Cellular and molecular immuno therapeutics derived from the bone marrow stroma

Thesis (Ph. D.)--Harvard-MIT Division of Health Sciences and Technology, 2008.Includes bibliographical references (p. 155-174).The bone marrow contains a multipotent stromal cell, commonly referred to as a mesenchymal stem cell (MSC). There has been recent interest in the clinical use of MSCs for cell-based therapy because: (1) bone marrow aspiration is a routine method used in medicine thereby allowing for easy accessibility to human MSCs; (2) MSCs are easily isolated and can expand to clinical scales in a relatively short period of time; (3) MSCs can be biopreserved without loss of potency and stored for point-of-care delivery; and (4) human trials of MSCs thus far have shown no adverse reactions to allogeneic versus autologous MSC transplants suggesting that therapy can cross histocompatibility barriers. This thesis describes the development of new modalities and indications for MSC-based treatments by leveraging the endogenous functions of these cells for therapeutic purposes. First, it is known that marrow stromal cells support hematopoiesis by secreting bioactive molecules that aid in the growth, differentiation, function and migration of hematopoietic cells within the marrow cavity. We show that these same secreted molecules derived from MSCs ex vivo can be formulated as an intravenous drug. In a D-galactosamine model of acute liver failure, a bolus injection of a concentrated form of MSC conditioned medium (MSC-CM) led to a significant survival benefit with a one week study endpoint. We employed in vitro and in vivo assays to demonstrate the effect of MSC-CM on leukocytes and resident liver cells. Traditional biochemical approaches were performed to identify active fractions within MSC-CM that were responsible for its therapeutic efficacy. As a corollary to an injectable drug, we developed MSCbased extracorporeal devices to serve as a dynamic source of MSC-CM in a dialysis-like setting.(cont.) Liver injured rats supported by extracorporeal bioreactors seeded with MSCs had significant improvements in liver serologies and survival in the short-term, whereas a composite device containing both MSCs and hepatocytes was shown to have a long-term survival benefit after 30 days. The second natural function of MSCs that was exploited for therapy concerns recent evidence that stromal cells can present antigens in lymphoid organs. We discovered that MSCs can express peripheral tissue antigens similar to other specialized antigen presenting cells in the thymus and lymph nodes - a process known to induce tolerance to self-reactive T cells in vivo. We show that MSC transplantation can be an effective treatment of intestinal autoimmunity in a chemically-induced model of colitis and a mouse model deficient in regulatory T cells. In addition, we demonstrate that MSC grafts increase the endogenous population of suppressor cells in vivo, which can potentially amplify and sustain the immunosuppression of the original transplant. The proposed work is significant, as development of such therapies for acute liver failure and inflammatory bowel disease would potentially treat an estimated 100,000+ newly diagnosed patients or ones who are refractory or contraindicated to standard-of-care medical/surgical procedures. These studies may empower the future use of MSCs in other organ failure syndromes and autoimmune conditions. Finally, exploration of the therapeutic functions of MSCs is expected to enhance our understanding of the mechanisms involved in cell therapy and give further insight to the natural functions of MSCs during health and disease.by Biju Parekkadan.Ph.D

Multiple genetically engineered humanized microenvironments in a single mouse

Background Immunodeficient mouse models that accept human cell and tissue grafts can contribute greater knowledge to human stem cell research. In this technical report, we used biomaterial implants seeded with genetically engineered stromal cells to create several unique microenvironments in a single mouse. The scope of study was focused on human CD34 hematopoietic stem/progenitor cell (HSPC) engraftment and differentiation within the engineered microenvironment. Results A mouse model system was created using subdermal implant sites that overexpressed a specific human cytokines (Vascular Endothelial Growth Factor A (hVEGFa), Stromal Derived Factor 1 Alpha (hSDF1a), or Tumor Necrosis Factor Alpha (hTNFa)) by stromal cells in a three-dimensional biomaterial matrix. The systemic exposure of locally overexpressed cytokines was minimized by controlling the growth of stromal cells, which led to autonomous local, concentrated sites in a single mouse for study. This biomaterial implant approach allowed for the local analysis of each cytokine on hematopoietic stem cell recruitment, engraftment and differentiation in four different tissue microenvironments in the same host. The engineered factors were validated to have bioactive effects on human CD34+ hematopoietic progenitor cell differentiation. Conclusions This model system can serve as a new platform for the study of multiple human proteins and their local effects on hematopoietic cell biology for in vivo validation studies

