Haemogenic Gastruloids Recapitulate Developmental Haematopoiesis and Provide an Ontogeny-Relevant Context to Dissect the Origins of Infant Leukemia

Meeting abstract presented at the 64th ASH Annual Meeting and Exposition, New Orleans, LA, USA, 10-13 Dec 2022..Modelling of developmental hematopoiesis has historically been challenging due to the inability to produce hematopoietic stem cells (HSC) and recapitulate microenvironment interactions ex vivo. Gastruloids are 3D aggregates of embryonic stem (ES) cells which display developmentally-specific spatial and temporal organization that recapitulate gastrulation. We adapted the gastruloid protocol to introduce hematopoietic signalling cues, and generated an in vitro model of embryonic hematopoiesis that sequentially recapitulates the formation of hemogenic endothelium, hematopoietic progenitors, and pre-HSC, over a culture period of 216 hours. Flow cytometry analysis detected the presence of c-Kit+ endothelium at 120h, followed by emergence of CD41+ hematopoietic progenitors at 144h, and the appearance of CD45+ cells from 192h. CD45+ cells were observed in small clusters adjoining endothelium-lined structures, reminiscent of developmental hemogenic-to-endothelial transition and intra-aortic clusters. Single-cell RNA sequencing revealed specification of pre-definitive and definitive waves of embryonic hematopoiesis, aligning 144h-CD41+ cells with erythro-myeloid progenitors (EMP), and late CD45+ with lympho-myeloid progenitors and pre-HSC, altogether supporting the hemogenic gastruloid as a model that is temporally and topographically congruous with the embryo. The close recapitulation of developmental ontogeny led us to explore hemogenic gastruloids to understand cell and stage-specific susceptibility to forms of Acute Myeloid Leukaemia exclusively observed in infants. The chromosomal translocation t(7;12)(q36;p13), characterized by the ectopic overexpression of the MNX1 gene, is found in up to one third of infant AML cases, but has been challenging to model using conventional strategies, largely due to the inability of MNX1 to transform adult hematopoietic cells. The age-selectivity of t(7;12) has been proposed to reflect a transient developmental window for a target cell of origin absent in adult life, but its nature is yet to be defined. In order to identify the context of MNX1-driven leukemogenesis, we produced hemogenic gastruloids using lentiviral-transduced mouse ES cells in which we overexpressed MNX1 as a proxy of t(7;12). Although MNX1 did not interfere with ES cell pluripotent cultures, it primed incipient hemogenic programmes and promoted hemogenic gastruloid formation. Critically, expression of MNX1 resulted in transformation of gastruloid-derived hematopoietic cells, as assessed by serial colony-forming cell replating, with expansion of a phenotypic myeloid cell, a phenomenon not observed in adult tissues. Detailed analysis of the cellular composition of MNX1-overexpressing hemogenic gastruloids revealed a significant effect in the output of CD41+ and c-Kit+ populations at 144h, but no effect in CD45+ cells at 192-216h, suggesting that the target of MNX1 lies within the EMP stage, an observation supported by single-cell RNA-seq analysis of MNX1 vs control gastruloids. Systematic comparison of the temporal transcriptional profiles of hemogenic gastruloids, MNX1-overexpressing gastruloids, and t(7;12) patients, pinpoints the target cell of MNX1 at the HE-to-EMP transition. In summary, we propose a novel model of embryonic hematopoiesis capable of capturing developmentally-relevant cellularity and topography of the early hematopoietic microenvironment, with the ability to mechanistically elucidate developmental associations of infant leukemia

Matrix metalloproteinases in human melanoma cell lines and xenografts: increased expression of activated matrix metalloproteinase-2 (MMP-2) correlates with melanoma progression

Matrix metalloproteinases (MMPs) and their tissue inhibitors (TIMPs) are involved in tumour progression and metastasis. In this study, we investigated the in vitro and in vivo expression patterns of MMP-1, MMP-2, MMP-3, MMP-9, TIMP-1 and TIMP-2 mRNA and protein in a previously described human melanoma xenograft model. This model consists of eight human melanoma cell lines with different metastatic behaviour after subcutaneous (s.c.) injection into nude mice. MMP-1 mRNA was detectable in all cell lines by reverse transcription polymerase chain reaction (RT-PCR), but the expression was too low to be detected by Northern blot analysis. No MMP-1 protein could be found using Western blotting. MMP-2 mRNA and protein were present in all cell lines, with the highest expression of both latent and active MMP-2 in the highest metastatic cell lines MV3 and BLM. MMP-3 mRNA was expressed in MV3 and BLM, and in the non-metastatic cell line 530, whereas MMP-3 protein was detectable only in MV3 and BLM. None of the melanoma cell lines expressed MMP-9. TIMP-1 and TIMP-2 mRNA and protein, finally, were present in all cell lines. A correlation between TIMP expression level and metastatic capacity of cell lines, however, was lacking. MMP and TIMP mRNA and protein expression levels were also studied in s.c. xenograft lesions derived from a selection of these cell lines. RT-PCR analysis revealed that MMP-1 mRNA was present in MV3 and BLM xenografts, and to a lesser extent in 530. Positive staining for MMP-1 protein was found in xenograft lesions derived from both low and high metastatic cell lines, indicating an in vivo up-regulation of MMP-1. MMP-2 mRNA was detectable only in xenografts derived from the highly metastatic cell lines 1F6m, MV3 and BLM. In agreement with the in vitro results, the highest levels of both latent and activated MMP-2 protein were observed in MV3 and BLM xenografts. With the exception of MMP-9 mRNA expression in 530 xenografts, MMP-3, MMP-9, and TIMP-1 mRNA and protein were not detectable in any xenograft, indicating a down-regulated expression of MMP-3 and TIMP-1 in vivo. TIMP-2 mRNA and protein were present in all xenografts; interestingly, the strongest immunoreactivity of tumour cells was found at the border of necrotic areas. Our study demonstrates that of all tested components of the matrix metalloproteinase system, only expression of activated MMP-2 correlates with increased malignancy in our melanoma xenograft model, corroborating an important role of MMP-2 in human melanoma invasion and metastasis. © 1999 Cancer Research Campaig

Standards of reporting in open and endovascular aortic surgery (STORAGE guidelines)

The number of patients undergoing surgery on the thoracic and thoraco-abdominal aorta has been steadily increasing over the past decade. This document aims to give guidance to authors reporting on results in aortic surgery by clarifying definitions of aortic pathologies, open and endovascular techniques and by listing clinical parameters that should be provided for full presentation of patients' clinical profile and in particular, their outcome. The aim is to help find a common language in the treatment of aortic disease and to contribute to a better understanding of this patient population

Multispectral Mueller matrix imaging dark-field microscope for biological sample observation

Mueller polarimetric imaging in dark-field observation shows a contrast enhancement between healthy and cancerous human colon tissue in some reports. We have developed a Mueller-matrix microscope system that combines a dark-field polarization illuminator with an imaging polarimeter to measure the polarization characteristics of scattered light from human colon tissue samples. A multichannel light source permits the acquisition of multispectral Mueller matrices of the sample. The wavelength and polarization state selections are automated, as is the Mueller matrix measurement. The imaging polarimeter permits the system to perform fast, stable measurements. Calibration allows us to reduce the error associated with the illumination and imaging optics in the microscope system. Our system indicates a clear difference between the average Mueller matrix measurements of healthy and cancerous human colon tissue, which agrees well with previously reported results.This item from the UA Faculty Publications collection is made available by the University of Arizona with support from the University of Arizona Libraries. If you have questions, please contact us at [email protected]