Identification and validation of multiple cell surface markers of clinical-grade adipose-derived mesenchymal stromal cells as novel release criteria for good manufacturing practice-compliant production

Background: Clinical translation of mesenchymal stromal cells (MSCs) necessitates basic characterization of the cell product since variability in biological source and processing of MSCs may impact therapeutic outcomes. Although expression of classical cell surface markers (e.g., CD90, CD73, CD105, and CD44) is used to define MSCs, identification of functionally relevant cell surface markers would provide more robust release criteria and options for quality control. In addition, cell surface expression may distinguish between MSCs from different sources, including bone marrow-derived MSCs and clinical-grade adipose-derived MSCs (AMSCs) grown in human platelet lysate (hPL). Methods: In this work we utilized quantitative PCR, flow cytometry, and RNA-sequencing to characterize AMSCs grown in hPL and validated non-classical markers in 15 clinical-grade donors. Results: We characterized the surface marker transcriptome of AMSCs, validated the expression of classical markers, and identified nine non-classical markers (i.e., CD36, CD163, CD271, CD200, CD273, CD274, CD146, CD248, and CD140B) that may potentially discriminate AMSCs from other cell types. More importantly, these markers exhibit variability in cell surface expression among different cell isolates from a diverse cohort of donors, including freshly prepared, previously frozen, or proliferative state AMSCs and may be informative when manufacturing cells. Conclusions: Our study establishes that clinical-grade AMSCs expanded in hPL represent a homogeneous cell culture population according to classical markers,. Additionally, we validated new biomarkers for further AMSC characterization that may provide novel information guiding the development of new release criteria

Loss of histone methyltransferase Ezh2 stimulates an osteogenic transcriptional program in chondrocytes but does not affectcartilage development

Ezh2 is a histone methyltransferase that suppresses osteoblast maturation and skeletal development. We evaluated the roleof Ezh2 in chondrocyte lineage differentiation and endochondral ossification. Ezh2 was genetically inactivated in the mesenchymal, osteoblastic, and chondrocytic lineages in mice using the Prrx1-Cre,Osx1-Cre, and Col2a1-Cre drivers, respectively. Wild-type and conditional knockout mice were phenotypically assessed by grossmorphology, histology, and micro-CT imaging. Ezh2-deficient chondrocytes in micromass culture models were evaluated usingRNA-sequencing, histologic evaluation, and western blotting. Aged mice with Ezh2 deficiency were also evaluated for prematuredevelopment of osteoarthritis using radiographic analysis. Ezh2 deficiency in murine chondrocytes reduced bone density at 4 weeks of age, although caused no other gross developmentaleffects. Knockdown of Ezh2 in chondrocyte micromass cultures resulted in a global reduction in trimethylation of histone 3lysine 27 (H3K27me3) and altered differentiation in vitro. RNA-seq analysis revealed enrichment of an osteogenic gene expressionprofile in Ezh2 deficient chondrocytes. Joint development proceeded normally in the absence of Ezh2 in chondrocytes withoutinducing excessive hypertrophy or premature osteoarthritis in vivo. In summary, loss of Ezh2 reduced H3K27me3 levels, increased expression of osteogenic genes in chondrocytes, and resulted ina transient post-natal bone phenotype. Remarkably, Ezh2 activity is dispensable for normal chondrocyte maturation and endochondralossification in vivo, even though it appears to have a critical role during early stages of mesenchymal lineage-commitment

Human mesenchymal stem cells retain multilineage differentiation capacity including neural marker expression after extended in vitro expansion

The suitability of human mesenchymal stem cells (hMSCs) in regenerative medicine relies on retention of their proliferative expansion potential in conjunction with the ability to differentiate toward multiple lineages. Successful utilisation of these cells in clinical applications linked to tissue regeneration requires consideration of biomarker expression, time in culture and donor age, as well as their ability to differentiate towards mesenchymal (bone, cartilage, fat) or non-mesenchymal (e.g., neural) lineages. To identify potential therapeutic suitability we examined hMSCs after extended expansion including morphological changes, potency (stemness) and multilineage potential. Commercially available hMSC populations were expanded in vitro for > 20 passages, equating to > 60 days and > 50 population doublings. Distinct growth phases (A-C) were observed during serial passaging and cells were characterised for stemness and lineage markers at representative stages (Phase A: P+5, approximately 13 days in culture; Phase B: P+7, approximately 20 days in culture; and Phase C: P+13, approximately 43 days in culture). Cell surface markers, stem cell markers and lineage-specific markers were characterised by FACS, ICC and Q-PCR revealing MSCs maintained their multilineage potential, including neural lineages throughout expansion. Co-expression of multiple lineage markers along with continued CD45 expression in MSCs did not affect completion of osteogenic and adipogenic specification or the\ud formation of neurospheres. Improved standardised isolation and characterisation of MSCs may facilitate the identification of biomarkers to improve therapeutic efficacy to ensure increased reproducibility and routine production of MSCs for therapeutic applications including neural repair

Molecular Validation of Chondrogenic Differentiation and Hypoxia Responsiveness of Platelet-Lysate Expanded Adipose Tissue–Derived Human Mesenchymal Stromal Cells

