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Abstract While poly(ethylene glycol) (PEG) hydrogels are generally regarded as biologically inert blank slates, concerns over PEG immunogenicity are growing, and the implications for tissue engineering are unknown. Here, we investigate these implications by immunizing mice against PEG to stimulate anti-PEG antibody production and evaluating bone defect regeneration after treatment with bone morphogenetic protein-2-loaded PEG hydrogels. Quantitative analysis reveals that PEG sensitization increases bone formation compared to naive controls, whereas histological analysis shows that PEG sensitization induces an abnormally porous bone morphology at the defect site, particularly in males. Furthermore, immune cell recruitment is higher in PEG-sensitized mice administered the PEG-based treatment than their naive counterparts. Interestingly, naive controls that were administered a PEG-based treatment also develop anti-PEG antibodies. Sex differences in bone formation and immune cell recruitment are also apparent. Overall, these findings indicate that anti-PEG immune responses can impact tissue engineering efficacy and highlight the need for further investigation.

Abstract Extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) have been recognized as promising cytotherapeutics due to their demonstrated immunomodulatory effects in various preclinical models. The immunomodulatory capabilities of EVs stem from the proteins and genetic materials they carry from parent cells, but the cargo contents of EVs are significantly influenced by MSC tissues and donors, cellular age and culture conditions, resulting in functional variations. However, there are no surrogate assays available to validate the immunomodulatory potency of MSC‐EVs before in vivo administration. In previous work, we discovered that microcarrier culture conditions enhance the immunomodulatory function of MSC‐EVs, as well as the levels of immunosuppressive molecules such as TGF‐β1 and let‐7b in MSC‐EVs. Building on these findings, we investigated whether TGF‐β1 levels in MSC‐EVs could serve as a surrogate biomarker for predicting their potency in vivo. Our studies revealed a strong correlation between TGF‐β1 and let‐7b levels in MSC‐EVs, as well as their capacity to suppress IFN‐γ secretion in stimulated splenocytes, establishing biopotency and surrogate assays for MSC‐EVs. Subsequently, we validated MSC‐EVs generated from monolayer cultures (ML‐EVs) or microcarrier cultures (MC‐EVs) using murine models of experimental autoimmune uveoretinitis (EAU) and additional in vitro assays reflecting the Mode of Action of MSC‐EVs in vivo. Our findings demonstrated that MC‐EVs carrying high levels of TGF‐β1 exhibited greater efficacy than ML‐EVs in halting disease progression in mice with EAU as well as inducing apoptosis and inhibiting the chemotaxis of retina‐reactive T cells. Additionally, MSC‐EVs suppressed the MAPK/ERK pathway in activated T cells, with treatment using TGF‐β1 or let‐7b showing similar effects on the MAPK/ERK pathway. Collectively, our data suggest that MSC‐EVs directly inhibit the infiltration of retina‐reactive T cells toward the eyes, thereby halting the disease progression in EAU mice, and their immunomodulatory potency in vivo can be predicted by their TGF‐β1 levels.

Abstract Human mesenchymal stem cells (hMSCs) are effective in treating disorders resulting from an inflammatory or heightened immune response. The hMSCs derived from induced pluripotent stem cells (ihMSCs) share the characteristics of tissue derived hMSCs but lack challenges associated with limited tissue sources and donor variation. To meet the expected future demand for ihMSCs, there is a need to develop scalable methods for their production at clinical yields while retaining immunomodulatory efficacy. Herein, we describe a platform for the scalable expansion and rapid harvest of ihMSCs with robust immunomodulatory activity using degradable gelatin methacryloyl (GelMA) microcarriers. GelMA microcarriers were rapidly and reproducibly fabricated using a custom microfluidic step emulsification device at relatively low cost. Using vertical wheel bioreactors, 8.8 to 16.3‐fold expansion of ihMSCs was achieved over 8 days. Complete recovery by 5‐minute digestion of the microcarriers with standard cell dissociation reagents resulted in >95% viability. The ihMSCs matched or exceeded immunomodulatory potential in vitro when compared with ihMSCs expanded on monolayers. This is the first description of a robust, scalable, and cost‐effective method for generation of immunomodulatory ihMSCs, representing a significant contribution to their translational potential.

