Joni Ylostalo

Human Mesenchymal Stem/Stromal Cells Cultured as Spheroids are Self‐activated to Produce Prostaglandin E2 that Directs Stimulated Macrophages into an Anti‐inflammatory Phenotype

Culturing cells in three dimension (3D) provides an insight into their characteristics in vivo. We previously reported that human mesenchymal stem/stromal cells (hMSCs) cultured as 3D spheroids acquire enhanced anti‐inflammatory properties. Here, we explored the effects of hMSC spheroids on macrophages that are critical cells in the regulation of inflammation. Conditioned medium (CM) from hMSC spheroids inhibited lipopolysaccharide‐stimulated macrophages from secreting proinflammatory cytokines TNFα, CXCL2, IL6, IL12p40, and IL23. CM also increased the secretion of anti‐inflammatory cytokines IL10 and IL1ra by the stimulated macrophages, and augmented expression of CD206, a marker of alternatively activated M2 macrophages. The principal anti‐inflammatory activity in CM had a small molecular weight, and microarray data suggested that it was prostaglandin E2 (PGE2). This was confirmed by the observations that PGE2 levels were markedly elevated in hMSC spheroid‐CM, and that the anti‐inflammatory activity was abolished by an inhibitor of cyclooxygenase‐2 (COX‐2), a silencing RNA for COX‐2, and an antibody to PGE2. The anti‐inflammatory effects of the PGE2 on stimulated macrophages were mediated by the EP4 receptor. Spheroids formed by human adult dermal fibroblasts produced low levels of PGE2 and displayed negligible anti‐inflammatory effects on stimulated macrophages, suggesting the features as unique to hMSCs. Moreover, production of PGE2 by hMSC spheroids was dependent on the activity of caspases and NFκB activation in the hMSCs. The results indicated that hMSCs in 3D‐spheroid cultures are self‐activated, in part by intracellular stress responses, to produce PGE2 that can change stimulated macrophages from a primarily proinflammatory M1 phenotype to a more anti‐inflammatory M2 phenotype. STEM Cells2012;30:2283–2296

Anti-inflammatory protein TSG-6 reduces inflammatory damage to the cornea following chemical and mechanical injury

Previous reports demonstrated that adult stem/progenitor cells from bone marrow (multipotent mesenchymal stem cells; MSCs) can repair injured tissues with little evidence of engraftment or differentiation. In exploring this phenomenon, our group has recently discovered that the therapeutic benefits of MSCs are in part explained by the cells being activated by signals from injured tissues to express an anti-inflammatory protein TNF-α–stimulated gene/protein 6 (TSG-6). Therefore, we elected to test the hypothesis that TSG-6 would have therapeutic effects in inflammatory but noninfectious diseases of the corneal surface. We produced a chemical and mechanical injury of the cornea in rats by brief application of 100% ethanol followed by mechanical debridement of corneal and limbal epithelium. Recombinant human TSG-6 or PBS solution was then injected into the anterior chamber of the eye. TSG-6 markedly decreased corneal opacity, neovascularization, and neutrophil infiltration. The levels of proinflammatory cytokines, chemokines, and matrix metalloproteinases were also decreased. The data indicated that TSG-6, a therapeutic protein produced by MSCs in response to injury signals, can protect the corneal surface from the excessive inflammatory response following injury.

Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension

Infusion of bone marrow stem or progenitor cells may provide powerful therapies for injured tissues such as the lung and heart. We examined the potential of bone marrow‐derived (BMD) progenitor cells to contribute to repair and remodeling of lung and heart in a rat monocrotaline (MCT) model of pulmonary hypertension. Bone marrow from green fluorescent protein (GFP)‐transgenic male rats was transplanted into GFP‐negative female rats. The chi‐meric animals were injected with MCT to produce pulmonary hypertension. Significant numbers of male GFP‐positive BMD cells engrafted in the lungs of MCT‐treated rats. Microarray analyses and double‐im‐munohistochemistry demonstrated that many of the cells were interstitial fibroblasts or myofibroblasts, some of the cells were hematopoietic cells, and some were pulmonary epithelial cells (Clara cells), vascular endothelial cells, and smooth muscle cells. A few BMD cells fused with pulmonary cells from the host, but the frequency was low. In the hypertrophied hearts of MCT‐treated rats, we found a significant increase in the relative numbers of BMD cells in the right ventricle wall as compared with the left ventricle. Some of the BMD cells in the right ventricle were vascular cells and cardiomyocytes. We report BMD cardiomyocytes with a normal chromosome number, fusion of BMD cells with host cardiomyocytes, and, in some cases, nuclear fusion. Spees, J. L., Whitney, M. J., Sullivan, D. E., Lasky, J. A., Laboy, M., Ylostalo, J., Prockop, D. J. Bone marrow progenitor cells contribute to repair and remodeling of the lung and heart in a rat model of progressive pulmonary hypertension. FASEB J. 22, 1226–1236 (2008)

