Human multipotent mesenchymal stromal cells - bone differentiation and hematopoietic support

Lidské mezenchymové stromální buňky (hMSC) jsou dospělé kmenové či progenitorové buňky, jejichž fyziologickou úlohou je napomáhat reparaci poškozených tkání. To se děje především sekrecí trofických, angiopoetických a imunomodulačních faktorů. Kromě toho mají hMSC potenciál diferencovat se in vitro do různých specializovaných buněčných typů, převážně mezodermové linie. Lidské MSC rovněž významně podporují krvetvorbu v rámci hematopoetické niche. Tyto poznatky vzbudily velké naděje na terapeutické využití hMSC, zejména v oblasti regenerativní medicíny a léčby autoimunních onemocnění, včetně reakce štěpu proti hostiteli (GvHD). Jako důkaz správnosti tohoto konceptu posloužily zpočátku hrubé preparáty mononukleárních buněk kostní dřeně (BMMC), které malá množství hMSC buněk obsahují. Na našem pracovišti byly provedeny pilotní klinické studie s BMMC v léčbě akutního infarktu myokardu (předčasně ukončená neúspěšná studie) a v léčbě ischemické choroby dolních končetin (studie se slibnými výsledky). Další výzkum probíhal s cílem optimalizace metodiku kultivace hMSC pro klinické užití, tedy dosažení co největších výtěžků a zbavení postupu xenogenních bílkovin. Lidské MSC byly totiž klasicky kultivovány v research grade médiích (např. alfa-MEM) s fetálním telecím sérem (FCS), které může vést k imunizaci...Human mesenchymal stromal cells (hMSC) are adult stem or progenitor cells, which physiological role is reparation of damaged tissues. This is achieved mostly by secretion of trophic, angiopoietic and immunomodulatory factors. Beside this, hMSC have potential to differentiate in vitro into specialized cells, especially of the mesodermal lineages. Human MSC also significantly support hematopoiesis in hematopoietic niche. This knowledge raised high hopes for therapeutic use of hMSC, especially in regenerative medicine and treatment of autoimmune diseases, including graft versus host disease (GvHD). As a proof of concept served initially crude preparations of bone marrow mononuclear cells (BMMC), which contain small numbers of hMSC. In our hospital, two pilot clinical studies with BMMC were performed: study of treatment of acute myocardial infarction (negative, prematurely terminated) and study of treatment of peripheral leg arthery disease (promising results). Further research was aimed on optimalisation of hMSC cultivation method for clinical use to obtain highest possible yield and get free from animal proteins. Human MSC were traditionally cultivated in research-grade media with fetal calf serum (FCS), which can lead to immunization of patients after repeated application of hMSC. We achieved...1st Department of Medicine - Clinical Department of Haematology First Faculty of MedicineI. interní klinika-klinika hematologie 1. LF UK a VFNFirst Faculty of Medicine1. lékařská fakult

Human multipotent mesenchymal stromal cells - bone differentiation and hematopoietic support

Human mesenchymal stromal cells (hMSC) are adult stem or progenitor cells, which physiological role is reparation of damaged tissues. This is achieved mostly by secretion of trophic, angiopoietic and immunomodulatory factors. Beside this, hMSC have potential to differentiate in vitro into specialized cells, especially of the mesodermal lineages. Human MSC also significantly support hematopoiesis in hematopoietic niche. This knowledge raised high hopes for therapeutic use of hMSC, especially in regenerative medicine and treatment of autoimmune diseases, including graft versus host disease (GvHD). As a proof of concept served initially crude preparations of bone marrow mononuclear cells (BMMC), which contain small numbers of hMSC. In our hospital, two pilot clinical studies with BMMC were performed: study of treatment of acute myocardial infarction (negative, prematurely terminated) and study of treatment of peripheral leg arthery disease (promising results). Further research was aimed on optimalisation of hMSC cultivation method for clinical use to obtain highest possible yield and get free from animal proteins. Human MSC were traditionally cultivated in research-grade media with fetal calf serum (FCS), which can lead to immunization of patients after repeated application of hMSC. We achieved..

Mesenchymal Stem Cells Isolated from the Human Bone Marrow: Cultivation, Phenotypic Analysis and Changes in Proliferation Kinetics

Mesenchymal Stem Cells (MSCs) are rare elements living in various organs (e.g., bone marrow), able to differentiate into specialized tissues, such as bone, cartilage, tendon, and myocardium. Since the first description of MSCs by Fridenshtein, several investigators have shown that these cells can also differentiate into chondrocytes, adipocytes, and, at least, in rodents into skeletal myoblasts. Later on, more primitive progenitor cells were characterized, able to give rise not only to limb-bud mesoderm, but also to cells of visceral mesoderm. Those cells were named mesodermal progenitor cells (MPCs). The aim of our study was to characterize and compare the biological properties and spontaneous differentiation potential of two different cell types (MSCs and MPCs) isolated from the human vertebral body bone marrow. The results of our experiments proved that the MPCs can be expanded beyond Hayflick’s limit and differed from MSCs in morphology, biological and phenotypic characteristics. Because of their high proliferative and differentiation potential, MPCs can become more attractive source of adult stem cells for therapeutic purposes

Characterization of Dental Pulp Stem Cells from Impacted Third Molars Cultured in Low Serum-Containing Medium

