HoloMonitor i Nature (igen)

Idag 7/7 har PHI återigen äran att omnämnas i en helt ny forskningsrapport.Det är 8 forskare från The University of Leeds (7 st) och tyska universitet Tübingen (1 st) som fått sina studier kring ledsjukdomen Artros i ben publicerade i Natures edition Scientific Reports.

The osteogenic commitment of CD271+CD56+ bone marrow stromal cells (BMSCs) in osteoarthritic femoral head bone

Published: 07 July 2020

Abstract

Osteoarthritis (OA), the most common joint disorder, is characterised by progressive structural changes in both the cartilage and the underlying subchondral bone. In late disease stages, subchondral bone sclerosis has been linked to heightened osteogenic commitment of bone marrow stromal cells (BMSCs). This study utilised cell sorting and immunohistochemistry to identify a phenotypically-distinct, osteogenically-committed BMSC subset in human OA trabecular bone. Femoral head trabecular bone tissue digests were sorted into CD45-CD271+CD56+CD146-, CD45-CD271+CD56-CD146+ and CD45-CD271+CD56-CD146-(termed double-negative, DN) subsets, and CD45+CD271-hematopoietic-lineage cells served as control. Compared to the CD146+ subset, the CD56+ subset possessed a lower-level expression of adipocyte-associated genes and significantly over 100-fold higher-level expression of many osteoblast-related genes including osteopontin and osteocalcin, whilst the DN subset presented a transcriptionally ‘intermediate’ BMSC population. All subsets were tri-potential following culture-expansion and were present in control non-OA trabecular bone. However, while in non-OA bone CD56+ cells only localised on the bone surface, in OA bone they were additionally present in the areas of new bone formation rich in osteoblasts and newly-embedded osteocytes. In summary, this study reveals a distinct osteogenically-committed CD271+CD56+ BMSC subset and implicates it in subchondral bone sclerosis in hip OA. CD271+CD56+ subset may represent a future therapeutic target for OA and other bone-associated pathologies.

Methods (urval)

Cell motility and differentiation assays

Following cell sorting, the BMSC subsets were placed in culture in StemMACS media for subsequent expansion and in vitro functional assays. For morphological assessment, images of individual cells from each subset were taken at 24 h after sorting and cell circularity was calculated using ImageJ software (version 1.8.0_172 National Institutes of Health, USA available at https://imagej.nih.gov/ij/) using the formula for circularity (4π(area/perimeter²) where 0 represented a straight line and 1 represented a perfect circle³⁶. Separate experiments were then performed for cell motility analysis. For this, freshly-sorted BMSC subsets from N = 4 OA donors were seeded in a 24 well Ibidi culture plates (Ibidi, Germany) at the seeding density of 1 × 10⁴ cells/cm² in 2 ml of StemMACS media and allowed to expand over one week. The cell attachment, spreading and movement were then tracked using Holomonitor M4 microscope (Phase Holographic Imaging AB, Sweden).
After automatic calibration of the background and microscope objective, one field of each sample was focused on by the digital autofocus feature. The cells were then imaged with photo captures every 30 min for 24 h. For the evaluation, at each time-lapse sequence, 25 cells were identified and selected by minimum error histogram-based threshold algorithm of the software (HoloStudio M4 version 2.6.2 available at https://phiab.com/product/holomonitor-app-suite-software). By tracking the cells, the motility of each subset was automatically analysed over time, as previously described⁶⁶. Genes’ selections were based on our previous studies^66,67,68 as well as other studies⁶⁹.

Supplementary Figure 3. Experimental design for CD271+ BMSCs subsets isolation and characterisation from OA femoral head trabecular bone samples.  For histological analysis, femoral heads from n=4 OA donors were slowly decalcified in EDTA solution, bisected using a standard pathology knife and tissue sections were used for IHC, IF or Picrosirius Red staining. To obtain  trabecular bone-resident live cells, soft tissue (ligament, periosteum) and cartilage were first removed from femoral heads using a scalpel then  cortical bone  was taken out using rongeur. The remaining trabecular bone was then mechanically broken down into small fragments using rongeur and enzymatically digested. From the obtained cell suspension, the BMSCs were isolated by FACS and used for subsequent gene expression analysis and functional in vitro assays. For gene expression analysis a total of n=6 OA donors were used for analysis by qPCR using a 96a gene panel TaqMan low-density array card. Each of the three subsets (CD56+, CD146+ and DN) isolated by FACS from n=3 OA donors were induced towards osteogenic, adipogenic and chondrogenic lineages and assessed by standard in-vitro quantitative and qualitative assays. The three subsets from different n=3 OA donors were used to study cell motility using the holographic imaging (Holomonitor)

Min kommentar
Värt att notera är 2 detaljer. I methods ser vi att PHI`s nya kompis,amerikanska NIH,har haft en fot inne i denna studie. Detalj nr 2 är att forskarna är ytterst generösa mot PHI då de bifogar en länk i rapporten direkt till Bolaget. Mycket gentilt agerat då andra forskare som kommer läsa denna rapport får en direktväg in till instrumentet och dess mjukvaror. VD Egelberg bör bjuda dessa forskare på varsin pint nästa gång han är i England.

Den här studien är en riktigt god karamell att suga på.
Men...det finns 1 forskningsrapport till som offentliggjorts idag. Den kommer ni få läsa om i morgon, för nån måtta på sötsakerna för en dag får det vara. 😎

                                            Mvh the99