Human multipotent stromal cells from bone marrow and microRNA: Regulation of differentiation and leukemia inhibitory factor expression

We observed that microRNAs (miRNAs) that regulate differentiation in a variety of simpler systems also regulate differentiation of human multipotent stromal cells (hMSCs) from bone marrow. Differentiation of hMSCs into osteoblasts and adipocytes was inhibited by using lentiviruses expressing shRNAs to decrease expression of Dicer and Drosha, two enzymes that process early transcripts to miRNA. Expression analysis of miRNAs during hMSC differentiation identified 19 miRNAs that were up-regulated during osteogenic differentiation and 20 during adipogenic differentiation, 11 of which were commonly up-regulated in both osteogenic and adipogenic differentiation. In silico models predicted that five of the up-regulated miRNAs targeted leukemia inhibitory factor (LIF) expression. The prediction was confirmed for two of the miRNAs, hsa-mir 199a and hsa-mir346, in that over-expression of the miRNAs decreased LIF secretion by hMSCs. The results demonstrate that differentiation of hMSCs is regulated by miRNAs and that several of these miRNAs target LIF.

Altered Integration of Matrilin-3 into Cartilage Extracellular Matrix in the Absence of Collagen IX

ABSTRACT The matrilins are a family of four noncollagenous oligomeric extracellular matrix proteins with a modular structure. Matrilins can act as adapters which bridge different macromolecular networks. We therefore investigated the effect of collagen IX deficiency on matrilin-3 integration into cartilage tissues. Mice harboring a deleted Col9a1 gene lack synthesis of a functional protein and produce cartilage fibrils completely devoid of collagen IX. Newborn collagen IX knockout mice exhibited significantly decreased matrilin-3 and cartilage oligomeric matrix protein (COMP) signals, particularly in the cartilage primordium of vertebral bodies and ribs. In the absence of collagen IX, a substantial amount of matrilin-3 is released into the medium of cultured chondrocytes instead of being integrated into the cell layer as in wild-type and COMP-deficient cells. Gene expression of matrilin-3 is not affected in the absence of collagen IX, but protein extraction from cartilage is greatly facilitated. Matrilin-3 interacts with collagen IX-containing cartilage fibrils, while fibrils from collagen IX knockout mice lack matrilin-3, and COMP-deficient fibrils exhibit an intermediate integration. In summary, the integration of matrilin-3 into cartilage fibrils occurs both by a direct interaction with collagen IX and indirectly with COMP serving as an adapter. Matrilin-3 can be considered as an interface component, capable of interconnecting macromolecular networks and mediating interactions between cartilage fibrils and the extrafibrillar matrix.

Pharmaceutical modulation of canonical Wnt signaling in multipotent stromal cells for improved osteoinductive therapy

Human mesenchymal stem cells (hMSCs) from bone marrow are regarded as putative osteoblast progenitors in vivo and differentiate into osteoblasts in vitro. Positive signaling by the canonical wingless (Wnt) pathway is critical for the differentiation of MSCs into osteoblasts. In contrast, activation of the peroxisome proliferator-activated receptor-γ (PPARγ)-mediated pathway results in adipogenesis. We therefore compared the effect of glycogen-synthetase-kinase-3β (GSK3β) inhibitors and PPARγ inhibitors on osteogenesis by hMSCs. Both compounds altered the intracellular distribution of β-catenin and GSK3β in a manner consistent with activation of Wnt signaling. With osteogenic supplements, the GSK3β inhibitor 6-bromo-indirubin-3′-oxime (BIO) and the PPARγ inhibitor GW9662 (GW) enhanced early osteogenic markers, alkaline phosphatase (ALP), and osteoprotegerin (OPG) by hMSCs and transcriptome analysis demonstrated up-regulation of genes encoding bone-related structural proteins. At higher doses of the inhibitors, ALP levels were attenuated, but dexamethasone-induced biomineralization was accelerated. When hMSCs were pretreated with BIO or GW and implanted into experimentally induced nonself healing calvarial defects, GW treatment substantially increased the capacity of the cells to repair the bone lesion, whereas BIO treatment had no significant effect. Further investigation indicated that unlike GW, BIO induced cell cycle inhibition in vitro. Furthermore, we found that GW treatment significantly reduced expression of chemokines that may exacerbate neutrophil- and macrophage-mediated cell rejection. These data suggest that use of PPARγ inhibitors during the preparation of hMSCs may enhance the capacity of the cells for osteogenic cytotherapy, whereas adenine analogs such as BIO can adversely affect the viability of hMSC preparations in vitro and in vivo.

