Hematopoietic Bone Marrow Research Articles

First reported case of thymoma-associated multiorgan autoimmunity induced by COVID-19.

Thymoma-associated multiorgan autoimmunity (TAMA) presents with skin symptoms similar to those of graft-versus-host disease (GVHD), liver dysfunction, and enteritis, in the absence of a history of hematopoietic stem cell or bone marrow transplantation. TAMA is a type of paraneoplastic syndrome associated with thymoma. Its etiology is unclear but is thought to be a result of breakdown of immune tolerance. Histopathologically, TAMA is characterized by epidermal acanthosis with parakeratosis, individual cell keratinization, liquefaction degeneration, and intraepidermal infiltration of CD8-positive lymphocytes. A 64-year-old female patient with a history of myasthenia gravis and thymoma treated with prednisolone (10 mg/day) and cyclosporine (150 mg/day) experienced erythema on her trunk after coronavirus disease 2019 (COVID-19) onset. A psoriatic drug eruption was suspected and the possible causative drug was discontinued, but the skin rash failed to improve. A skin biopsy demonstrated GVHD-like histopathological findings. Diarrhea, abdominal pain, and duodenal perforation occurred concurrently, leading to the diagnosis of TAMA. Thereafter, the patient continued prednisolone and cyclosporine in the same doses as the TAMA treatment and added topical steroids. During the disease course, candida fungemia and cytomegalovirus infection developed, resulting in the patient's death. The TAMA was considered to have been caused by the release of inflammatory cytokines, autoreactive T cell activation, and regulatory T cell dysfunction induced by COVID-19.

Limited Alleviation of Lysosomal Acid Lipase Deficiency by Deletion of Matrix Metalloproteinase 12

Lysosomal acid lipase (LAL) is the only known enzyme that degrades cholesteryl esters and triglycerides at an acidic pH. In LAL deficiency (LAL-D), dysregulated expression of matrix metalloproteinase 12 (MMP-12) has been described. The overexpression of MMP-12 in myeloid lineage cells causes an immune cell dysfunction resembling that of Lal knockout (Lal KO) mice. Both models develop progressive lymphocyte dysfunction and expansion of myeloid-derived suppressor (CD11b+ Gr-1+) cells. To study whether MMP-12 might be a detrimental contributor to the pathology of LAL-D, we have generated Lal/Mmp12 double knockout (DKO) mice. The phenotype of Lal/Mmp12 DKO mice closely resembled that of Lal KO mice, while the weight and morphology of the thymus were improved in Lal/Mmp12 DKO mice. Cytological examination of blood smears showed a mildly reversed lymphoid-to-myeloid shift in DKO mice. Despite significant decreases in CD11b+ Ly6G+ cells in the peripheral blood, bone marrow, and spleen of Lal/Mmp12 DKO mice, the hematopoietic bone marrow progenitor compartment and markers for neutrophil chemotaxis were unchanged. Since the overall severity of LAL-D remains unaffected by the deletion of Mmp12, we conclude that MMP-12 does not represent a viable target for treating the inflammatory pathology in LAL-D.

Mesenchymal Stem Cell and Hematopoietic Stem and Progenitor Cell Co-Culture in a Bone-Marrow-on-a-Chip Device toward the Generation and Maintenance of the Hematopoietic Niche.

Bone marrow has raised a great deal of scientific interest, since it is responsible for the vital process of hematopoiesis and is affiliated with many normal and pathological conditions of the human body. In recent years, organs-on-chips (OoCs) have emerged as the epitome of biomimetic systems, combining the advantages of microfluidic technology with cellular biology to surpass conventional 2D/3D cell culture techniques and animal testing. Bone-marrow-on-a-chip (BMoC) devices are usually focused only on the maintenance of the hematopoietic niche; otherwise, they incorporate at least three types of cells for on-chip generation. We, thereby, introduce a BMoC device that aspires to the purely in vitro generation and maintenance of the hematopoietic niche, using solely mesenchymal stem cells (MSCs) and hematopoietic stem and progenitor cells (HSPCs), and relying on the spontaneous formation of the niche without the inclusion of gels or scaffolds. The fabrication process of this poly(dimethylsiloxane) (PDMS)-based device, based on replica molding, is presented, and two membranes, a perforated, in-house-fabricated PDMS membrane and a commercial poly(ethylene terephthalate) (PET) one, were tested and their performances were compared. The device was submerged in a culture dish filled with medium for passive perfusion via diffusion in order to prevent on-chip bubble accumulation. The passively perfused BMoC device, having incorporated a commercial poly(ethylene terephthalate) (PET) membrane, allows for a sustainable MSC and HSPC co-culture and proliferation for three days, a promising indication for the future creation of a hematopoietic bone marrow organoid.

