Identification and characterisation of novel CAR-T cells to target IL13Rα2 positive human glioma in vitro and in vivo.

BACKGROUND: Previously, we discovered that human solid tumours, but not normal human tissues, preferentially overexpress interleukin-13Receptor alpha2, a high binding receptor for IL-13. To develop novel anti-cancer approaches, we constructed a chimeric antigen receptor construct using a high binding and codon optimised scFv-IL-13Rα2 fragment fused with CD3ζ and co-stimulatory cytoplasmic domains of CD28 and 4-1BB. METHODS: We developed a scFv clone, designated 14-1, by biopanning the bound scFv phages using huIL-13Rα2Fc chimeric protein and compared its binding with our previously published clone 4-1. We performed bioinformatic analyses for complementary determining regions (CDR) framework and residue analyses of the light and heavy chains. This construct was packaged with helper plasmids to produce CAR-lentivirus and transduced human Jurkat T or activated T cells from peripheral blood mononuclear cells (PBMCs) to produce CAR-T cells and tested for their quality attributes in vitro and in vivo. Serum enzymes including body weight from non-tumour bearing mice were tested for assessing general toxicity of CAR-T cells. RESULTS: The binding of 14-1 clone is to IL-13Rα2Fc-chimeric protein is ∼5 times higher than our previous clone 4-1. The 14-1-CAR-T cells grew exponentially in the presence of cytokines and maintained phenotype and biological attributes such as cell viability, potency, migration and T cell activation. Clone 14-1 migrated to IL-13Rα2Fc and cell free supernatants only from IL-13Rα2+ve confluent glioma tumour cells in a chemotaxis assay. scFv-IL-13Rα2-CAR-T cells specifically killed IL-13Rα2+ve but not IL-13Rα2-ve tumour cells in vitro and selectively caused significant release of IFN-γ only from IL-13Rα2+ve co-cultures. These CAR-T cells regressed IL-13Rα2+ve glioma xenografts in vivo without any general toxicity. In contrast, the IL-13Rα2 gene knocked-down U251 and U87 xenografts failed to respond to the CAR-T therapy. CONCLUSION: Taken together, we conclude that the novel scFv-IL-13Rα2 CAR-T cell therapy may offer an effective therapeutic option after designing a careful pre-clinical and clinical study.

Experimental and computational approach to establish fit-for-purpose cell viability assays.

Aim: Cell viability assays are critical for cell-based products. Here, we demonstrate a combined experimental and computational approach to identify fit-for-purpose cell assays that can predict changes in cell proliferation, a critical biological response in cell expansion. Materials & methods: Jurkat cells were systematically injured using heat (45 ± 1°C). Cell viability was measured at 0 h and 24 h after treatment using assays for membrane integrity, metabolic function and apoptosis. Proliferation kinetics for longer term cultures were modeled using the Gompertz distribution to establish predictive models between cell viability results and proliferation. Results & conclusion: We demonstrate an approach for ranking these assays as predictors of cell proliferation and for setting cell viability specifications when a particular proliferation response is required.

In recent years, there has been a surge in the amount of cellular therapy products which have been engineered to treat patients with severe diseases. These cellular products use living cells to treat the disease, and the quality of these cell products is critical for ensuring product safety and effectiveness. Throughout the process of engineering and manufacturing these cell products, many cells can die or be in the process of dying, and the amount of dead cells in the product can impact product yield and quality. In any given cell product at any given time during the manufacturing process, cells are exposed to stresses, and these stresses can injure the cells through several mechanisms, leading to a range of cell death events that can follow different timelines. There are many existing assays which evaluate the health of the cells, known as cell viability assays, and these assays can be based on many different cell features that indicate a cell has been injured (i.e., cell membrane permeability, changes in cell metabolism, molecular markers for cell death). These cell viability assays provide different insights into the state of cell health/injury based on what cell features are being evaluated and the timing at which the viability measurements are taken, and some viability assays may be more appropriate than others for specific applications. Therefore, a method is needed to appropriately select cell viability assays that are designed to evaluate injuries to cells that occur in specific bioprocess. In this series of studies, we used a range of analytical methods to study the number of living and dead cells in a series of cell populations that we treated to induce damage to the cells, reducing their ability to grow. We then used mathematical models to determine the relationship between cell viability measurements and cell growth over time, and used the results to determine the sensitivity of the viability assays to changes in cell growth. We used a specific cell line in this example, but this technique can be applied to any cell line or cell sample population and different types of injuries can be applied to the cells. This approach can be used by manufacturers of cell-based products and therapies to identify cell viability assays that are meaningful for monitoring the production of cells and characterizing product quality.

