DTA

Digital Theses Archive

 

Tesi etd-09302020-181145

Type of thesis
Dottorato
Author
PANVINI, FRANCESCA MARGHERITA
URN
etd-09302020-181145
Title
Ex vivo study of the human haematopoietic stem cell niche and generation of humanized ossicles in mice
Scientific disciplinary sector
BIO/17
Course
Istituto di Scienze della Vita - TRANSLATIONAL MEDICINE
Committee
Presidente Prof. EMDIN, MICHELE
Membro Prof. PAOLICCHI, ALDO
Membro Dott. MÉNDEZ-FERRER, SIMÓN
Membro Prof. PETRINI, MARIO
Membro Dott.ssa ANGELONI, DEBORA
Membro Prof. PASSINO, CLAUDIO
Keywords
  • bone marrow
  • hematopoietic stem-cell niche
  • capillaries
  • humanized ossicle
  • Mesangiogenic progenitor cells
Exam session start date
22/12/2020;
Availability
parziale
Abstract
Haematopoiesis is hosted, supported and regulated by special bone marrow (BM) microenvironments known as “niches”, consisting of distinct cell types communicating by a unique dense vascular network. Although the BM microenvironment has been widely studied in mice, our knowledge about the human BM architecture is much more limited. We designed a double approach to study the human hematopoietic niche: the histological analysis of human BM trephine biopsies (BMTs) by confocal microscopy and the evaluation of isolated human BM cells as candidates to generate a niche model when transplanted into nude mice. In particular, we used a combination of endothelial and perivascular markers to dissect the blood vessel network in healthy BMTs, reporting the expression of NESTIN in BM endothelial cells (BMECs) of small arteries (A) and endosteal arterioles (EA) while sinusoids were NESTIN negative. Besides, NESTIN was detected in very small vessels that we named NESTIN+ capillary-like tubes (NCLTs) and in a few clustered or single NESTIN+ cells (NCs), not surrounded by perivascular cells. Interestingly, NCLTs were detected within 40 μm from bone trabeculae similarly to EA but more frequently found in direct contact to the bone line. Considering their peculiar characteristics and micro-anatomical localisation, NCLTs were proposed as part of transitional vessels in the BM arterio-venous junctions. Interestingly, data distribution analysis revealed significant spatial correlation among NCLTs and haematopoietic stem-progenitor cells, suggesting a possible involvement of these micro-vessels in the regulation of haematopoietic cell fate in humans. A deeper understanding of these mechanisms may be greatly relevant for clinical stem cell transplantation and mobilization protocols and in describing the perturbation of the BM homeostasis that leads to haematological diseases. Additionally, following the recently proposed methods to generate humanized ossicles as haematopoietic niche study models, we evaluated the ability of NESTIN-positive Mesangiogenic Progenitors cells (MPCs) to build an ectopic BM when implanted subcutaneously in nude mice. We treated mice with cell suspensions and with MPCs-derived sprouted spheroids. Implants of MPCs showed osteoid-like histology, rich in collagen fibres and NESTIN positive vessels. By contrast, MPCs sprouted spheroids implants displayed immature BM-like histology characterized by unilocular adipocytes, osteoid-like tissue and the presence of ENDOMUCIN blood vessels. Taken together, these findings could suggest a distinct behaviour of MPCs depending on the strategy chosen to inoculate the mice. Indeed, cell suspension treatment could have relevance as a novel clinical application for improving bone fracture healing protocols. Instead, MPCs-derived sprouted spheroids implants may be good candidates for the generation of an ectopic humanized BM in mice.
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