Germline Predisposition to Hematopoietic Malignancies: An Overview

Deleterious germline variants are now recognized as common drivers of hematopoietic malignancies (HMs) and bone marrow failure syndromes. With the increasing use of personalized medicine and the application of tumor-based profiling via next-generation sequencing, diagnosis of HM predisposition occurs with increasing frequency. Although deleterious germline variants can be readily identified by comprehensive clinical testing, numerous barriers exist for many clinicians. Observations regarding particular germline predisposition disorders challenge widely held assumptions about these conditions. Here, we review approaches to germline genetic testing, highlighting key points in a typical patient's course that present challenges for testing and interpreting results. Increasing awareness by health care providers of these conditions and improvements in testing platforms are crucial for enabling a proactive approach to tailoring a suitable treatment plan and surveillance program for the patient and their family members.

Novel Small Molecule, UTS-1401, as a Radioprotector for Total-Body Irradiation.

We report on a new radioprotector, UTS-1401, a small molecule that was synthesized (by one of us, JS) and evaluated here for its radioprotective effect against total-body irradiation (TBI). Female and male NIH Swiss mice were subjected to TBI at doses of 6.5, 7.5 and 8.5 Gy either with or without a 24 h pretreatment of UTS-1401 given ip and observed for 30 days. Survival rates were significantly increased when mice were treated with UTS-1401 compared to those not treated. The radioprotective effect of UTS-1401 was drug-dose dependent for male mice exposed to 8.5 Gy TBI with 150 mg/kg of UTS-1401 as the optimal dose. The radioprotective effect of UTS-1401 on female mice exposed to 8.5 Gy TBI was observed at 50, 100, and 150 mg/kg, with no dose response relationship noted. Female mice were more radioresistant than male mice with LD50/30 values of 7.8 Gy vs. 6.8 Gy, respectively. Weight changes after UTS-1401 alone showed a significant body weight increase at 150 mg/kg. Both the ip and iv route for UTS-1401 were similarly effective for male mice exposed to 8 Gy TBI. Further analysis using an endogenous spleen colony assay demonstrated that pretreatment of UTS-1401 for up to 72h prior to TBI protected both spleen weight and hematopoietic stem cells with a treated/untreated ratio between 2.0 and 3.2 for the latter for times between 0.5 h and 72 h. A separate invivo study showed that pretreatment of UTS-1401 protected bone marrow CFU-GM for mice exposed to TBI. In summary, UTS-1401 is a promising small-molecule radioprotective agent as demonstrated by whole animal, hematopoietic stem cell and bone marrow myeloid progenitor cell survival.

Systemic immunological responses are dependent on sex and ovarian hormone presence following acute inhaled woodsmoke exposure

BackgroundRural regions of the western United States have experienced a noticeable surge in both the frequency and severity of acute wildfire events, which brings significant challenges to both public safety and environmental conservation efforts, with impacts felt globally. Identifying factors contributing to immune dysfunction, including endocrinological phenotypes, is essential to understanding how hormones may influence toxicological susceptibility.MethodsThis exploratory study utilized male and female C57BL/6 mice as in vivo models to investigate distinct responses to acute woodsmoke (WS) exposure with a focus on sex-based differences. In a second set of investigations, two groups were established within the female mouse cohort. In one group, mice experienced ovariectomy (OVX) to simulate an ovarian hormone-deficient state similar to surgical menopause, while the other group received Sham surgery as controls, to investigate the mechanistic role of ovarian hormone presence in driving immune dysregulation following acute WS exposure. Each experimental cohort followed a consecutive 2-day protocol with daily 4-h exposure intervals under two conditions: control HEPA-filtered air (FA) and acute WS to simulate an acute wildfire episode.ResultsMetals analysis of WS particulate matter (PM) revealed significantly increased levels of 63Cu, 182W, 208Pb, and 238U, compared to filtered air (FA) controls, providing insights into the specific metal components most impacted by the changing dynamics of wildfire occurrences in the region. Male and female mice exhibited diverse patterns in lung mRNA cytokine expression following WS exposure, with males showing downregulation and females displaying upregulation, notably for IL-1β, TNF-α, CXCL-1, CCL-5, TGF-β, and IL-6. After acute WS exposure, there were notable differences in the responses of macrophages, neutrophils, and bronchoalveolar lavage (BAL) cytokines IL-10, IL-6, IL-1β, and TNF-α. Significant diverse alterations were observed in BAL cytokines, specifically IL-1β, IL-10, IL-6, and TNF-α, as well as in the populations of immune cells, such as macrophages and polymorphonuclear leukocytes, in both Sham and OVX mice, following acute WS exposure. These findings elucidated the profound influence of hormonal changes on inflammatory outcomes, delineating substantial sex-related differences in immune activation and revealing altered immune responses in OVX mice due to ovarian hormone deficiency. In addition, the flow cytometry analysis highlighted the complex interaction between OVX surgery, acute WS exposure, and their collective impact on immune cell populations within the hematopoietic bone marrow niche.ConclusionsIn summary, both male and female mice, alongside females subjected to OVX and those who had sham surgery, exhibit significant variations in the expression of proinflammatory cytokines, chemokines, lung mRNA gene expression, and related functional networks linked to signaling pathways. These differences potentially act as mediators of sex-specific and hormonal influences in the systemic inflammatory response to acute WS exposure during a wildfire event. Understanding the regulatory roles of genes expressed differentially under environmental stressors holds considerable implications, aiding in identifying sex-specific therapeutic targets for addressing acute lung inflammation and injury.