Objective: To determine the optimal environmental conditions for chondrogenic differentiation of human adipose tissue–derived mesenchymal stromal/stem cells (AMSCs). In this investigation we specifically investigate the role of oxygen tension and 3-dimensional (3D) culture systems. Design: Both AMSCs and primary human chondrocytes were cultured for 21 days in chondrogenic media under normoxic (21% oxygen) or hypoxic (2% oxygen) conditions using 2 distinct 3D culture methods (high-density pellets and poly-ε-caprolactone [PCL] scaffolds). Histologic analysis of chondro-pellets and the expression of chondrocyte-related genes as measured by reverse transcriptase quantitative polymerase chain reaction were used to evaluate the efficiency of differentiation. Results: AMSCs are capable of expressing established cartilage markers including COL2A1, ACAN, and DCN when grown in chondrogenic differentiation media as determined by gene expression and histologic analysis of cartilage markers. Expression of several cartilage-related genes was enhanced by low oxygen tension, including ACAN and HAPLN1. The pellet culture environment also promoted the expression of hypoxia-inducible cartilage markers compared with cells grown on 3D scaffolds. Conclusions: Cell type–specific effects of low oxygen and 3D environments indicate that mesenchymal cell fate and differentiation potential is remarkably sensitive to oxygen. Genetic programming of AMSCs to a chondrocytic phenotype is effective under hypoxic conditions as evidenced by increased expression of cartilage-related biomarkers and biosynthesis of a glycosaminoglycan-positive matrix. Lower local oxygen levels within cartilage pellets may be a significant driver of chondrogenic differentiation

Neural marker proteins examined by FACS and WB in MSCs during expansion.

<p>Positive expression of neural markers is demonstrated. <b>A-C:</b> Populations average more than 30% positive for the neural stem cell marker, Nestin; and <b>D-F:</b> more than 50% positive for Sox2. Variation was observed between populations in percentages of the population positive for each maker, with the greatest variation seen at Phase B. Dark grey histogram is marker of interest. Light grey histogram is secondary antibody only control. <b>G:</b> Neural proteins TUBB3 and SOX2 were detected in undifferentiated hMSCs by WB during expansion.</p

hMSCs are positive for neural markers throughout expansion.

<p>Representative ICC images showing positive neural marker staining for <b>A-F:</b> neural markers in each of the three MSC populations examined at each of the three passages investigated. Cells stained positive for: <b>A-C:</b> neural stem cell markers, donkey anti-mouse-Nestin (1/300); and <b>D-F:</b> donkey anti-rabbit-Sox2 (1/1000); as well as <b>G-I:</b> lineage markers donkey anti-rabbit-glial fibrillary acidic protein (astrocyte; GFAP; 1/500); <b>J-L:</b> donkey anti-mouse-microtubule associated protein 2 (neuronal; MAP2; 1/300); and <b>M-O:</b> donkey anti-mouse IgM-O1 (oligodendrocyte; 1/500). Secondary antibodies anti-mouse-FITC (green; 1/1000), anti-rabbit-Cy3 (yellow; 1/1000), anti-mouse IgM-AlexaFluor594 (red; 1/500). Cells were mounted in mounting media containing DAPI (blue) to counterstain nuclei. Scale bar represents 70μm.</p

Total days in culture and population doublings (PD) for individual hMSC populations.

<p>Total number of days in culture and population doublings calculated for each population at each passage representing each phase of growth. Average days in culture and population doublings for the three hMSC populations examined are presented.</p

Characterisation of Phase A neurospheres.

<p><b>A:</b> Phase contrast image (20x magnification, scale bar = 90 uM) of hMSC Phase A neurospheres <b>B:</b> FDA/PI stain indicating the presence of live (= green) and dead (= red) cells in hMSC neurospheres (40x magnification, scale bar = 90 uM). Bar graphs indicating <b>C:</b> the average sphere diameter for each hMSC population and <b>D:</b> the ratio of live/dead cells indicated by FDA and PI stain signal intensity. <b>E:</b> Q-PCR expression of self-renewal, neural and MSC lineage markers in undifferentiated Phase A hMSCs compared to neurospheres with significant differences in expression observed in multiple markers (*** p<0.001). F: ICC (40x magnification, scale bar = 90 uM) of Nestin, SOX2, CD44 and TUBB3 in neurospheres along with a bar graph indicating relative signal intensity of each of the markers when compared to DAPI.</p

Primary and secondary antibody concentrations for; Immunocytochemistry (ICC) and Fluorescence Assisted Cell Sorting (FACS).

<p>Primary and secondary antibody concentrations for; Immunocytochemistry (ICC) and Fluorescence Assisted Cell Sorting (FACS).</p

Neural transcription factors and cell surface CD marker expression.

<p>Undifferentiated hMSCs express transcription factors and cell surface CD markers including stemness (CD73, CD271, HES1, KLF4, SOX5/6/9) and neuronal (CD24, CD56, CD200, CD304, MEF2C, PAX3/9) markers reported to influence neural differentiation. With the exception of CD73 and CD304, expressed at levels equal to CD44 (a cell surface marker of MSCs), these neural markers were expressed at levels 10-10000X lower than CD44.</p