Dynamic compaction of human mesenchymal stem/precursor cells into spheres self‐activates caspase‐dependent IL1 signaling to enhance secretion of modulators of inflammation and immunity (PGE2, TSG6, and STC1)

Human mesenchymal stem/precursor cells (MSC) are similar to some other stem/progenitor cells in that they compact into spheres when cultured in hanging drops or on nonadherent surfaces. Assembly of MSC into spheres alters many of their properties, including enhanced secretion of factors that mediate inflammatory and immune responses. Here we demonstrated that MSC spontaneously aggregated into sphere‐like structures after injection into a subcutaneous air pouch or the peritoneum of mice. The structures were similar to MSC spheres formed in cultures demonstrated by the increased expression of genes for inflammation‐modulating factors TSG6, STC1, and COX2, a key enzyme in production of PGE2. To identify the signaling pathways involved, hanging drop cultures were used to follow the time‐dependent changes in the cells as they compacted into spheres. Among the genes upregulated were genes for the stress‐activated signaling pathway for IL1α/β, and the contact‐dependent signaling pathway for Notch. An inhibitor of caspases reduced the upregulation of IL1A/B expression, and inhibitors of IL1 signaling decreased production of PGE2, TSG6, and STC1. Also, inhibition of IL1A/B expression and secretion of PGE2 negated the anti‐inflammatory effects of MSC spheres on stimulated macrophages. Experiments with γ‐secretase inhibitors suggested that Notch signaling was also required for production of PGE2 but not TSG6 or STC1. The results indicated that assembly of MSC into spheres triggers caspase‐dependent IL1 signaling and the secretion of modulators of inflammation and immunity. Similar aggregation in vivo may account for some of the effects observed with administration of the cells in animal models. Stem Cells 2013;31:2443–2456

The Fibril-associated Collagen IX Provides a Novel Mechanism for Cell Adhesion to Cartilaginous Matrix

Collagen IX is the prototype fibril-associated collagen with interruptions in triple helix. In human cartilage it covers collagen fibrils, but its putative cellular receptors have been unknown. The reverse transcription-PCR analysis of human fetal tissues suggested that based on their distribution all four collagen receptor integrins, namely α1β1, α2β1, α10β1, and α11β1, are possible receptors for collagen IX. Furthermore primary chondrocytes and chondrosarcoma cells express the four integrins simultaneously. Chondrosarcoma cells, as well as Chinese hamster ovary cells transfected to express α1β1, α2β1, or α10β1 integrin as their only collagen receptor, showed fast attachment and spreading on human recombinant collagen IX indicating that it is an effective cell adhesion protein. To further study the recognition of collagen IX we produced recombinant αI domains in Escherichia coli. For each of the four αI domains, collagen IX was among the best collagenous ligands, making collagen IX exceptional compared with all other collagen subtypes tested so far. Rotary shadowing electron microscopy images of both α1I- and α2I-collagen IX complexes unveiled only one binding site located in the COL3 domain close to the kink between it and the COL2 domain. The recognition of collagen IX by α2I was considered to represent a novel mechanism for two reasons. First, collagen IX has no GFOGER motif, and the identified binding region lacks any similar sequences. Second, the α2I domain mutations D219R and H258V, which both decreased binding to collagen I and GFOGER, had very different effects on its binding to collagen IX. D219R had no effect, and H258V prevented type IX binding. Thus, our results indicate that collagen IX has unique cell adhesion properties when compared with other collagens, and it provides a novel mechanism for cell adhesion to cartilaginous matrix.

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.

Structural and functional characterization of recombinant matrilin-3 A-domain and implications for human genetic bone diseases.

Mutations in matrilin-3 result in multiple epiphyseal dysplasia, which is characterized by delayed and irregular bone growth and early onset osteoarthritis. The majority of disease-causing mutations are located within the β-sheet of the single A-domain of matrilin-3, suggesting that they disrupt the structure and/or function of this important domain. Indeed, the expression of mutant matrilin-3 results in its intracellular retention within the rough endoplasmic reticulum of cells, where it elicits an unfolded protein response. To understand the folding characteristics of the matrilin-3 A-domain we determined its structure using CD, analytical ultracentrifugation, and dual polarization interferometry. This study defined novel structural features of the matrilin-3 A-domain and identified a conformational change induced by the presence or the absence of Zn2+. In the presence of Zn2+ the A-domain adopts a more stable “tighter” conformation. However, after the removal of Zn2+ a potential structural rearrangement of the metal ion-dependent adhesion site motif occurs, which leads to a more “relaxed” conformation. Finally, to characterize the interactions of the matrilin-3 A-domain we performed binding studies on a BIAcore using type II and IX collagen and cartilage oligomeric matrix protein. We were able to demonstrate that it binds to type II and IX collagen and cartilage oligomeric matrix protein in a Zn2+-dependent manner. Furthermore, we have also determined that the matrilin-3 A-domain appears to bind exclusively to the COL3 domain of type IX collagen and that this binding is abolished in the presence of a disease causing mutation in type IX collagen.