Current and Future Therapeutics for Treating Patients with Sickle Cell Disease.

Sickle cell disease (SCD) is the most common genetic blood disorder in the United States, with over 100,000 people suffering from this debilitating disease. SCD is caused by abnormal hemoglobin (Hb) variants that interfere with normal red blood cell (RBC) function. Research on SCD has led to the development and approval of several new SCD therapies in recent years. The recent FDA-approved novel gene therapies are potentially curative, giving patients an additional option besides a hematopoietic bone marrow transplant. Despite the promise of existing therapies, questions remain regarding their long-term pharmacological effects on adults and children. These questions, along with the exorbitant cost of the new gene therapies, justify additional research into more effective therapeutic options. Continual research in this field focuses on not only developing cheaper, more effective cures/treatments but also investigating the physiological effects of the current therapies on SCD patients, particularly on the brain and kidneys. In this article, we undertake a comprehensive review of ongoing clinical trials with completion dates in 2024 or later. Our exploration provides insights into the landscape of current therapeutics and emerging novel therapies designed to combat and potentially eradicate SCD, including the latest FDA-approved gene therapies.

Therapeutic use of autologous hematopoietic bone marrow transplant combined with concentrated platelet in infected surgical wounds: single-center prospective study

The healing wound is the consecutive process of maintaining tissue integrity including inflammatory, proliferative, and remodeling processes. Stem cells can self-renew, and also to differentiate into all cell types, and are involved in physiological regeneration, use of mesenchymal stem cells deriving from the bone marrow with PRP has potentiated the healing process. We conducted a single-arm pilot prospective analytical study was performed on 15 patients whose mean age was 31 years (range: 29–51 years), with refractory abdominal wounds at a private hospital in Iraq from March 30, 2022, to March 2023. Ten days after the stem cell transplant the re-enforcement with PRP subcutaneous injection around wounds. Small papules of granulation tissue appeared over the wound, and tissue granulation increased from 80% (30.26–80.00%) to 100% (81–100%). At the same time, the wound size was reduced by 60% (52.10–78.23%). The follow-up period was 7–24 months, during which no recurrence of the wound was found. In conclusion, the current study is the first to characterize the safety and efficacy of bone marrow-derived stem cells in combination with PRP in the treatment of refractory abdominal wound infection. All patients achieved a 100% healing rate in a short time, and no treatment-related adverse reactions occurred during the treatment.

Lessons Learned From Ocular Graft versus Host Disease: An Ocular Surface Inflammatory Disease of Known Time of Onset.