Extracellular vesicles (EVs) play key roles in cell biology and may provide new clinical diagnostics and therapies. However, it has proven difficult to develop protocols for their purification and characterization. One of the major barriers in the field has been a lack of convenient assays for their bioactivity. Developing assays has not been a trivial matter, because of the heterogeneity of EVs, the multiple activities they demonstrate, and the uncertainty about their modes of action. Therefore, it is likely that multiple assays for their activities are needed. One important assay will be for the anti-inflammatory activity observed in mice after administration of the small EVs commonly referred to as exosomes. We developed an assay for the anti-inflammatory activity of exosomes with a line of mouse macrophages. The assay makes it possible to rank different preparations of exosomes by their anti-inflammatory activity, and their ranking predicts their efficacy in suppressing LPS-stimulated inflammation in mice. The assay is convenient for comparing multiple samples and, therefore, should be useful in developing protocols for the purification and characterization of anti-inflammatory exosomes. Keywords: in-process assay, bioactivity, extracellular vesicles

Accumulating evidence shows that extracellular vesicles (EVs) produced by mesenchymal stem/stromal cells (MSCs) exert their therapeutic effects in several disease models. We previously demonstrated that MSCs suppress autoimmunity in models of type 1 diabetes (T1D) and experimental autoimmune uveoretinitis (EAU). Therefore, here, we investigated the therapeutic potential of MSC-derived EVs using our established mouse models for autoimmune diseases affecting the pancreas and the eye: T1D and EAU. The data demonstrate that MSC-derived EVs effectively prevent the onset of disease in both T1D and EAU. In addition, the mixed lymphocyte reaction assay with MSC-derived EVs indicated that EVs inhibit activation of antigen-presenting cells and suppress development of T helper 1 (Th1) and Th17 cells. These results raise the possibility that MSC-derived EVs may be an alternative to cell therapy for autoimmune disease prevention.

No effective treatment for Sjögren’s syndrome (SS), a chronic autoimmune disease affecting mainly salivary and lacrimal glands, is available now. Systemic infusion of allogeneic mesenchymal stem cells (MSCs) isolated from tissues such as bone marrow (BM) alleviated SS in mouse models and a small clinical trial, but further research and application of this MSC therapy were hindered by limited expandability, significant donor variations, and safety concerns of tissue-derived MSCs. To circumvent these issues, we derived MSCs from human iPSCs using an optimized protocol that can be easily scaled up to produce a huge amount of standardized MSCs. Our iPSC-MSCs inhibited the onset of lymphocyte infiltration into salivary glands in the NOD mouse model of SS in the same way as BM-MSCs. Extracellular vesicles (EVs) carry bioactive molecules in the same way as their originating cells and are more stable and considered much safer than cells for therapies. We found that EVs derived from BM-MSCs and iPSC-MSCs suppressed activation of immune cells and expression of proinflammation factors essential for SS progression in vitro and that infusion of iPSC-MSC EVs at the predisease stage decreased the lymphocyte infiltration in salivary glands and serum autoantibody levels in the same way as infusion of BM-MSCs and iPSC-MSCs. These data suggested that iPSC-MSC EVs have the potential to prevent the progression of SS before the onset of sialadenitis.

