Ex vivo expansion of human umbilical cord blood hematopoietic stem and progenitor cells

The goal of this thesis research is to establish ex vivo expansion conditions for HSCs derived from UCB. To realize the expansion of HSCs, CD34+ or ACJ33+ UCB cells were cultured in the absence or presence of various cocktails of early acting cytokines including Flt3-L, Tpo, SCF or IL6 under stroma-free or stroma-supported conditions. The HSC and progenitor expansion was assessed using in vitro and in vivo long-term repopulating cells. First, the experiments were designed to test whether HSC expansion would alter the in vivo long-term engraftment potential of CD34+ UCB cells in the presence of EMderived stromal cells during two weeks. Also the cytokines required for expansion of HSCs and progenitors in either the presence or absence of stroma have been evaluated. The experiments described in chapter 3 are closely linked to the work described in the previous chapter. They were designed to investigate whether HSC expansion could be improved when cultured for more than two weeks, and whether the presence of EMderived stromal cells, and combinations of specific cytokines could affect the HSC and progenitor maintenance or expansion. In chapter 4 the effect of a new fusion protein ofiL6 and the soluble IL6R, H-IL6, has been evaluated on the long-term ex vivo expansion ofHCSs derived from AC133+ UCB cells. To do this, we used stroma-free and stroma-supported LTCs and compared several cytokine combinations in the presence or absence of this chimeric protein, or IL6, and estimated the HSC and progenitor output by multiparameter FACS analysis and CAFC assays. Following these experiments, nineteen newly established murine embryonic stromal clones have been investigated for their ability to sustain human HSCs and progenitors in extended LTCs of CD34+ UCB cells in the absence or presence of the cytokines Flt3- L and Tpo for periods as long as twelve weeks. A significant proportion of HSC and progenitor subsets was found in the non-adherent compartment of these co-cultures. With an interest to elucidate the factors that determine the proportion of adherent and non-adherent compartments, we evaluated in chapter 6 the chemoattractive activity of different stromal cells and the effect of exogenously added cytokines herein

A Pathophysiological Perspective on the SARS-CoV-2 Coagulopathy

Abstract: Recent evidence is focusing on the presence of a hypercoagulable state with development of both venous and arterial thromboembolic complications in patients infected with SARS-CoV-2. The ongoing activation of coagulation related to the severity of the illness is further characterized by thrombotic microangiopathy and endotheliitis. These microangiopathic changes cannot be classified as classical disseminated intravascular coagulation (DIC). In this short review we describe the interaction between coagulation and inflammation with focus on the possible mechanisms that might be involved in SARS-CoV-2 infection associated coagulopathy in the critically ill

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

Long-Term Outcome of Patients With a Hematologic Malignancy and Multiple Organ Failure Admitted at the Intensive Care

Objectives: Historically, patients with a hematologic malignancy have one of the highest mortality rates among cancer patients admitted to the ICU. Therefore, physicians are often reluctant to admit these patients to the ICU. The aim of our study was to examine the survival of patients who have a hematologic malignancy and multiple organ failure admitted to the ICU. Design: This retrospective cohort study, part of the HEMA-ICU study group, was designed to study the survival of patients with a hematologic malignancy and organ failure after admission to the ICU. Patients were followed for at least 1 year. Setting: Five university hospitals in the Netherlands. Patients: One-thousand ninety-seven patients with a hematologic malignancy who were admitted at the ICU. Interventions: None. Measurements and Main Results: Primary outcome was 1-year survival. Organ failure was categorized as acute kidney injury, respiratory failure, hepatic failure, and hemodynamic failure; multiple organ failure was defined as failure of two or more organs. The World Health Organization performance score measured 3 months after discharge from the ICU was used as a measure of functional outcome. The 1-year survival rate among these patients was 38%. Multiple organ failure was inversely associated with long-term survival, and an absence of respiratory failure was the strongest predictor of 1-year survival. The survival rate among patients with 2, 3, and 4 failing organs was 27%, 22%, and 8%, respectively. Among all surviving patients for which World Health Organization scores were available, 39% had a World Health Organization performance score of 0-1 3 months after ICU discharge. Functional outcome was not associated with the number of failing organs. Conclusions: Our results suggest that multiple organ failure should not be used as a criterion for excluding a patient with a hematologic malignancy from admission to the ICU.</p

Thrombosis pathways in COVID-19 vs. influenza-associated ARDS: a targeted proteomics approach