The ocular surface inflammatory disorders (OSIDs) comprise a group of conditions characterized by persistent inflammation of the ocular surface and adnexal tissues. Systemic autoimmune diseases and hypersensitivity reactions cause them, and, if left untreated, can result in severe inflammatory dry eye, corneal damage, and vision loss. Ocular graft-versus-host disease (oGVHD) forms part of the ocular surface inflammatory disease umbrella. It is a condition occurring after allogeneic hematopoietic stem cell or bone marrow transplantation, usually in chronic graft-versus-host disease. oGVHD can virtually affect any ocular adnexal tissue, especially the meibomian glands, and cause persistent inflammation, tissue fibrosis, and subsequent chronic, severe dry eye disease. Among the OSIDs, oGVHD has the particularity that it has a "time zero," meaning we know when the disease started. As such, preclinical models have leveraged this to investigate the molecular mechanisms involved in the damage oGVHD causes to the ocular surface. In oGVHD, establishing a "time zero" allows for predicting the clinical course and establishing adequate treatment. This is also possible because the inflammatory infiltration occurs in ocular surface tissues, which are readily accessible. Using oGVHD, we might be able to understand the immune response mechanisms in other OSIDs better (i.e., Sjögren syndrome, Stevens-Johnson syndrome, among others). This review presents an up-to-date overview of the pathogenesis, clinical presentation, and treatment of oGVHD. In addition, we will discuss the value of the "time zero" concept in the study of oGVHD.

LARYNGEAL PLASMACYTOMA IN KAHLERS DISEASE: A CASE REPORT

Multiple myeloma or Kahlers disease is a malignant hematological disorder due to a monoclonal plasma cell proliferation primarily invading the hematopoietic bone marrow. Extra-medullar involvement occurs in 18% of patients with multiple myeloma. Laryngeal Localization remains rare and has been described in a few cases after a literature review.We report the case of a 44-year-old woman who presented an inspiratory dyspnea caused by a laryngeal tumor. The pathological study and bone marrow examination confirmed the laryngeal localization of multiple myeloma. Treatment consisted of chemotherapy with good clinical outcomes over a 2-year follow-up period.

Generation of complex bone marrow organoids from human induced pluripotent stem cells

The human bone marrow (BM) niche sustains hematopoiesis throughout life. We present a method for generating complex BM-like organoids (BMOs) from human induced pluripotent stem cells (iPSCs). BMOs consist of key cell types that self-organize into spatially defined three-dimensional structures mimicking cellular, structural and molecular characteristics of the hematopoietic microenvironment. Functional properties of BMOs include the presence of an in vivo-like vascular network, the presence of multipotent mesenchymal stem/progenitor cells, the support of neutrophil differentiation and responsiveness to inflammatory stimuli. Single-cell RNA sequencing revealed a heterocellular composition including the presence of a hematopoietic stem/progenitor (HSPC) cluster expressing genes of fetal HSCs. BMO-derived HSPCs also exhibited lymphoid potential and a subset demonstrated transient engraftment potential upon xenotransplantation in mice. We show that the BMOs could enable the modeling of hematopoietic developmental aspects and inborn errors of hematopoiesis, as shown for human VPS45 deficiency. Thus, iPSC-derived BMOs serve as a physiologically relevant in vitro model of the human BM microenvironment to study hematopoietic development and BM diseases.

Cross-Modal Imaging Reveals Nanoparticle Uptake Dynamics in Hematopoietic Bone Marrow during Inflammation.

Nanoparticles have been employed to elucidate the innate immune cell biology and trace cells accumulating at inflammation sites. Inflammation prompts innate immune cells, the initial responders, to undergo rapid turnover and replenishment within the hematopoietic bone marrow. Yet, we currently lack a precise understanding of how inflammation affects cellular nanoparticle uptake at the level of progenitors of innate immune cells in the hematopoietic marrow. To bridge this gap, we aimed to develop imaging tools to explore the uptake dynamics of fluorescently labeled cross-linked iron oxide nanoparticles in the bone marrow niche under varying degrees of inflammation. The inflammatory models included mice that received intramuscular lipopolysaccharide injections to induce moderate inflammation and streptozotocin-induced diabetic mice with additional intramuscular lipopolysaccharide injections to intensify inflammation. In vivo magnetic resonance imaging (MRI) and fluorescence imaging revealed an elevated level of nanoparticle uptake at the bone marrow as the levels of inflammation increased. The heightened uptake of nanoparticles within the inflamed marrow was attributed to enhanced permeability and retention with increased nanoparticle intake by hematopoietic progenitor cells. Moreover, intravital microscopy showed increased colocalization of nanoparticles within slowly patrolling monocytes in these inflamed hematopoietic marrow niches. Our discoveries unveil a previously unknown role of the inflamed hematopoietic marrow in enhanced storage and rapid deployment of nanoparticles, which can specifically target innate immune cells at their production site during inflammation. These insights underscore the critical function of the hematopoietic bone marrow in distributing iron nanoparticles to innate immune cells during inflammation. Our findings offer diagnostic and prognostic value, identifying the hematopoietic bone marrow as an imaging biomarker for early detection in inflammation imaging, advancing personalized clinical care.

