Mesenchymal Stem Cell (MSC) Aggregate Formation in vivo

Human mesenchymal stem/progenitor cells (MSCs) isolated from various adult tissues show remarkable therapeutic potential and are being employed in clinical trials for the treatment of numerous diseases (Prockop et al., 2010). While routes of cell administration vary, profound beneficial effects of MSCs in animal models have been observed following intraperitoneal injections of the cells (Roddy et al., 2011). Similar to MSC spheres formed in culture under conditions where attachment to plastic is not permitted (Bartosh et al., 2010), MSCs injected into the peritoneum of mice spontaneously aggregate into 3D sphere-like structures (Bartosh et al., 2013). During the process of sphere assembly and compaction, MSCs upregulate expression of numerous therapeutic anti-inflammatory and immune modulatory factors. Here we describe the method we previously used for the generation of human bone marrow-derived MSC aggregates/spheres in vivo (Bartosh et al., 2013). By tagging the MSCs with green fluorescent protein (GFP), the aggregates formed can be easily visualized, collected and analyzed for changes in cellular properties and interactions with host immune cells

Efficacy of 3D Culture Priming is Maintained in Human Mesenchymal Stem Cells after Extensive Expansion of the Cells

The use of non-optimal preparations of mesenchymal stem cells (MSCs), such as extensively expanded cells, might be necessary to obtain the large numbers of cells needed for many clinical applications. We previously demonstrated that minimally expanded (early passage) MSCs can be pre-activated as spheroids to produce potentially therapeutic factors in 3D cultures. Here, we used extensively expanded (late passage) MSCs and studied their 3D-culture activation potential. MSCs were culture-expanded as 2D monolayers, and cells from various passages were activated by 3D culture in hanging drops with either fetal bovine serum (FBS)-containing media or a more clinically-applicable animal product-free (xeno-free) media. Gene expression analyses demonstrated that MSC spheroids prepared from passage 3, 5, and 7 cells were similar to each other but different from 2D MSCs. Furthermore, the expression of notable anti-inflammatory/immune-modulatory factors cyclooxygenase-2 (PTGS2), TNF alpha induced protein 6 (TNFAIP6), and stanniocalcin 1 (STC-1) were up-regulated in all spheroid preparations. This was confirmed by the detection of secreted prostaglandin E2 (PGE-2), tumor necrosis factor-stimulated gene 6 (TSG-6, and STC-1. This study demonstrated that extensively expanded MSCs can be activated in 3D culture through spheroid formation in both FBS-containing and xeno-free media. This work highlights the possibility of activating otherwise less useable MSC preparations through 3D culture generating large numbers of potentially therapeutic MSCs

Sox11 Is Expressed in Early Progenitor Human Multipotent Stromal Cells and Decreases with Extensive Expansion of the Cells

There has been considerable interest in developing new therapies with adult multipotent progenitor stromal cells or mesenchymal stem cells (MSCs) in organ replacement and repair. To be effectively seeded into scaffolds for therapy, large numbers of cells are needed, but concerns remain regarding their chromatin stability in long-term culture. We therefore expanded four donors of human MSCs (hMSCs) from bone marrow aspirates with a protocol that maintains the cells at low density. MSCs initially proliferated at average doubling times of 24 h and then gradually reached senescence after 8–15 passages (33–55 population doublings) without evidence of immortalization. Comparative genomic hybridization assays of two preparations revealed no abnormalities through 33 population doublings. One preparation had a small amplification of unknown significance in chromosome 7 (7q21:11) after 55 population doublings. Microarray assays demonstrated progressive changes in the transcriptome of the cells. However, the transcriptomes clustered more closely over time within a single passage, rather than with passage number, indicating a partial reversibility of the patterns of gene expression. One of the largest changes was a decrease in mRNA for Sox11, a transcription factor previously identified in neural progenitor cells. Knockdown of Sox11 with siRNA decreased the proliferation and osteogenic differentiation potential of hMSCs. The results suggested that assays for Sox11 may provide a biomarker for early progenitor hMSCs

Cross-talk between human mesenchymal stem/progenitor cells (MSCs) and rat hippocampal slices in LPS-stimulated cocultures: The MSCs are activated to secrete prostaglandin E2

Mesenchymal stem/progenitor cells (MSCs) improve functional outcome in a number of disease models through suppression of inflammation. However, their effects on neuroinflammation are unknown. In this study, we show that MSCs suppress endotoxin-induced glial activation in organotypic hippocampal slice cultures (OHSCs). Lipopolysaccharide-stimulated OHSCs activated MSCs to increase the expression of cyclo-oxygenase-2 and produce prostaglandin E2. MSC-derived prostaglandin E2, then suppressed pro-inflammatory cytokine production by the OHSCs. Together, the results suggest the potential anti-inflammatory mechanism of MSCs in models of disease and support earlier observations that MSCs may offer a therapy for neuroinflammation produced by trauma or disease. © 2011 The Authors. Journal of Neurochemistry © 2011 International Society for Neurochemistry.Y