TNF-α stimulated gene/protein 6 (TNFAIP6/TSG-6) is a multifunctional protein that has a number of potential therapeutic applications. Experiments and clinical trials with TSG-6, however, have been limited by the technical difficulties of producing the recombinant protein. We prepared stable clones of CHO cells that expressed recombinant human TSG-6 (rhTSG-6) as a secreted glycoprotein. Paradoxically, both cell number and protein production decreased dramatically when the clones were expanded. The decreases occurred because the protein aggregated the synthesizing CHO cells by binding to the brush border of hyaluronan that is found around many cultured cells. In addition, the rhTSG-6 readily self-aggregated. To address these problems, we added to the medium an inhibitor of hyaluronan synthesis and heparin to compete with the binding of TSG-6 to hyaluronan. Also, we optimized the composition of the culture medium, and transferred the CHO cells from a spinner culture system to a bioreactor that controlled pH and thereby decreased pH-dependent binding properties of the protein. With these and other improvements in the culture conditions, we obtained 57.0 mg ± 9.16 S.D. of rhTSG-6 in 5 or 6 liter of medium. The rhTSG-6 accounted for 18.0% ± 3.76 S.D. of the total protein in the medium. We then purified the protein with a Ni-chelate column that bound the His tag engineered into the C-terminus of the protein followed by an anion exchange column. The yield of the purified monomeric rhTSG-6 was 4.1 mg to 5.6 mg per liter of culture medium. After intravenous injection into mice, the protein had a longer plasma half-life than commercially available rhTSG-6 isolated from a mammalian cell lysate, apparently because it was recovered as a secreted glycoprotein. The bioactivity of the rhTSG-6 in suppressing inflammation was demonstrated in a murine model.

Recent research indicated that extracellular vesicles (EVs) derived from mesenchymal stem/stromal cells (MSCs) are a promising alternative to MSCs for immunomodulatory therapy. However, the contents of MSC-EVs would change as their parent MSCs change, hence the therapeutic efficacy of MSC-derived EVs (MSC-EVs) would largely depend on donors, tissue sources and culture conditions of MSCs. To overcome limitations of tissue-derived MSCs, we previously used MSCs derived from human induced pluripotent stem cells (iMSCs) to produce EVs and demonstrated their therapeutic potential in a mouse model of secondary Sjögren's Syndrome. Here, we further found that EVs from early-passage iMSCs had better immunomodulatory potency than EVs from late-passage iMSCs in TLR4-stimulated splenocytes and in a mouse model of primary Sjögren's syndrome. Comparative molecular profiling using proteomics and microRNA sequencing revealed distinctive molecular profiles of iMSC-EVs with or without immunomodulation capacity. Amongst them, manipulation of TGF-ß1, miR-21 and miR-125b levels in iMSC-EVs significantly affected their immunosuppressive effects. These findings would help improve our understanding of the molecular mechanism underlying iMSC-EV-mediated immunomodulation and further provide strategies to improve regulatory function of EVs for the treatment of immune-mediated diseases. [ABSTRACT FROM AUTHOR]

The past decade has seen an explosion of research directed toward better understanding of the mechanisms of mesenchymal stem/stromal cell (MSC) function during rescue and repair of injured organs and tissues. In addition to delineating cell-cell signaling and molecular controls for MSC differentiation, the field has made particular progress in defining several other mechanisms through which administered MSCs can promote tissue rescue/repair. These include: 1) paracrine activity that involves secretion of proteins/peptides and hormones; 2) transfer of mitochondria by way of tunneling nanotubes or microvesicles; and 3) transfer of exosomes or microvesicles containing RNA and other molecules. Improved understanding of MSC function holds great promise for the application of cell therapy and also for the development of powerful cell-derived therapeutics for regenerative medicine. Focusing on these three mechanisms, we discuss MSC-mediated effects on immune cell responses, cell survival, and fibrosis and review recent progress with MSC-based or MSC-derived therapeutics. [ABSTRACT FROM AUTHOR]

Mesenchymal stem or stromal cells (MSCs) have many potential therapeutic applications including therapies for cancers and tissue damages caused by cancers or radical cancer treatments. However, tissue-derived MSCs such as bone marrow MSCs (BM-MSCs) may promote cancer progression and have considerable donor variations and limited expandability. These issues hinder the potential applications of MSCs, especially those in cancer patients. To circumvent these issues, we derived MSCs from transgene-free human induced pluripotent stem cells (iPSCs) efficiently with a modified protocol that eliminated the need of flow cytometric sorting. Our iPSC-derived MSCs were readily expandable, but still underwent senescence after prolonged culture and did not form teratomas. These iPSC-derived MSCs homed to cancers with efficiencies similar to BM-MSCs but were much less prone than BM-MSCs to promote the epithelial-mesenchymal transition, invasion, stemness, and growth of cancer cells. The observations were probably explained by the much lower expression of receptors for interleukin-1 and TGFβ, downstream protumor factors, and hyaluronan and its cofactor TSG6, which all contribute to the protumor effects of BM-MSCs. The data suggest that iPSC-derived MSCs prepared with the modified protocol are a safer and better alternative to BM-MSCs for therapeutic applications in cancer patients. The protocol is scalable and can be used to prepare the large number of cells required for "off-the-shelf" therapies and bioengineering applications. [ABSTRACT FROM AUTHOR]