Differentiation Potential Analysis of Bone Marrow Mesenchymal Stem Cells from Guizhou Miniature Pigs

In this study, different growth characteristics of hematopoietic stem cells and bone marrow mesenchymal stem cells were used to isolate and culture BMSCs by whole bone marrow adhesion method, and RT-PCR was used to detect cell surface markers CD29, CD31, CD34, CD44, CD71, CD73, CD90. The differentiation of osteocytes into adipocytes and osteoblasts was induced in vitro, and the differentiation ability was observed by staining with oil red O and rubinin. To investigate the isolation and culture methods of BMSCs from Guizhou miniature pigs (Sussa lvanius) in vitro, and to study their biological characteristics and potential for multi-differentiation. The results showed that a small number of adherent cells could be seen in the primary BMSCs cultured in vitro within 48 hours, and the confluent cells reached 90% in 5~6 days. The cell growth state was good, and the shape was spindle and polygonal. RT-PCR was used to detect the positive expression of CD29, CD44, CD71, CD73 and CD90 on the cell surface, and the negative expression of CD31 and CD34. Oil red O staining showed the formation of lipid droplets in the cells, and alizarine red staining showed calcium nodules in the cells, indicating that the cells isolated and cultured in this experiment had the ability of multidirection differentiation. It is consistent with the characteristics of BMSCs. The growth of BMSCs cultured in vitro was stable, and the expanded BMSCs had multidirectional differentiation potential, which was an ideal seed cell for tissue engineering.

Thrombopoietin mimetic therapy alleviates radiation-induced bone marrow vascular injury in a bone marrow transplant mouse model.

There is a need for therapies that can mitigate bone marrow dysfunction and organ toxicity that occur following myeloablative injury and reduced intensity conditioning regimens used in patients undergoing bone marrow transplantation (BMT). The pathogenesis of adverse effects from BMT conditioning has been linked to injury to the vascular endothelium, bone marrow (BM), and other organs. To evaluate the impact of the thrombopoietin mimetic drug JNJ-26366821 (TPOm) on BM vascular recovery in mice undergoing myeloablative radiation conditioning followed by BMT. TPOm (doses: 0 µg, 300 µg, 1000 µg per Kg body weight) was administered on Days 0 and 7 after BMT, in mice receiving a total body irradiation (TBI) conditioning regimen (5.5 Gy x 2) before congenic BMT. BM donner cell engraftment was analyzed using flow cytometry on Days 7, 14, and 30 post-BMT. The morphological and biophysical properties of the BM vasculature were evaluated by intravital multiphoton microscopy (MPM) and immunofluorescence confocal imaging. Herein, morphological properties involve microvascular density (MVD), vessel diameter, and vascular area, while biophysical properties include transfer rate (Ktrans) of contrast within the BM vascular niche, as well as the fractional volume (vec ) of extracellular extravascular tissue (EES). No significant difference in donor chimerism was observed at days 7, 14, and 30 post-BMT, between TPOm and PBS-treated mice. TPOm intervention improved BM vasculature regeneration in transplanted mice. The MVD, Ktrans, and BM vasculature as well as vascular endothelial growth factor receptor-2 (VEGFR2) in the BM, showed a dose dependent improvement in mice treated with TPOm. On day 14 post-BMT, the group receiving 1000 µg/Kg TPOm showed significant shifts (p-value < 0.05) in MVD, Ktrans, and VEGFR2 expression from their corresponding control types (TPOm dose 0 µg) towards levels comparable to healthy controls. TPOm intervention augments BM vascular structure and function, which may be important for hematopoietic recovery and bone marrow function in radiation conditioned hematopoietic stem cell transplant patients, in addition to enhancing platelet recovery.

Lipotoxicity-Related Hematological Disorders in Obesity.

Lipotoxicity can mediate endothelial dysfunction in obesity. Altered endothelial cell phenotype during the pathobiological course of the lipotoxicity may lead to hemostatic abnormalities, which is a hallmark of several hematological disorders. Impaired hemostasis could also be directly related to numerous metabolic diseases such as hypertension, diabetes, and atherosclerosis. On the other hand, the local hematopoietic bone marrow (BM) renin-angiotensin system (RAS) contributes to the development of atherosclerosis via acting on the lipotoxicity processes. Local BM RAS, principally an autocrine/paracrine/intracrine hematological system, is located at the crossroads of cellular regulation, molecular interactions, and lipotoxicity-mediated vascular endothelial dysfunction. The positive regulatory role of plasma LDL on AT1 receptor-mediated hematopoietic stem cell (HSC) differentiation and the production of pro-atherogenic monocytes have been described. LDL-regulated HSC function may explain in part hypercholesterolemia-induced inflammation as well as the anti-inflammatory and anti-atherosclerotic effects of AT1 receptor blockers. The role of local adipose tissue RAS is directly related to the pathogenesis of metabolic derangements in obesity. There may be a crosstalk between local BM RAS and local adipose tissue RAS at the genomics and transcriptomics levels. This chapter aims to review hematological alterations propagating the pathological influences of lipotoxicity on the vascular endothelium.