Action at a Distance: Systemically Administered Adult Stem/Progenitor Cells (MSCs) Reduce Inflammatory Damage to the Cornea Without Engraftment and Primarily by Secretion of TNF-alpha Stimulated Gene/Protein 6

Previous reports demonstrated that the deleterious effects of chemical injury to the cornea were ameliorated by local or systemic administration of adult stem/progenitor cells from bone marrow referred to as mesenchymal stem or stromal cells (MSCs). However, the mechanisms for the beneficial effects of MSCs on the injured cornea were not clarified. Herein, we demonstrated that human MSCs (hMSCs) were effective in reducing corneal opacity and inflammation without engraftment after either intraperitoneal (i.p.) or intravenous (i.v.) administration following chemical injury to the rat cornea. A quantitative assay for human mRNA for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) demonstrated that less than 10 hMSCs were present in the corneas of rats 1-day and 3 days after i.v. or i.p. administration of 1 × 10 7 hMSCs. In vitro experiments using a transwell coculture system demonstrated that chemical injury to corneal epithelial cells activated hMSCs to secrete the multipotent anti-inflammatory protein TNF-α stimulated gene/protein 6 (TSG-6). In vivo, the effects of i.v. injection of hMSCs were largely abrogated by knockdown of TSG-6. Also, the effects of hMSCs were essentially duplicated by either i.v. or topical administration of TSG-6. Therefore, the results demonstrated that systemically administered hMSCs reduce inflammatory damage to the cornea without engraftment and primarily by secretion of the anti-inflammatory protein TSG-6 in response to injury signals from the cornea. © AlphaMed Press.Y

Leukemia Inhibitory Factor Secretion is a Predictor and Indicator of Early Progenitor Status in Adult Bone Marrow Stromal Cells

Bone marrow–derived stromal cells (BMSCs) are defined by their ability to self-renew and differentiate into at least three mesenchymal cell types (bone, adipose, and cartilage). The inability to isolate a reliably efficacious and homogeneous population of early progenitor cells has limited efforts to increase their therapeutic potential. In this study, we focused on identifying protein markers that may be employed to predict the efficacy of a cultured BMSC population. Markers of progenitor status were identified by comparing BMSCs at early and late passage, donor-matched skin fibroblasts, and commercially available dermal fibroblast cell lines. Differentiation potential was determined according to in vitro assays of osteogenesis, adipogenesis, and chondrogenesis. Early-passage BMSCs differentiated into all three lineages, whereas late-passage BMSCs and both fibroblast preparations did not. To identify novel markers of early progenitors, microarray transcript analysis between early-passage BMSCs and fibroblasts was performed. Messenger RNA encoding the cytokine leukemia inhibitory factor (LIF) was identified as differentially expressed. Enzyme-linked immunosorbent assay on conditioned media confirmed that LIF secretion was much higher from early progenitor BMSCs than donor-matched or commercial lines of fibroblasts and dropped with extensive expansion or induction of differentiation. In clonally expanded BMSCs, colonies that retained progenitor status expressed significantly higher levels of LIF than those that failed to differentiate. Our results indicate that LIF expression may represent a marker to quantify the differentiation potential of BMSCs and may be especially suited for the rapid, noninvasive quality control of clinical preparations

Stanniocalcin-1 rescued photoreceptor degeneration in two rat models of inherited retinal degeneration

Oxidative stress and photoreceptor apoptosis are prominent features of many forms of retinal degeneration (RD) for which there are currently no effective therapies. We previously observed that mesenchymal stem/stromal cells reduce apoptosis by being activated to secrete stanniocalcin-1 (STC-1), a multifunctional protein that reduces oxidative stress by upregulating mitochondrial uncoupling protein-2 (UCP-2). Therefore, we tested the hypothesis that intravitreal injection of STC-1 can rescue photoreceptors. We first tested STC-1 in the rhodopsin transgenic rat characterized by rapid photoreceptor loss. Intravitreal STC-1 decreased the loss of photoreceptor nuclei and transcripts and resulted in measurable retinal function when none is otherwise present in this rapid degeneration. We then tested STC-1 in the Royal College of Surgeons (RCS) rat characterized by a slower photoreceptor degeneration. Intravitreal STC-1 reduced the number of pyknotic nuclei in photoreceptors, delayed the loss of photoreceptor transcripts, and improved function of rod photoreceptors. Additionally, STC-1 upregulated UCP-2 and decreased levels of two protein adducts generated by reactive oxygen species (ROS). Microarrays from the two models demonstrated that STC-1 upregulated expression of a similar profile of genes for retinal development and function. The results suggested that intravitreal STC-1 is a promising therapy for various forms of RD including retinitis pigmentosa and atrophic age-related macular degeneration (AMD). © The American Society of Gene & Cell Therapy.Y