Pharmacokinetics of Natural and Engineered Secreted Factors Delivered by Mesenchymal Stromal Cells

Transient cell therapy is an emerging drug class that requires new approaches for pharmacological monitoring during use. Human mesenchymal stem cells (MSCs) are a clinically-tested transient cell therapeutic that naturally secrete anti-inflammatory factors to attenuate immune-mediated diseases. MSCs were used as a proof-of-concept with the hypothesis that measuring the release of secreted factors after cell transplantation, rather than the biodistribution of the cells alone, would be an alternative monitoring tool to understand the exposure of a subject to MSCs. By comparing cellular engraftment and the associated serum concentration of secreted factors released from the graft, we observed clear differences between the pharmacokinetics of MSCs and their secreted factors. Exploration of the effects of natural or engineered secreted proteins, active cellular secretion pathways, and clearance mechanisms revealed novel aspects that affect the systemic exposure of the host to secreted factors from a cellular therapeutic. We assert that a combined consideration of cell delivery strategies and molecular pharmacokinetics can provide a more predictive model for outcomes of MSC transplantation and potentially other transient cell therapeutics

Enriched protein screening of human bone marrow mesenchymal stromal cell secretions reveals MFAP5 and PENK as novel IL-10 modulators

The secreted proteins from a cell constitute a natural biologic library that can offer significant insight into human health and disease. Discovering new secreted proteins from cells is bounded by the limitations of traditional separation and detection tools to physically fractionate and analyze samples. Here, we present a new method to systematically identify bioactive cell-secreted proteins that circumvent traditional proteomic methods by first enriching for protein candidates by differential gene expression profiling. The bone marrow stromal cell secretome was analyzed using enriched gene expression datasets in combination with potency assay testing. Four proteins expressed by stromal cells with previously unknown anti-inflammatory properties were identified, two of which provided a significant survival benefit to mice challenged with lethal endotoxic shock. Greater than 85% of secreted factors were recaptured that were otherwise undetected by proteomic methods, and remarkable hit rates of 18% in vitro and 9% in vivo were achieved

Genome-wide Analyses Identify KIF5A as a Novel ALS Gene

To identify novel genes associated with ALS, we undertook two lines of investigation. We carried out a genome-wide association study comparing 20,806 ALS cases and 59,804 controls. Independently, we performed a rare variant burden analysis comparing 1,138 index familial ALS cases and 19,494 controls. Through both approaches, we identified kinesin family member 5A (KIF5A) as a novel gene associated with ALS. Interestingly, mutations predominantly in the N-terminal motor domain of KIF5A are causative for two neurodegenerative diseases: hereditary spastic paraplegia (SPG10) and Charcot-Marie-Tooth type 2 (CMT2). In contrast, ALS-associated mutations are primarily located at the C-terminal cargo-binding tail domain and patients harboring loss-of-function mutations displayed an extended survival relative to typical ALS cases. Taken together, these results broaden the phenotype spectrum resulting from mutations in KIF5A and strengthen the role of cytoskeletal defects in the pathogenesis of ALS.Peer reviewe

A comparison of adipose and bone marrow-derived mesenchymal stromal cell secreted factors in the treatment of systemic inflammation

Background: Bone marrow-derived mesenchymal stromal cells (BMSCs) are a cell population of intense exploration for therapeutic use in inflammatory diseases. Secreted factors released by BMSCs are responsible for the resolution of inflammation in several pre-clinical models. New studies have uncovered that adipose tissue also serves as a reservoir of multipotent, non-hematopoietic stem cells, termed adipose-derived stromal/stem cells (ASCs), with many common characteristics to BMSCs. We hypothesized that ASC and BMSC secreted factors would lead to a comparable benefit in the context of generalized inflammation. Findings: Proteomic profiling of conditioned media revealed that BMSCs express significantly higher levels of sVEGFR1 and sTNFR1, two soluble cytokine receptors with known therapeutic activity in sepsis. In a prophylactic study of endotoxin-induced inflammation in mice, we observed that BMSC secreted factors provided a greater survival benefit and tissue protection of endotoxemic mice compared to ASCs. Neutralization of sVEGFR1 and sTNFR1 did not significantly affect the survival benefit experienced by mice treated with BMSC secreted factors. Conclusions: Our findings suggest that BMSCs may be more effective as a cell therapeutic for use in endotoxic shock and that ASCs may be positioned for continued exploration in immunomodulatory diseases. Soluble cytokine receptors can distinguish stromal cells from different tissue origins, though they may not be the sole contributors to the therapeutic benefit of BMSCs. Furthermore, other secreted factors not discussed in this study may also differentiate these stromal cell populations from one another.National Institutes of Health (U.S.) (R01EB012521)National Institutes of Health (U.S.) (K01DK087770)Broad Foundations (Broad Medical Research Program BMRP498382)Shriners Hospital for Childre

Premature polyadenylation of MAGI3 is associated with diminished N[superscript 6]-methyladenosine in its large internal exon