Genetic and immunological screening for type 1 diabetes has led to the possibility of preventing disease in susceptible individuals. Here, we show that human mesenchymal stem/stromal cells (hMSCs) and tumor necrosis factor-α-stimulated gene 6 (TSG-6), a protein produced by hMSCs in response to signals from injured tissues, delayed the onset of spontaneous autoimmune diabetes in NOD mice by inhibiting insulitis and augmenting regulatory T cells (Tregs) within the pancreas. Importantly, hMSCs with a knockdown of tsg-6 were ineffective at delaying insulitis and the onset of diabetes in mice. TSG-6 inhibited the activation of both T cells and antigen-presenting cells (APCs) in a CD44-dependent manner. Moreover, multiple treatments of TSG-6 rendered APCs more tolerogenic, capable of enhancing Treg generation and delaying diabetes in an adoptive transfer model. Therefore, these results could provide the basis for a novel therapy for the prevention of type 1 diabetes. [ABSTRACT FROM AUTHOR]

Previous reports suggested that culture as 3D aggregates or as spheroids can increase the therapeutic potential of the adult stem/progenitor cells referred to as mesenchymal stem cells or multipotent mesenchymal stromal cells (MSCs). Here we used a hanging drop protocol to prepare human MSCs (hMSCs) as spheroids that maximally expressed TNFa stimulated gene/protein 6 (TSG-6), the antiinflammatory protein that was expressed at high levels by hMSCs trapped in the lung after i.v. infusion and that largely explained the beneficial effects of hMSCs in mice with myocardial infarcts. The properties of spheroid hMSCs were found to depend critically on the culture conditions. Under optimal conditions for expression of TSG-6, the hMSCs also expressed high levels of stanniocalcin-1, a protein with both antiinflammatory and antiapoptotic properties. In addition, they expressed high levels of three anticancer proteins: IL-24, TNFa-related apoptosis inducing ligand, and CD82. The spheroid hMSCs were more effective than hMSCs from adherent monolayer cultures in suppressing inflammatory responses in a coculture system with LPS-activated macrophages and in a mouse model for peritonitis. In addition, the spheroid hMSCs were about one-fourth the volume of hMSCs from adherent cultures. Apparently as a result, larger numbers of the cells trafficked through the lung after i.v. infusion and were recovered in spleen, liver, kidney, and heart. The data suggest that spheroid hMSCs may be more effective than hMSCs from adherent cultures in therapies for diseases characterized by sterile tissue injury and unresolved inflammation and for some cancers that are sensitive to antiinflammatory agents. [ABSTRACT FROM AUTHOR]

Multipotent mesenchymal stromal cells (MSCs) home to damaged tissue by processes partly regulated by integrins. Integrin subunits expressed by MSCs were identified by flow cytometry (FC), immunocytochemistry (IC), and a panel of integrin-binding antibodies. In subconfluent cultures, over 80% of MSCs expressed integrin subunits β1, β2, and α3, 20%–55% expressed α1, α2, α4, α5, α6, and αV, and about 10% expressed β3 when assayed by FC. None of the cells expressed significant levels of 13 other integrins as assayed by FC, but seven of the 13 integrins were detected by IC: β5, α7, α8, α9, α11, αX, and αD. Expression of some integrins changed with MSC confluency: integrins β3, α1, α3, α5, and αV increased, and α6 decreased. Furthermore, α4 was the only integrin to vary among preparations of MSCs from different donors. The results resolved some discrepancies in the literature concerning integrin expression by MSCs. We also investigated the role of specific integrins in MSC adhesion to endothelial cells (ECs) from the pulmonary artery (HPAEC), cardiac-derived microvasculature (HMVEC-C), and umbilical veins (HUVEC). In experiments with blocking antibodies to beta integrins, anti-β5 reduced MSC adhesion to all types of ECs, anti-β1 to both HUVEC and HPAEC, anti-β3 to HUVEC, and anti-β2 to HMVEC-C. With blocking antibodies to alpha integrins, anti-αX reduced adhesion to HPAEC and HMVEC-C, anti-αV to HPAEC, and both anti-α7 and anti-αD to HMVEC-C. Thus, MSCs use diverse integrins to adhere to EC from various blood vessels in vitro. [ABSTRACT FROM AUTHOR]

