known to harbor HSCs (i.e. aortagonads-mesonephros, yolk sac, liver), we generated numerous stromal cell clones from these mid-gestational sites. Owing to the limited cell numbers, isolations were performed with tissues from transgenic embryos containing the ts SV40 Tag gene (tsA58) under the transcriptional control of constitutive and ubiquitously expressing promoters. We report here that the growth and cloning efficiency of embryonic cells (with the exception of the aorta) is increased in the presence of the tsA58 transgene. Furthermore, our results show that the large panel of stromal clones isolated from the different embryonal subregions exhibit heterogeneity in their ability to promote murine and human hematopoietic differentiation. Despite our findings of heterogeneity in hematopoietic growth factor gene expression profiles, high-level expression of some factors may influence hematopoietic differentiation. Interestingly, a few of these stromal clones express a recently described chordin-like protein, which is an inhibitor of bone morphogenic proteins and is preferentially expressed in cells of the mesenchymal lineage.