In cancer, tumor suppressor genes (TSGs) are frequently truncated, causing their encoded products to be non-functional or dominant-negative. We previously showed that premature polyadenylation (pPA) of MAGI3 truncates the gene, switching its functional role from a TSG to a dominant-negative oncogene. Here we report that MAGI3 undergoes pPA at the intron immediately downstream of its large internal exon, which is normally highly modified by N[superscript 6]-methyladenosine (m[superscript 6]A). In breast cancer cells that upregulate MAGI3[superscript pPA], m[superscript 6]A levels in the large internal exon of MAGI3 are significantly reduced compared to cells that do not express MAGI3[superscript pPA]. We further find that MAGI3[superscript pPA] transcripts are significantly depleted of m[superscript 6]A modifications, in contrast to highly m6A-modified full-length MAGI3 mRNA. Finally, we analyze public expression data and find that other TSGs, including LATS1 and BRCA1, also undergo intronic pPA following large internal exons, and that m[superscript 6]A levels in these exons are reduced in pPA-activated breast cancer cells relative to untransformed mammary cells. Our study suggests that m6A may play a role in regulating intronic pPA of MAGI3 and possibly other TSGs, warranting further investigation

Role for Egr1 in the Transcriptional Program Associated with Neuronal Differentiation of PC12 Cells

PC12 cells are a well-established model to study how differences in signal transduction duration can elicit distinct cell behaviors. Epidermal growth factor (EGF) activates transient ERK signaling in PC12 cells that lasts 30–60 min, which in turn promotes proliferation; nerve growth factor (NGF) activates more sustained ERK signaling that lasts 4–6 h, which in turns induces neuronal differentiation. Data presented here extend a previous study by Mullenbrock et al. (2011) that demonstrated that sustained ERK signaling in response to NGF induces preferential expression of a 69-member gene set compared to transient ERK signaling in response to EGF and that the transcription factors AP-1 and CREB play a major role in the preferential expression of several genes within the set. Here, we examined whether the Egr family of transcription factors also contributes to the preferential expression of the gene set in response to NGF. Our data demonstrate that NGF causes transient induction of all Egr family member transcripts, but a corresponding induction of protein was detected for only Egr1 and 2. Chromatin immunoprecipitation experiments provided clearest evidence that, after induction, Egr1 binds 12 of the 69 genes that are preferentially expressed during sustained ERK signaling. In addition, Egr1 expression and binding upstream of its target genes were both sustained in response to NGF versus EGF within the same timeframe that its targets are preferentially expressed. These data thus provide evidence that Egr1 contributes to the transcriptional program activated by sustained ERK signaling in response to NGF, specifically by contributing to the preferential expression of its target genes identified here

Sustained Egr1 expression and binding upstream of target genes in response to NGF versus EGF.

<p><b>(a)</b> PC12 cell cultures were treated with 50 ng/ml NGF or 25 ng/ml EGF for 0–4 h. Total cell lysates were harvested and subjected to SDS-PAGE and Western blot for Egr1. Egr1 levels were quantified by densitometry and converted to % maximum levels for ten independent experiments and plotted (average ± S.E.). <b>(b)</b> PC12 cultures were treated with or without 25 ng/ml EGF for 1 h and subjected to ChIP assay using antibodies against Egr1 or an IgG control. Real-time PCR was then conducted on the immunoprecipitated DNA using primers within 250 bp of predicted Egr binding sites (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170076#pone.0170076.g001" target="_blank">Fig 1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0170076#pone.0170076.s003" target="_blank">S2 Table</a> for primer locations and sequences). Primers to amplify a region approximately 100 bp upstream of the <i>Myog</i> gene were used as a negative control for Egr1 binding. Data are plotted as % input and are averages from three to six independent experiments ± S.E. *, One-tailed Student’s <i>t</i> tests were conducted comparing the % input value for <i>Myog</i> after Egr1 IP to the % input values for each predicted Egr target after Egr1 IP, which yielded <i>p</i> values ≤ 0.05. <b>(c)</b> PC12 cell cultures were treated with 50 ng/ml NGF or 25 ng/ml EGF for 0–4 h and subjected to ChIP with anti-Egr1 antibody. Immunoprecipitated DNA was then subjected to real-time PCR with primers to detect Egr1 binding to a subset of its target genes, which was quantified as % input. Percent input values were then converted into % maximum values, which were plotted. *, One-tailed Student’s <i>t</i> tests were conducted comparing the % input values at each time point after NGF versus EGF treatment, which yielded <i>p</i> values ≤ 0.05 at the 3-hour time point for <i>Arc</i>, <i>Kctd11</i>, <i>Trib1</i>, <i>Tph1</i>, and <i>Vgf</i>.</p

Predicted Egr binding sites and ChIP primer locations upstream of genes preferentially expressed during sustained ERK signaling in response to NGF.

<p>Of the 69 genes that Mullenbrock et al. (2011) determined are preferentially expressed during sustained ERK signaling in response to NGF, 21 contained putative Egr binding sites within 5 kb upstream of their transcription start site (TSS). The locations of each Egr binding site are denoted by the vertical blue lines. The red horizontal lines denote the relative locations of primer sets used for real time PCR to detect Egr binding to nearby Egr binding site(s). For genes with multiple dispersed Egr binding sites, multiple primers sets were designed (denoted P1, P2, etc.) to detect Egr binding to the nearest predicted Egr binding site.</p

AP-1, CREB, and Egr1 cooperatively regulate 28 genes during their preferential expression in response to NGF and sustained ERK signaling.

<p>The Venn diagram summarizes genes bound by AP-1, CREB, and/or Egr1 during their preferentially expression in response to NGF and sustained ERK signaling, as detected by Mullenbrock et al. (2011) and the present study.</p