GLI1+ perivascular, renal, progenitor cells: The likely source of spontaneous neoplasia that created the AGMK1-9T7 cell line.

The AGMK1-9T7 cell line has been used to study neoplasia in tissue culture. By passage in cell culture, these cells evolved to become tumorigenic and metastatic in immunodeficient mice at passage 40. Of the 20 x 106 kidney cells originally plated, less than 2% formed the colonies that evolved to create this cell line. These cells could be the progeny of some type of kidney progenitor cells. To characterize these cells, we documented their renal lineage by their expression of PAX-2 and MIOX, detected by indirect immunofluorescence. These cells assessed by flow-cytometry expressed high levels of CD44, CD73, CD105, Sca-1, and GLI1 across all passages tested; these markers have been reported to be expressed by renal progenitor cells. The expression of GLI1 was confirmed by immunofluorescence and western blot analysis. Cells from passages 13 to 23 possessed the ability to differentiate into adipocytes, osteoblasts, and chondrocytes; after passage 23, their ability to form these cell types was lost. These data indicate that the cells that formed the AGMK1-9T7 cell line were GLI1+ perivascular, kidney, progenitor cells.

Characterization of chimeric antigen receptor modified T cells expressing scFv-IL-13Rα2 after radiolabeling with 89Zirconium oxine for PET imaging.

BACKGROUND: Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Enabling in vitro methods to allow meaningful, sensitive in vivo biodistribution studies is needed to better understand CAR-T cell disposition and its relationship to both effectiveness and safety of these products. METHODS: To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with 89Zirconium-oxine (89Zr-oxine) and characterized and compared their product attributes with non-labeled CAR-T cells. The 89Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, T cell subtype characterization and product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells. RESULTS: We observed that radiolabeling of CAR-T cells with 89Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells and subtypes such as CD4 + , CD8 + and scFV-IL-13Rα2 transgene positive T cell population were characterized and found similar to that of unlabeled cells as determined by TUNEL assay, caspase 3/7 enzyme and granzyme B activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion (PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells. CONCLUSIONS: Importantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with 89Zr-oxine retain critical product attributes and suggest 89Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.

Cytocentric measurement for regenerative medicine.

Any Regenerative Medicine (RM) business requires reliably predictable cell and tissue products. Regulatory agencies expect control and documentation. However, laboratory tissue production is currently not predictable or well-controlled. Before conditions can be controlled to meet the needs of cells and tissues in culture for RM, we have to know what those needs are and be able to quantify them. Therefore, identification and measurement of critical cell quality attributes at a cellular or pericellular level is essential to generating reproducible cell and tissue products. Here, we identify some of the critical cell and process parameters for cell and tissue products as well as technologies available for sensing them. We also discuss available and needed technologies for monitoring both 2D and 3D cultures to manufacture reliable cell and tissue products for clinical and non-clinical use. As any industry matures, it improves and standardizes the quality of its products. Cytocentric measurement of cell and tissue quality attributes are needed for RM.

Characterization of Chimeric Antigen Receptor Modified T Cells Expressing scFv-IL-13Rα2 after Radiolabeling with 89Zirconium Oxine for PET Imaging.