Особливості клінічного перебігу анапластичної крупноклітинної лімфоми в дітей. Клінічний випадок

Anaplastic large cell lymphoma (ALCL) is a rare type of T-cell non-Hodgkin lymphoma with a characteristic primary localized, mainly extra-bone marrow tumor growth of lymphoid tissue, the clinical picture of which includes lesions of peripheral lymph nodes and extranodal organs, such as bones, skin, lungs, liver, muscles which tissues and bone marrow. Purpose - to acquaint practicing doctors with the features of clinical manifestations, differential diagnosis and course of ALCL in children. Features of the clinical course of ALCL in a child are described. The main diagnostic value of the anamnesis, clinical picture, laboratory, instrumental, immunohistochemical and microscopic research methods, in particular trepan biopsy of this tumor, is highlighted. Microscopic examination of the tissue trepanbioptate show that in half of the cells of the bone marrow, its normal structure is revealed, hematopoietic bone marrow is present with the preservation of all sprouts, with a slightly increased number of megakaryocytes. Half of the cells are filled with solid layers of sharply atypical lymphocyte-like cells with a moderate amount of eosinophilic cytoplasm, the nuclei of tumor cells are large, vesicular, with the formation of 1-2 basophilic nuclei. In the tumor cells there are figures of mitosis, including atypical ones. Single hematopoietic cells of various germs are found between these cells. The conducted immunohistochemical study of tumor cells show that they are positive for common leukocyte antigen (CD4), negative for common cytokeratins, S-100, myogenin, MuoD1, which indicates their hematopoietic origin. Upon further examination, the tumor cells were positive for CD 30, negative for CD3, CD20, CD 43, CD 68, CD11c, tdt, myeloperoxidase, CD 1a. Staining for CD246 reveals a granular cytoplasmic reaction in tumor cells. The histological pattern and immunophenotype of tumor cells correspond to ALCL. The research was carried out in accordance with the principles of the Helsinki Declaration. The informed consent of the patient was obtained for conducting the studies. No conflict of interests was declared by the authors.

Machine Learning Algorithm: Texture Analysis in CNO and Application in Distinguishing CNO and Bone Marrow Growth-Related Changes on Whole-Body MRI.

The purpose of this study is to analyze the texture characteristics of chronic non-bacterial osteomyelitis (CNO) bone lesions, identified as areas of altered signal intensity on short tau inversion recovery (STIR) sequences, and to distinguish them from bone marrow growth-related changes through Machine Learning (ML) and Deep Learning (DL) analysis. We included a group of 66 patients with confirmed diagnosis of CNO and a group of 28 patients with suspected extra-skeletal systemic disease. All examinations were performed on a 1.5 T MRI scanner. Using the opensource 3D Slicer software version 4.10.2, the ROIs on CNO lesions and on the red bone marrow were sampled. Texture analysis (TA) was carried out using Pyradiomics. We applied an optimization search grid algorithm on nine classic ML classifiers and a Deep Learning (DL) Neural Network (NN). The model's performance was evaluated using Accuracy (ACC), AUC-ROC curves, F1-score, Positive Predictive Value (PPV), Mean Absolute Error (MAE) and Root-Mean-Square Error (RMSE). Furthermore, we used Shapley additive explanations to gain insight into the behavior of the prediction model. Most predictive characteristics were selected by Boruta algorithm for each combination of ROI sequences for the characterization and classification of the two types of signal hyperintensity. The overall best classification result was obtained by the NN with ACC = 0.91, AUC = 0.93 with 95% CI 0.91-0.94, F1-score = 0.94 and PPV = 93.8%. Between classic ML methods, ensemble learners showed high model performance; specifically, the best-performing classifier was the Stack (ST) with ACC = 0.85, AUC = 0.81 with 95% CI 0.8-0.84, F1-score = 0.9, PPV = 90%. Our results show the potential of ML methods in discerning edema-like lesions, in particular by distinguishing CNO lesions from hematopoietic bone marrow changes in a pediatric population. The Neural Network showed the overall best results, while a Stacking classifier, based on Gradient Boosting and Random Forest as principal estimators and Logistic Regressor as final estimator, achieved the best results between the other ML methods.