We isolated and expanded stem cells from dental pulp from extracted third molars using an innovative culture method consisting of low serum-containing medium supplemented with epidermal growth factor and platelet-derived growth factor BB. We evaluated the differentiation potential of these cells when they were growing either adherently or as micromass/spheroid cultures in various media. Undifferentiated and differentiated cells were analyzed by flow cytometry, immunocytochemistry and immunoblotting. The flow cytometry results showed that the dental pulp stem cells (DPSCs) were positive for mesenchymal stromal cell markers, but negative for hematopoietic markers. Immunocytochemical and/or immunoblotting analyses revealed the expression of numerous stem cell markers, including nanog, Sox2, nestin, Musashi-1 and nucleostemin, whereas they were negative for markers associated with differentiated neural, vascular and hepatic cells. Surprisingly, the cells were only slightly positive for α-smooth muscle actin, and a heterogeneous expression of CD146 was observed. When cultured in osteogenic media, they expressed osteonectin, osteopontin and procollagen I, and in micromass cultures, they produced collagen I. DPSCs cultured in TGF-β1/3-supplemented media produced extracellular matrix typical of cartilaginous tissue. The addition of vascular endothelial growth factor to serum-free media resulted in the expression of endothelial markers. Interestingly, when cultured in neurogenic media, DPSCs exhibited de novo or upregulated markers of undifferentiated and differentiated neural cells. Collectively, our data show that DPSCs are self-renewing and able to express markers of bone, cartilage, vascular and neural tissues, suggesting their multipotential capacity. Their easy accessibility makes these cells a suitable source of somatic stem cells for tissue engineering.Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG-geförderten) Allianz- bzw. Nationallizenz frei zugänglich

Human Dental Pulp Stem Cells – Isolation and Long Term Cultivation

Human adult mesenchymal stem cells (MSCs) are rare elements living in various organs (e.g. bone marrow, skeletal muscle), with capability to differentiate in various cell types (e.g. chondrocytes, adipocytes and osteoblasts). In the year 2000, Gronthos and co-workers isolated stem cells from the human dental pulp (DPSCs). Later on, stem cells from exfoliated tooth were also obtained. The aims of our study were to establish protocol of DPSCs isolation and to cultivate DPSCs either from adult or exfoliated tooth, and to compare these cells with mesenchymal progenitor cell (MPCs) cultures. MPCs were isolated from the human bone marrow of proximal femur. DPSCs were isolated from deciduous and permanent teeth. Both cell types were cultivated under the same conditions in the media with 2 % of FCS supplemented with PDGF and EGF growth factors. We have cultivated undifferentiated DPSCs for long time, over 60 population doublings in cultivation media designed for bone marrow MPCs. After reaching Hayflick’s limit, they still have normal karyotype. Initial doubling time of our cultures was from 12 to 50 hours for first 40 population doublings, after reaching 50 population doublings, doubling time had increased to 60–90 hours. Regression analysis of uncumulated population doublings proved tight dependence of population doublings on passage number and slow decrease of proliferation potential. In comparison with bone marrow MPCs, DPSCs share similar biological characteristics and stem cell properties. The results of our experiments proved that the DPSCs and MPCs are highly proliferative, clonogenic cells that can be expanded beyond Hayflick’s limit and remain cytogenetically stable. Moreover we have probably isolated two different populations of DPSCs. These DPSCs lines differed one from another in morphology. Because of their high proliferative and differentiation potential, DPSCs can become more attractive, easily accessible source of adult stem cells for therapeutic purposes

A new prognostic score for elderly patients with diffuse large B-cell lymphoma treated with R-CHOP: the prognostic role of blood monocyte and lymphocyte counts is absent.

BACKGROUND: Absolute lymphocyte count (ALC) and absolute monocyte count (AMC) have been documented as independent predictors of survival in patients with newly diagnosed Diffuse Large B-cell Lymphoma (DLBCL). Analysis of the prognostic impact of ALC and AMC in the context of International Prognostic Index (IPI) and other significant variables in elderly population treated in the R-CHOP regime has not been carried out yet. METHODOLOGY/PRINCIPAL FINDINGS: In this retrospective study, a cohort of 443 newly diagnosed DLBCL patients with age ≥ 60 was analyzed. All patients were treated with the R-CHOP therapy. An extensive statistical analysis was performed to identify risk factors of 3-year overall survival (OS). In multivariate analysis, only three predictors proved significant: Eastern Cooperative Oncology Group performance status (ECOG), age and bulky disease presence. These predictors were dichotomized (ECOG ≥ 1, age ≥ 70, bulk ≥ 7.5) to create a novel four-level score. This score predicted 3-year OS of 94.0%, 77.4%, 62.7% and 35.4% in the low-, low-intermediate, high-intermediate and high-risk groups, respectively (P<0.001). Further, a three-level score was tested which stratifies the population better (3-year OS: 91.9%, 67.2%, 36.2% in the low, intermediate and high-risk groups, respectively) but is more difficult to interpret. Both the 3- and 4-level scores were compared to standard scoring systems and, in our population, were shown to be superior in terms of patients risk stratification with respect to 3-year OS prediction. The results were successfully validated on an independent cohort of 162 patients of similar group characteristics. CONCLUSIONS: The prognostic role of baseline ALC, AMC or their ratio (LMR) was not confirmed in the multivariate context in elderly population with DLBCL treated with R-CHOP. The newly proposed age-specific index stratifies the elderly population into risk groups more precisely than the conventional IPI and its existing variants