Background Chimeric antigen receptor (CAR) T cell therapy is an exciting cell-based cancer immunotherapy. Unfortunately, CAR-T cell therapy is associated with serious toxicities such as cytokine release syndrome (CRS) and neurotoxicity. The mechanism of these serious adverse events (SAEs) and how homing, distribution and retention of CAR-T cells contribute to toxicities is not fully understood. Methods To determine if radiolabelling of CAR-T cells could support positron emission tomography (PET)-based biodistribution studies, we labeled IL-13Rα2 targeting scFv-IL-13Rα2-CAR-T cells (CAR-T cells) with 89 Zirconium-oxine ( 89 Zr-oxine), and characterized and compared their product attributes with non-labeled CAR-T cells. The 89 Zr-oxine labeling conditions were optimized for incubation time, temperature, and use of serum for labeling. In addition, product attributes of radiolabeled CAR-T cells were studied to assess their overall quality including cell viability, proliferation, phenotype markers of T-cell activation and exhaustion, cytolytic activity and release of interferon-γ upon co-culture with IL-13Rα2 expressing glioma cells. Results We observed that radiolabeling of CAR-T cells with 89 Zr-oxine is quick, efficient, and radioactivity is retained in the cells for at least 8 days with minimal loss. Also, viability of radiolabeled CAR-T cells was similar to that of unlabeled cells as determined by TUNEL assay and caspase 3/7 enzyme activity assay. Moreover, there were no significant changes in T cell activation (CD24, CD44, CD69 and IFN-γ) or T cell exhaustion(PD-1, LAG-3 and TIM3) markers expression between radiolabeled and unlabeled CAR-T cells. In chemotaxis assays, migratory capability of radiolabeled CAR-T cells to IL-13Rα2Fc was similar to that of non-labeled cells. Conclusions Importantly, radiolabeling has minimal impact on biological product attributes including potency of CAR-T cells towards IL-13Rα2 positive tumor cells but not IL-13Rα2 negative cells as measured by cytolytic activity and release of IFN-γ. Thus, IL-13Rα2 targeting CAR-T cells radiolabeled with 89 Zr-oxine retain critical product attributes and suggest 89 Zr-oxine radiolabeling of CAR-T cells may facilitate biodistribution and tissue trafficking studies in vivo using PET.

3D bioprinting optimization of human mesenchymal stromal cell laden gelatin-alginate-collagen bioink.

3D bioprinting technology has gained increased attention in the regenerative medicine and tissue engineering communities over the past decade with their attempts to create functional living tissues and organsde novo. While tissues such as skin, bone, and cartilage have been successfully fabricated using 3D bioprinting, there are still many technical and process driven challenges that must be overcome before a complete tissue engineered solution is realized. Although there may never be a single adopted bioprinting process in the scientific community, adherence to optimized bioprinting protocols could reduce variability and improve precision with the goal of ensuring high quality printed constructs. Here, we report on the bioprinting of a gelatin-alginate-collagen bioink containing human mesenchymal stromal cells (hMSCs) which has been optimized to ensure printing consistency and reliability. The study consists of three phases: a pre-printing phase which focuses on bioink characterization; a printing phase which focuses on bioink extrudability/printability, construct stability, and printing accuracy; and a post-processing phase which focuses on the homogeneity and bioactivity of the encapsulated hMSC printed constructs. The results showed that eight identical constructs containing hMSCs could be reliably and accurately printed into stable cross-hatched structures with a single material preparation, and that batch-to-batch consistency was accurately maintained across all preparations. Analysis of the proliferation, morphology, and differentiation of encapsulated hMSCs within the printed constructs showed that cells were able to form large,interconnected colonies and were capable of robust adipogenic differentiation within 14 d of culturing.

Morphological landscapes from high content imaging reveal cytokine priming strategies that enhance mesenchymal stromal cell immunosuppression.

Successful clinical translation of mesenchymal stromal cell (MSC) products has not been achieved in the United States and may be in large part due to MSC functional heterogeneity. Efforts have been made to identify "priming" conditions that produce MSCs with consistent immunomodulatory function; however, challenges remain with predicting and understanding how priming impacts MSC behavior. The purpose of this study was to develop a high throughput, image-based approach to assess MSC morphology in response to combinatorial priming treatments and establish morphological profiling as an effective approach to screen the effect of manufacturing changes (i.e., priming) on MSC immunomodulation. We characterized the morphological response of multiple MSC lines/passages to an array of Interferon-gamma (IFN-γ) and tumor necrosis factor-⍺ (TNF-⍺) priming conditions, as well as the effects of priming on MSC modulation of activated T cells and MSC secretome. Although considerable functional heterogeneity, in terms of T-cell suppression, was observed between different MSC lines and at different passages, this heterogeneity was significantly reduced with combined IFN-γ/TNF-⍺ priming. The magnitude of this change correlated strongly with multiple morphological features and was also reflected by MSC secretion of immunomodulatory factors, for example, PGE2, ICAM-1, and CXCL16. Overall, this study further demonstrates the ability of priming to enhance MSC function, as well as the ability of morphology to better understand MSC heterogeneity and predict changes in function due to manufacturing.

