Immunoglobulin light-chain (AL) amyloidosis is a disorder caused by a misfolded light chain, produced by a clonal population of plasma cells in the bone marrow, that deposits in tissues and causes organ dysfunction. It is recently reported that microenvironment defect may either lead to or favor the pathogenesis of different myelodysplastic syndromes. Since Mesenchymal Stromal Cells (MSC) represent one of the key components of the BM niche, in the present study we evaluated MSC of AL patients. MSC from Bone Marrow (BM) of 40 AL patients and from BM of 3 Healthy Donors (HD) were expanded following standard in vitro culture procedure. MSC were characterized by plastic adhesion, morphology, clonogenic capacity (defined as CFU-F/106 plated MNC at P0), immunophenotype by flow-cytometry, adipogenic and osteogenic differentiation and proliferation capacity defined as Population Doubling (PD) [log(harvested cells/seeded cells)/log2]. BM Mononuclear cells (MNC) from 25 AL patients were cultured both before (AL-MSC) and after CD138 immunomagnetic depletion (AL-CD138neg-MSC). Statistical analysis was not performed due to the low number of patients considered nowadays. MSC were isolated from all patients and HD. Morphology, immunophenotype, osteogenic and adipogenic differentiation, and clonogenic capacity were comparable in MSC from AL patients and HD. On the contrary, AL-MSC display a reduced proliferative capacity compared to HD-MSC. Moreover, AL-CD138neg-MSC showed lower PD than AL-MSC. AL-MSC arrested their growth (number of harvested cells ≤ number of plated cells) at earlier passages compared to HD-MSC. Our data suggest some discrepancies between AL-MSC and HD-MSC; whether these features are involved in the pathophysiology of the disease or are caused by the disease itself, deserves further investigation.
L’amiloidosi da catene leggere (AL) è una patologia causata dalla proliferazione incontrollata di un clone di plasmacellule nel midollo osseo (MO) che secerne catene leggere. Queste catene si depositano sotto forma di fibrille, compromettendo la funzionalità di numerosi organi e tessuti. L’ obiettivo della tesi consiste nella caratterizzazione di Cellule Stromali Mesenchimali (MSC) derivanti da MO di pazienti affetti da amiloidosi AL, al fine di evidenziarne eventuali alterazioni morfologiche e funzionali che potrebbero essere coinvolte nella patogenesi della malattia. Le MSC sono state espanse in vitro dal MO di 40 pazienti e di 3 soggetti sani (HD-MSC) secondo procedure standard. In 25 campioni le MSC sono state espanse sia prima (AL-MSC) che dopo immuno-deplezione delle plasmacellule CD138+ (AL-CD138neg-MSC). Le AL-MSC e AL-CD138neg-MSC sono state caratterizzate per morfologia, fenotipo, capacità clonogenica (CFU-F/106 MNC al passaggio P0), proliferativa (cPD) e differenziativa, risultando simili alle HD-MSC. Le MSC sono state espanse fino alla senescenza (n.cellule staccate ≤ n.cellule piastrate). Le AL-MSC hanno invece mostrato una capacità clonogenica significativamente più elevata rispetto alle AL-CD138neg-MSC. Inoltre, le AL-MSC e AL-CD138neg-MSC raggiungono lo stato di senescenza a passaggi significativamente più precoci rispetto alle HD-MSC. I risultati ottenuti indicano l’esistenza di alcune differenze tra le AL-MSC e le HD-MSC; sono tuttavia necessari ulteriori studi al fine di poter stabilire se tali discrepanze siano coinvolte nella patogenesi della malattia o siano causate dalla malattia stessa.
Microambiente midollare in pazienti affetti da amiloidosi da catene leggere: isolamento, espansione e caratterizzazione di cellule stromali mesenchimali
AMATO, MARIANNA
2017/2018
Abstract
Immunoglobulin light-chain (AL) amyloidosis is a disorder caused by a misfolded light chain, produced by a clonal population of plasma cells in the bone marrow, that deposits in tissues and causes organ dysfunction. It is recently reported that microenvironment defect may either lead to or favor the pathogenesis of different myelodysplastic syndromes. Since Mesenchymal Stromal Cells (MSC) represent one of the key components of the BM niche, in the present study we evaluated MSC of AL patients. MSC from Bone Marrow (BM) of 40 AL patients and from BM of 3 Healthy Donors (HD) were expanded following standard in vitro culture procedure. MSC were characterized by plastic adhesion, morphology, clonogenic capacity (defined as CFU-F/106 plated MNC at P0), immunophenotype by flow-cytometry, adipogenic and osteogenic differentiation and proliferation capacity defined as Population Doubling (PD) [log(harvested cells/seeded cells)/log2]. BM Mononuclear cells (MNC) from 25 AL patients were cultured both before (AL-MSC) and after CD138 immunomagnetic depletion (AL-CD138neg-MSC). Statistical analysis was not performed due to the low number of patients considered nowadays. MSC were isolated from all patients and HD. Morphology, immunophenotype, osteogenic and adipogenic differentiation, and clonogenic capacity were comparable in MSC from AL patients and HD. On the contrary, AL-MSC display a reduced proliferative capacity compared to HD-MSC. Moreover, AL-CD138neg-MSC showed lower PD than AL-MSC. AL-MSC arrested their growth (number of harvested cells ≤ number of plated cells) at earlier passages compared to HD-MSC. Our data suggest some discrepancies between AL-MSC and HD-MSC; whether these features are involved in the pathophysiology of the disease or are caused by the disease itself, deserves further investigation.È consentito all'utente scaricare e condividere i documenti disponibili a testo pieno in UNITESI UNIPV nel rispetto della licenza Creative Commons del tipo CC BY NC ND.
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https://hdl.handle.net/20.500.14239/20152