Phase 1 Trial Results for Patients with Advanced Hematologic Malignancies Undergoing Reduced Intensity Allogeneic HCT with Orca-T Donor Cell Therapy Product and Single Agent Tacrolimus

Introduction: Reduced intensity conditioning hematopoietic bone marrow stem cell transplantation (HSCT-RIC) is the only curative option for many hematological malignancies in older/unfit patients. Strategies to minimize complications such as graft versus host disease (GVHD) in HSCT include donor graft engineering but this approach has not been widely tested to the RIC setting due to a potential trade-off between engraftment and reduced GVHD with T cell depletion strategies. Here we present the interim results of our single center Phase I trial evaluating the T cell reduced Orca-T cell therapy product and single agent tacrolimus in the RIC setting. Methods: The trial is a single-center open-label phase 1 feasibility and safety study for dose escalation of conventional T cell (Tcon) infusion. Primary objectives included measuring the incidence of grade III-IV acute GVHD, time of engraftment and donor T cell chimerism at day +30. Secondary objectives are measuring relapse free survival, severity of GVHD and incidence of serious infections. While the study has three independent trial arms depending on donor type (HLA matched related and unrelated, 9/10 HLA matched or haploidentical matched), accrual has focused on the HLA-matched cohort which is reported here. The first 11 patients on this arm were conditioned with fludarabine 40mg/m 2, melphalan 50 mg/m 2 and 4 Gy of total body irradiation (TBI), an additional 4 patients have been treated with thiotepa 10 mg/kg replacing the melphalan. All patients received single agent tacrolimus with a target goal of 6-8 ng/ml. Results: A total of 15 HLA-matched patients have been recruited thus far in this interim dose escalation analysis. The median patient age is 68.0 years (range 62 to 72), with 73% male (11/15) patient distribution. Pre transplant, 53% had acute myeloid leukemia (8/15), 20% had myelodysplastic syndrome (3/15), 13% had acute lymphoblastic leukemia (2/15) and 13% had a myeloproliferative neoplasm (2/15). Prior to transplant, all acute leukemia patients were in complete remission (CR) or CR with incomplete count recovery. Median follow up was 10.4 months for the entire cohort (range 2 to 20 months). 14/15 patients had engraftment by day 20 and 1/15 at day 39 post-transplant. Based on these findings, no dose escalation of Tcon infusion was needed. None of the patients had infusion reactions on day of transplant nor engraftment syndrome. Median percent donor chimerism for CD15 was high, with 100% at day 30 (15/15, range 94% to 100%) and 99% (13/15, patients with available data, range 98% to 100%) at day 90. Median CD3 chimerism at days 30 and 90 of transplant were also high at 99% (15/15, range 68% to 100%) and 99% (for 13/15 patients with available data, range 78% to 100%) respectively. No patients developed grade III-IV acute GVHD ( Fig.), defined &amp;lt;100 days post-transplant. One patient developed grade II aGVHD. In 12 months, the incidence for moderate to severe chronic GVHD was 7%. The 12-month relapse free survival (RFS) is thus far 79% ( Fig.). The cumulative incidence for grade 2 and 3 infections in the first 90 days was 26.7%, where 2 patients died. One patient died at day 180 from liver biopsy complications for late onset hepatic sinusoidal obstruction syndrome. No surviving patient has had disease relapse. Conclusions: These early results demonstrate that Orca-T is a safe and feasible transplant strategy in the RIC setting for patients with advanced hematological malignancies. Patients showed robust and early donor myeloid and eventual full donor T cell engraftment. The low incidence of acute and chronic GVHD with low rates of disease relapse, suggest retention of graft versus leukemia effect. Few studies of donor graft-engineered products have been successfully evaluated in the RIC setting and merits larger multicenter studies evaluatingGVHD-free and RFS post-transplant to validate these findings.