Quantitative assays for human DNA and mRNA were used to examine the paradox that intravenously (i.v.) infused human multipotent stromal cells (hMSCs) can enhance tissue repair without significant engraftment. After 2 × 106 hMSCs were i.v. infused into mice, most of the cells were trapped as emboli in lung. The cells in lung disappeared with a half-life of about 24 hr, but <1000 cells appeared in six other tissues. The hMSCs in lung upregulated expression of multiple genes, with a large increase in the anti-inflammatory protein TSG-6. After myocardial infarction, i.v. hMSCs, but not hMSCs transduced with TSG-6 siRNA, decreased inflammatory responses, reduced infarct size, and improved cardiac function. l.v. administration of recombinant TSG-6 also reduced inflammatory responses and reduced infarct size. The results suggest that improvements in animal models and patients after i.v. infusions of MSCs are at least in part explained by activation of MSCs to secrete TSG-6. [ABSTRACT FROM AUTHOR]

We tested the hypothesis that multipotent stromal cells from human bone marrow (hMSCs) can provide a potential therapy for human diabetes mellitus. Severe but nonlethal hyperglycemia was produced in NOD/scid mice with daily low doses of streptozotocin on days 1–4, and hMSCs were delivered via intracardiac infusion on days 10 and 17. The hMSCs lowered blood glucose levels in the diabetic mice on day 32 relative to untreated controls (18.34 mM ± 1.12 SE vs. 27.78 mM ± 2.45 SE, P = 0.0019). ELISAs demonstrated that blood levels of mouse insulin were higher in the hMSC-treated as compared with untreated diabetic mice, but human insulin was not detected. PCR assays detected human Alu sequences in DNA in pancreas and kidney on day 17 or 32 but not in other tissues, except heart, into which the cells were infused. In the hMSC-treated diabetic mice, there was an increase in pancreatic islets and β cells producing mouse insulin. Rare islets contained human cells that colabeled for human insulin or PDX-1. Most of the β cells in the islets were mouse cells that expressed mouse insulin. In kidneys of hMSC-treated diabetic mice, human ceils were found in the glomeruli. There was a decrease in mesangial thickening and a decrease in macrophage infiltration. A few of the human cells appeared to differentiate into glomerular endothelial cells. Therefore, the results raised the possibility that hMSCs may be useful in enhancing insulin secretion and perhaps improving the renal lesions that develop in patients with diabetes mellitus. [ABSTRACT FROM AUTHOR]

Human mesenchymal stem cells (MSC), that have been reported to be present in bone marrow, adipose tissues, dermis, muscles and peripheral blood, have the potential to differentiate along different lineages including those forming bone, cartilage, fat, muscle and neuron. This differentiation potential makes MSC excellent candidates for cell-based tissue engineering. In this study, we have examined phenotypes and gene expression profile of the human adipose tissue-derived stromal cells (ATSC) in the undifferentiated states, and compared with that of bone marrow stromal cells (BMSC). ATSC were enzymatically released from adipose tissues from adult human donors and were expanded in monolayer with serial passages at confluence. BMSC were harvested from the metaphysis of adult human femur. Flowcytometric analysis showed that ATSC have a marker expression that is similar to that of BMSC. ATSC expressed CD29, CD44, CD90, CD105 and were absent for HLA-DR and c-kit expression. Under appropriate culture conditions, MSC were induced to differentiate to the osteoblast, adipocyte, and chondrogenic lineages. ATSC were superior to BMSC in respect to maintenance of proliferating ability, and microarray analysis of gene expression revealed differentially expressed genes between ATSC and BMSC. The proliferating ability and differentiation potential of ATSC were variable according to the culture condition. The similarities of the phenotypes and the gene expression profiles between ATSC and BMSC could have broad implications for human tissue engineering. [ABSTRACT FROM AUTHOR]