Background: Pulmonary embolism (PE) occurs in one-third of critically-ill COVID-19 patients. Although prior studies identified several pathways contributing to thrombogenicity, it is unknown whether this is COVID-19-specific or also occurs in ARDS patients with another infection. Objective: To compare pathway activity among patients having COVID-19 with PE (C19PE+), COVID-19 without PE (C19PE-), and influenza-associated ARDS (IAA) using a targeted proteomics approach. Methods: We exploited an existing biorepository containing daily plasma samples to carefully match C19PE+ cases to C19PE- and IAA controls on mechanical ventilation duration, PEEP, FiO2, and cardiovascular-SOFA (n = 15 per group). Biomarkers representing various thrombosis pathways were measured using proximity extension- and ELISA-assays. Summed z-scores of individual biomarkers were used to represent total pathway activity. Results: We observed no relevant between-group differences among 22 biomarkers associated with activation of endothelium, platelets, complement, coagulation, fibrinolysis or inflammation, except sIL-1RT2 and sST2, which were lower in C19PE- than IAA (log2-Foldchange −0.67, p =.022 and −1.78, p =.022, respectively). However, total pathway analysis indicated increased activation of endothelium (z-score 0.2 [−0.3–1.03] vs. 0.98 [−2.5–−0.3], p =.027), platelets (1.0 [−1.3–3.0] vs. −3.3 [−4.1–−0.6], p =.023) and coagulation (0.8 [−0.5–2.0] vs. −1.0 [−1.6–1.0], p =.023) in COVID-19 patients (C19PE+/C19PE- groups combined) compared to IAA. Conclusion: We observed only minor differences between matched C19PE+, C19PE-, and IAA patients, which suggests individual biomarkers mostly reflect disease severity. However, analysis of total pathway activity suggested upregulation of some distinct processes in COVID-19 could be etiologically related to increased PE-risk

Tacrolimus Variability and Clinical Outcomes in the Early Post-lung Transplantation Period: Oral Versus Continuous Intravenous Administration

Background and Objective: High variability in tacrolimus pharmacokinetics directly after lung transplantation (LuTx) may increase the risk for acute kidney injury (AKI) and transplant rejection. The primary objective was to compare pharmacokinetic variability in patients receiving tacrolimus orally versus intravenously early after LuTx. Methods: Pharmacokinetic and clinical data from 522 LuTx patients transplanted between 2010 and 2020 in two university hospitals were collected to compare orally administered tacrolimus to intravenous tacrolimus early post-transplantation. Tacrolimus blood concentration variability, measured as intrapatient variability (IPV%) and percentage of time within the therapeutic range (TTR%), was analyzed within the first 14 days after LuTx. Secondary outcomes were AKI, acute rejection, length of stay in the intensive care unit (ICU), and mortality in the ICU and during hospital admission. Results: We included 224 patients in the oral and 298 in the intravenous group. The mean adjusted IPV% was 10.8% (95% confidence interval [CI] 6.9–14.6; p < 0.001) higher in the oral group (27.2%) than the intravenous group (16.4%). The mean TTR% was 7.3% (95% CI − 11.3 to − 3.4; p < 0.001) lower in the oral group (39.6%) than in the intravenous group (46.9%). The incidence of AKI was 46.0% for oral and 42.6% for intravenous administration (adjusted odds ratio [OR] 1.2; 95% CI 0.8–1.8; p = 0.451). The frequencies of clinically diagnosed acute rejection in the oral and intravenous groups were nonsignificant (24.6% vs 17.8%; OR 1.5 [95% CI 1.0–2.3; p = 0.059]). ICU and hospital mortality rate and ICU length of stay were similar. Conclusions: Administering tacrolimus orally directly after LuTx leads to a higher variability in blood concentrations compared to intravenous administration. There was no difference in the occurrence of AKI or transplant rejection

Tacrolimus Variability and Clinical Outcomes in the Early Post-lung Transplantation Period: Oral Versus Continuous Intravenous Administration

Background and Objective: High variability in tacrolimus pharmacokinetics directly after lung transplantation (LuTx) may increase the risk for acute kidney injury (AKI) and transplant rejection. The primary objective was to compare pharmacokinetic variability in patients receiving tacrolimus orally versus intravenously early after LuTx. Methods: Pharmacokinetic and clinical data from 522 LuTx patients transplanted between 2010 and 2020 in two university hospitals were collected to compare orally administered tacrolimus to intravenous tacrolimus early post-transplantation. Tacrolimus blood concentration variability, measured as intrapatient variability (IPV%) and percentage of time within the therapeutic range (TTR%), was analyzed within the first 14 days after LuTx. Secondary outcomes were AKI, acute rejection, length of stay in the intensive care unit (ICU), and mortality in the ICU and during hospital admission. Results: We included 224 patients in the oral and 298 in the intravenous group. The mean adjusted IPV% was 10.8% (95% confidence interval [CI] 6.9–14.6; p < 0.001) higher in the oral group (27.2%) than the intravenous group (16.4%). The mean TTR% was 7.3% (95% CI − 11.3 to − 3.4; p < 0.001) lower in the oral group (39.6%) than in the intravenous group (46.9%). The incidence of AKI was 46.0% for oral and 42.6% for intravenous administration (adjusted odds ratio [OR] 1.2; 95% CI 0.8–1.8; p = 0.451). The frequencies of clinically diagnosed acute rejection in the oral and intravenous groups were nonsignificant (24.6% vs 17.8%; OR 1.5 [95% CI 1.0–2.3; p = 0.059]). ICU and hospital mortality rate and ICU length of stay were similar. Conclusions: Administering tacrolimus orally directly after LuTx leads to a higher variability in blood concentrations compared to intravenous administration. There was no difference in the occurrence of AKI or transplant rejection