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Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions.

ABSTRACT: Human mesenchymal stromal cells (MSCs) are being used in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3 dimensions (3D). We compared the activation of MSCs in 3D in fetal bovine serum containing medium and in multiple xeno-free media formulations.MSC aggregation and sphere formation was studied with the use of hanging drop cultures with medium containing fetal bovine serum or with various commercially available stem cell media with or without human serum albumin (HSA). Activation of MSCs was studied with the use of gene expression and protein secretion measurements and with functional studies with the use of macrophages and cancer cells.MSCs did not condense into tight spheroids and express a full complement of therapeutic genes in ?-minimum essential medium or several commercial stem-cell media. However, we identified a chemically defined xeno-free media, which, when supplemented with HSA from blood or recombinant HSA, resulted in compact spheres with high cell viability, together with high expression of anti-inflammatory (prostaglandin E2, TSG-6 TNF-alpha induced gene/protein 6) and anti-cancer molecules (TRAIL TNF-related apoptosis-inducing ligand, interleukin-24). Furthermore, spheres cultured in this medium showed potent anti-inflammatory effects in a lipopolysaccharide-stimulated macrophage system and suppressed the growth of prostate cancer cells by promoting cell-cycle arrest and cell death.We demonstrated that cell activation in 3D depends critically on the culture medium. The conditions developed in the present study for 3D culture of MSCs should be useful in further research on MSCs and their potential therapeutic applications.

SUBMITTER: Ylostalo JH

PROVIDER: S-EPMC4190045 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions.

Ylostalo Joni H JH Bartosh Thomas J TJ Tiblow April A Prockop Darwin J DJ

Cytotherapy 20140916 11

<h4>Background aims</h4>Human mesenchymal stromal cells (MSCs) are being used in clinical trials, but the best protocol to prepare the cells for administration to patients remains unclear. We previously demonstrated that MSCs could be pre-activated to express therapeutic factors by culturing the cells in 3 dimensions (3D). We compared the activation of MSCs in 3D in fetal bovine serum containing medium and in multiple xeno-free media formulations.<h4>Methods</h4>MSC aggregation and sphere format ...[more]

PMID: 25231893

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Project description:Carcinoma-associated fibroblasts (CAFs) are a dominant component of the tumor microenvironment with pro-tumorigenic properties. Despite this knowledge, their physiologic origins remain poorly understood. Mesenchymal stem cells (MSCs) can be recruited from the bone marrow to areas of tissue damage and inflammation, including prostate cancer. MSCs can generate and have many overlapping properties with CAFs in preclinical models.Multiparameter flow cytometry and multipotent differentiation assays used to define MSCs in primary prostate stromal cultures derived from young (<25?yrs) organ donors and prostate cancer patients compared with bone marrow-derived stromal cultures. Population doubling times, population doublings, cell size, and differentiation potential determined under multiple culture conditions, including normoxia, hypoxia, and a variety of media. TGF-? measured by ELISA.MSCs and stromal progenitors are not only present in normal and malignant prostate tissue, but are quickly selected for in primary stromal cultures derived from these tissues; becoming the dominant population within just a few passages. Growth potential inversely associated with TGF-? concentrations. All conditions generated populations with an average cell diameter >15?µm. All cultures tested had the ability to undergo osteogenic and chondrogenic differentiation, but unlike bone marrow-derived MSCs, primary stromal cultures derived from normal prostate tissue lack adipogenic differentiation potential. In contrast, a subset of stromal cultures derived from prostate cancer patients retain the ability to differentiate into adipocytes; a property that is significantly suppressed under hypoxic conditions in both bone marrow- and prostate-derived MSCs.Primary prostate stromal cultures are highly enriched in cells with an MSC or stromal progenitor phenotype. The use of primary cultures such as these to study CAFs raises interesting implications when considering their overlapping properties. The lack of adipogenesis in stromal cultures derived from normal prostates suggests they have a lineage-restricted progenitor phenotype. The retention of adipogenic differentiation in cultures from a subset of prostate cancer patients suggests the active recruitment of less committed progenitors or MSCs from the bone marrow as a function of disease progression. This recruitment can potentially be exploited for prognostic purposes or a cell-based platform for the systemic delivery of cytotoxic agents to sites of prostate cancer.

Project description:BackgroundSenescent cells are undesirable in cell therapy products due to reduced therapeutic activity and risk of aberrant cellular effects, and methods for assessing senescence are needed. Early-passage mesenchymal stromal cells (MSCs) are known to be small and spindle-shaped but become enlarged upon cell aging. Indeed, cell morphology is routinely evaluated during MSC production using subjective methods. We have therefore explored the possibility of utilizing automated imaging-based analysis of cell morphology in clinical cell manufacturing.MethodsAn imaging system was adopted for analyzing changes in cell morphology of bone marrow-derived MSCs during long-term culture. Cells taken from the cultures at the desired passages were plated at low density for imaging, representing morphological changes observed in the clinical-grade cultures. The manifestations of aging and onset of senescence were monitored by population doubling numbers, expression of p16INK4a and p21Cip1/Waf1, β-galactosidase activity, and telomeric terminal restriction fragment analysis.ResultsCell area was the most statistically significant and practical parameter for describing morphological changes, correlating with biochemical senescence markers. MSCs from passages 1 (p1) and 3 (p3) were remarkably uniform in size, with cell areas between 1800 and 2500 μm2. At p5 the cells began to enlarge resulting in a 4.8-fold increase at p6-9 as compared to p1. The expression of p16INK4a and activity of β-galactosidase had a strong correlation with the increase in cell area, whereas the expression of p21Cip1/Waf1 reached its maximum at the onset of growth arrest and subsequently decreased. Mean telomere length shortened at an apparently constant rate during culture, from 8.2 ± 0.3 kbp at p1, reaching 6.08 ± 0.6 kbp at senescence.ConclusionsImaging analysis of cell morphology is a useful tool for evaluating aging in cell cultures throughout the lifespan of MSCs. Our findings suggest that imaging analysis can reproducibly detect aging-related changes in cell morphology in MSC cultures. These findings suggest that cell morphology is still a supreme measure of cell quality and may be utilized to develop new noninvasive imaging-based methods to screen and quantitate aging in clinical-grade cell cultures.

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Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions.

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Unique characteristics of human mesenchymal stromal/progenitor cells pre-activated in 3-dimensional cultures under different conditions.

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