Cell-matrix interactions have major effects upon phenotypic features such as gene regulation, cytoskeletal structure, differentiation and aspects of cell growth control. Detachment from the matrix epithelial cells induces programmed cell death, and this cell detachment induced apoptosis has been referred to as ‘anoikis’. This study was undertaken to determine whether apoptosis is induced by inhibition of contact with extracellular matrix (ECM) in collecting duct cells and to investigate the signaling mechanisms of the process. Upon detachment from ECM, mouse inner medullary collecting duct cells (mIMCD-3) and mouse outer cortical collecting duct cells (M-1), which were derived from an SV40 transgenic mouse, entered into programmed cell death. Forced suspension of mIMCD-3 or M-1 cells did not affect the expression of Bcl-2-related proteins and did not activate c-Jun NH[sub 2] -terminal kinase. Detachment of cells from ECM activated p38 mitogen-activated protein kinase (p38), but its inhibition with SB203580 did not protect cells from anoikis. Detachment of cells from matrix inhibited NF-κB activity, and the inhibition of NF-κB activity by overexpression of nonphosphorylatable I-κB increased detachment-induced apoptotic cell death in M-1 cells. Forced suspension of M-1 cells still activated p53 activity. Caspase-8 was activated during anoikis, but the time course of its activation was in accordance with DNA fragmentation. These results indicate that detachment from ECM induces apoptosis in the kidney collecting duct cells. Changes in expression levels of Bcl-2-related proteins or activation of JNK/p38 kinase are not critical for anoikis. Decrease in NF-κB activity and activation of p53 induced by inhibition of interaction with ECM play roles in anoikis in SV-40-transformed collecting duct cells. Caspase-8 is activated during detachment-induced apoptosis, the mechanisms of which are independent of activation of cell death receptors.Copyright © 1999 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Background aims. Previously, we showed that human mesenchymal stromal cells (hMSCs) were activated to express tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) upon TNF-α stimulation, induced cell death in triple-negative breast cancer (TNBC) MDA-MB-231 cells (MDA), and RNA released from apoptotic MDA further increased TRAIL expression in hMSCs. This feed-forward stimulation increased apoptosis in MDA cells. Here, we tested whether TRAIL-expressing hMSCs, in combination with a sub-toxic-dose of a chemotherapy drug doxorubicin, would overcome TRAIL resistance and create synergistic effects on targeting metastatic TNBC. Methods. To optimize conditions for the combination treatment, we (i) selected an optimal condition to activate hMSCs for TRAIL expression, (ii) selected an optimal dose of doxorubicin treatment, (iii) examined underlying mechanisms in vitro and (iv) tested the efficacy of the optimized conditions in a xenograft mouse model of human breast cancer lung metastasis. Results. The results showed that DNA fragments from apoptotic MDA triggered hMSCs to increase further TRAIL expression in an absent in melanoma 2 (AIM2)--dependent manner, and thus higher TRAIL-expressing hMSCs stimulated with synthetic DNA, poly(deoxyadenylic-deoxythymidylic) acid [poly(dA:dT)], more effectively suppressed tumor progression in vivo. Furthermore, activated hMSCs increased apoptosis in MDA cells when combined with a sub-toxic dose of doxorubicin, which was mediated by up-regulating TRAIL and Fas-related pathways. When we combined the optimized conditions, pre-activated hMSCs with poly (dA:dT) synergistically reduced tumor burden even with minimal doxorubicin treatment in a xenograft mouse model of human breast cancer lung metastasis. Conclusions. These results suggest that the treatment of hMSCs with a sub-toxic dose of doxorubicin can overcome TRAIL resistance and be a potential novel therapy for TNBC metastasis treatment. [ABSTRACT FROM AUTHOR]