Mesenchymal Stem Cell-Derived Molecules Reverse Fulminant Hepatic Failure

Modulation of the immune system may be a viable alternative in the treatment of fulminant hepatic failure (FHF) and can potentially eliminate the need for donor hepatocytes for cellular therapies. Multipotent bone marrow-derived mesenchymal stem cells (MSCs) have been shown to inhibit the function of various immune cells by undefined paracrine mediators in vitro. Yet, the therapeutic potential of MSC-derived molecules has not been tested in immunological conditions in vivo. Herein, we report that the administration of MSC-derived molecules in two clinically relevant forms-intravenous bolus of conditioned medium (MSC-CM) or extracorporeal perfusion with a bioreactor containing MSCs (MSC-EB)-can provide a significant survival benefit in rats undergoing FHF. We observed a cell mass-dependent reduction in mortality that was abolished at high cell numbers indicating a therapeutic window. Histopathological analysis of liver tissue after MSC-CM treatment showed dramatic reduction of panlobular leukocytic infiltrates, hepatocellular death and bile duct duplication. Furthermore, we demonstrate using computed tomography of adoptively transferred leukocytes that MSC-CM functionally diverts immune cells from the injured organ indicating that altered leukocyte migration by MSC-CM therapy may account for the absence of immune cells in liver tissue. Preliminary analysis of the MSC secretome using a protein array screen revealed a large fraction of chemotactic cytokines, or chemokines. When MSC-CM was fractionated based on heparin binding affinity, a known ligand for all chemokines, only the heparin-bound eluent reversed FHF indicating that the active components of MSC-CM reside in this fraction. These data provide the first experimental evidence of the medicinal use of MSC-derived molecules in the treatment of an inflammatory condition and support the role of chemokines and altered leukocyte migration as a novel therapeutic modality for FHF

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

Long-adapter single-strand oligonucleotide probes for the massively multiplexed cloning of kilobase genome regions

As the catalogue of sequenced genomes and metagenomes continues to grow, massively parallel approaches for the comprehensive and functional analysis of gene products and regulatory elements are becoming increasingly valuable. Current strategies to synthesize or clone complex libraries of DNA sequences are limited by the length of the DNA targets, throughput and cost. Here, we show that long-adapter single-strand oligonucleotide (LASSO) probes can capture and clone thousands of kilobase DNA fragments in a single reaction. As a proof-of-principle, we simultaneously cloned >3,000 bacterial open reading frames (ORFs) from E. coli genomic DNA (spanning 400–5,000 bp targets). Targets were enriched up to a median of ~60-fold compared to non-targeted genomic regions. At a cutoff of 3 times the median non-target reads per kilobase of genetic element per million reads, ~75% of the targeted ORFs were successfully captured. We also show that LASSO probes can clone human ORFs from complementary DNA, and an ORF library from a human-microbiome sample. LASSO probes could be used for the preparation of long-read sequencing libraries and for massively multiplexed cloning

Additional file 1: Figure S1. of Multiple genetically engineered humanized microenvironments in a single mouse

Selection and conformation of lentivial transfected mouse stromal cells. (A) Flow cytometric analysis of GFP mBMSC, (B) Culture-expanded genetically engineered mBMSCs. (Scale bar, 200μm). Figure S2. Characterized secretion of human cytokines from genetically engineered stromal cells in 1 and 3 weeks in vitro culture. Figure S3. hSDF1a ELISA in mouse blood serum. Control mice without scaffold implantation showed a background level of SDF1a signal due to cross-reactivity. This level was used as a baseline and was also observed in growth arrested, which was concluded, as undetectable. The other groups showed measurable levels above background and were concluded to be true hSDF-1a detection. Figure S4. SEM images of growth-competent genetically engineered stromal cell-seeded scaffolds. (A) Cross-sectional images of human soluble factor secreting engineered stromal cell-seeded scaffolds after 6 weeks subcutaneous implantation. Except hTNFa, entire pores were completely filled with tissue cells with no hematopoietic components. (B) Closed-up image of growing engineered stromal cell-seeded scaffolds. Figure S5. Examples of semi-quantitative image analysis using ImageJ. (A) Collagen fiber area estimation from a Masson’s Trichrome staining image, (B) Vasculature area estimation from an immunohistostaining mCD31 and DAPI image. Figure S6. Long-term maintenance of inflammation-mimicking tissue microenvironment indirectly indicates survival and function of growth-arrested hTNFa secreting engineered stromal cells in the implanted scaffolds. (DOCX 2962 kb