Human Induced Pluripotent Stem Cell-Derived Bone Marrow Organoids to Model Hematopoietic Development

The human bone marrow niche plays an essential role in supporting and regulating hematopoiesis throughout life and inherited bone marrow failure syndromes are often linked to dysfunction of the niche. Recently, methods for generating induced pluripotent stem cell (iPSC)-derived hematopoietic organoids (Motazedian et al., 2020) and bone marrow organoids (Khan et al., 2022) have been developed. We recently presented our approach to generate a model of a complex human bone marrow microenvironment in vitro (ASH annual meeting 2022 ). Here, we characterize our model in greater detail and analyse functionality of bone marrow organoid (BMO)-derived HSPCs. We devised a feeder- and serum-free protocol to generate a complex human iPSC-derived BMO within three weeks. In comparison to the protocol by Khan et al., we incorporated a sequential combination of Wnt activation and Activin/Nodal inhibition to induce mesoderm formation and patterning. We replicated the method with five different iPSC lines, including fibroblast- and PBMC-derived iPSCs, as well as iPSCs generated from renal epithelial cells isolated from urine samples. Using confocal and two-photon microscopy we visualized the spatial architecture of differentiated BMOs and detected CD45 + cells embedded into a network of CD31 + vascular cells and CD271 + stromal cells. PDGFRβ expressing pericytes were enwrapping the vascular cells. Moreover, stromal cells expressed CXCL12, Leptin receptor and Nestin. Electron microscopy, HE staining of organoid sections and 3D surface rendering showed lumen-forming vascular structures containing hematopoietic cells. To further elucidate the cell type composition, we performed single-cell RNA sequencing (scRNA-seq) of differentiated BMOs. Clustering of the scRNA-seq data by marker gene expression yielded three main populations: Cells in the endothelial cluster expressed canonical genes of arterial ECs. In the mesenchymal cluster we could distinguish pericytes, vascular smooth muscle cells and osteochondrogenic precursors. In the hematopoietic compartment, we identified cells expressing molecular signatures of granulocytes, monocytes and their progenitors, but also megakaryocytic-like cells and lymphoid progenitors. Remarkably, a cluster of cells expressed genes of fetal HSCs. This suggests that BMOs may recapitulate definitive hematopoiesis. To test T-cell differentiation potential in vitro, we sorted CD34 + HSPCs from dissociated BMOs and differentiated them within an artificial thymic organoid system (ATO). After three weeks we detected CD45 +CD4 +CD8 + cells, some of them expressing CD3 +TCRαβ +. Thus, we postulate that T-cells phenotypically resembling conventional T-cells emerge from BMO-derived HSPCs. To assess functional properties of BMO-derived HSPCs in vivo, we transplanted sorted CD34 + HSPCs into immunodeficient NSG mice and are currently analysing their engraftment potential. In summary, we provide a novel bone marrow niche model to study hematopoietic development in a complex three-dimensional organoid derived from human iPSCs.

Proliferation and apoptosis after whole-body irradiation: longitudinal PET study in a mouse model

PurposeA reliable method for regional in vivo imaging of radiation-induced cellular damage would be of great importance for the detection of therapy-induced injury to healthy tissue and the choice of adequate treatment of radiation emergency patients in both civilian and military events. This study aimed to investigate in a mouse model if positron emission tomography (PET) imaging with proliferation and apoptosis markers is potentially suitable for this purpose.MethodsFour groups, including twenty mice (wild-type C57BL/6) each, were whole-body irradiated with 0 Gy, 0.5 Gy, 1 Gy, and 3 Gy and examined by PET over a six-month period at defined time points. 3'-[18F]fluoro-3'-deoxythymidine ([18F]FLT) and 2-(5-[18F]fluoropentyl)-2-methyl malonic acid ([18F]ML-10) were used to visualise proliferation and apoptosis. Regional standard uptake values were compared with respect to irradiation dose over time. Histologic data and peripheral blood cell values were correlated with the PET results.ResultsThe hematopoietic bone marrow showed a significantly increased [18F]FLT signal at early time points after radiation exposure (day 3 and day 7). This correlated with blood parameters, especially leukocytes, and histological data. A significantly increased [18F]FLT signal also occurred in the gastrointestinal tract and thymus at early time points. An increased [18F]ML-10 signal related to irradiation doses was observed in the bone marrow on day 8, but there was a high variability of standard uptake values and no correlation with histological data.Conclusion[18F]FLT showed potential to visualise the extent, regional distribution and recovery from radiation-induced cellular damage in the bone marrow, gastrointestinal tract and thymus. The potential of [18F]FLT imaging to assess the extent of bone marrow affected by irradiation might be especially useful to predict the subsequent severity of hematopoietic impairment and to adapt the therapy of the bone marrow reserve. [18F]ML-10 PET proved to be not sensitive enough for the reliable detection of radiation induced apoptosis.