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

An Analysis of Transcriptomic Burden Identifies Biological Progression Roadmaps for Hematological Malignancies and Solid Tumors

Biological paths of tumor progression are difficult to predict without time-series data. Using median shift and abacus transformation in the analysis of RNA sequencing data sets, natural patient stratifications were found based on their transcriptomic burden (TcB). Using gene-behavior analysis, TcB groups were evaluated further to discover biological courses of tumor progression. We found that solid tumors and hematological malignancies (n = 4179) share conserved biological patterns, and biological network complexity decreases at increasing TcB levels. An analysis of gene expression datasets including pediatric leukemia patients revealed TcB patterns with biological directionality and survival implications. A prospective interventional study with PI3K targeted therapy in canine lymphomas proved that directional biological responses are dynamic. To conclude, TcB-enriched biological mechanisms detected the existence of biological trajectories within tumors. Using this prognostic informative novel informatics method, which can be applied to tumor transcriptomes and progressive diseases inspires the design of progression-specific therapeutic approaches

Effects of intermittent T-cell cluster disaggregation on proliferative capacity and checkpoint marker expression

Background/aim: T-cell immunotherapies are rapidly gaining grounds in clinical success. Presently, there is first-to-market knowledge on the translation of research scale methods to clinical and commercial scales. Improved understanding can lead to more consistent and efficient production, scaling, and eventual potency. T-cell checkpoint markers, proliferation, and T-cell cluster size and disaggregation are one set of parameters that have yet to be explored. Methods: We herein activated T-cells and assessed various mechanical dissociation frequencies in relation to expression of checkpoint markers (measured by flow cytometry). Results: We herein find increased T-cell proliferation capacity with increased dissociation frequency. We also find that with increased cluster size and duration, lower proliferation, and increased expression of checkpoint markers. Conclusions: These findings reveal new translation findings with respect to T-cell handling and production and suggest that T-cell disaggregation may be important to improved cell yields and phenotype

Mesenchymal stem cell-derived molecules directly modulate hepatocellular death and regeneration in vitro and in vivo

Orthotopic liver transplantation is the only proven effective treatment for fulminant hepatic failure (FHF), but its use is limited because of organ donor shortage, associated high costs, and the requirement for lifelong immunosuppression. FHF is usually accompanied by massive hepatocellular death with compensatory liver regeneration that fails to meet the cellular losses. Therefore, therapy aimed at inhibiting cell death and stimulating endogenous repair pathways could offer major benefits in the treatment of FHF. Recent studies have demonstrated that mesenchymal stem cell (MSC) therapy can prevent parenchymal cell loss and promote tissue repair in models of myocardial infarction, acute kidney failure, and stroke through the action of trophic secreted molecules. In this study, we investigated whether MSC therapy can protect the acutely injured liver and stimulate regeneration. In a D-galactosamine-induced rat model of acute liver injury, we show that systemic infusion of MSC-conditioned medium (MSC-CM) provides a significant survival benefit and prevents the release of liver injury biomarkers. Furthermore, MSC-CM therapy resulted in a 90% reduction of apoptotic hepatocellular death and a three-fold increment in the number of proliferating hepatocytes. This was accompanied by a dramatic increase in the expression levels of 10 genes known to be up-regulated during hepatocyte replication. Direct antiapoptotic and promitotic effects of MSC-CM on hepatocytes were demonstrated using in vitro assays. Conclusion: These data provide the first dear evidence that MSC-CM therapy provides trophic support to the injured liver by inhibiting hepatocellular death and stimulating regeneration, potentially creating new avenues for the treatment of FHF