Establishing CD19 B-cell reference control materials for comparable and quantitative cytometric expression analysis.

In the field of cell-based therapeutics, there is a great need for high-quality, robust, and validated measurements for cell characterization. Flow cytometry has emerged as a critically important platform due to its high-throughput capability and its ability to simultaneously measure multiple parameters in the same sample. However, to assure the confidence in measurement, well characterized biological reference materials are needed for standardizing clinical assays and harmonizing flow cytometric results between laboratories. To date, the lack of adequate reference materials, and the complexity of the cytometer instrumentation have resulted in few standards. This study was designed to evaluate CD19 expression in three potential biological cell reference materials and provide a preliminary assessment of their suitability to support future development of CD19 reference standards. Three commercially available human peripheral blood mononuclear cells (PBMCs) obtained from three different manufacturers were tested. Variables that could potentially contribute to the differences in the CD19 expression, such as PBMCs manufacturing process, number of healthy donors used in manufacturing each PBMC lot, antibody reagent, operators, and experimental days were included in our evaluation. CD19 antibodies bound per cell (ABC) values were measured using two flow cytometry-based quantification schemes with two independent calibration methods, a single point calibration using a CD4 reference cell and QuantiBrite PE bead calibration. Three lots of PBMC from three different manufacturers were obtained. Each lot of PBMC was tested on three different experimental days by three operators using three different lots of unimolar anti-CD19PE conjugates. CD19 ABC values were obtained in parallel on a selected lot of the PBMC samples using mass spectrometry (CyTOF) with two independent calibration methods, EQ4 and bead-based calibration were evaluated with CyTOF-technology. Including all studied variabilities such as PBMC lot, antibody reagent lot, and operator, the averaged mean values of CD19 ABC for the three PBMC manufacturers (A,B, and C) obtained by flow cytometry were found to be: 7953 with a %CV of 9.0 for PBMC-A, 10535 with a %CV of 7.8 for PBMC-B, and 12384 with a %CV of 16 for PBMC-C. These CD19 ABC values agree closely with the findings using CyTOF. The averaged mean values of CD19 ABC for the tested PBMCs is 9295 using flow cytometry-based method and 9699 using CyTOF. The relative contributions from various sources of uncertainty in CD19 ABC values were quantified for the flow cytometry-based measurement scheme. This uncertainty analysis suggests that the number of antigens or ligand binding sites per cell in each PBMC preparation is the largest source of variability. On the other hand, the calibration method does not add significant uncertainty to the expression estimates. Our preliminary assessment showed the suitability of the tested materials to serve as PBMC-based CD19+ reference control materials for use in quantifying relevant B cell markers in B cell lymphoproliferative disorders and immunotherapy. However, users should consider the variabilities resulting from different lots of PBMC and antibody reagent when utilizing cell-based reference materials for quantification purposes and perform bridging studies to ensure harmonization between the results before switching to a new lot.

Stromal DLK1 promotes proliferation and inhibits differentiation of the intestinal epithelium during development.

The stem/progenitor cells of the developing intestine are biologically distinct from their adult counterparts. Here, we examine the microenvironmental cues that regulate the embryonic stem/progenitor population, focusing on the role of Notch pathway factor delta-like protein-1 (DLK1). mRNA-seq analyses of intestinal mesenchymal cells (IMCs) collected from embryonic day 14.5 (E14.5) or adult IMCs and a novel coculture system with E14.5 intestinal epithelial organoids were used. Following addition of recombinant DLK1 (rDLK) or Dlk1 siRNA (siDlk1), epithelial characteristics were compared using imaging, replating efficiency assays, qPCR, and immunocytochemistry. The intestinal phenotypes of littermate Dlk1+/+ and Dlk1-/- mice were compared using immunohistochemistry. Using transcriptomic analyses, we identified morphogens derived from the embryonic mesenchyme that potentially regulate the developing epithelial cells, to focus on Notch family candidate DLK1. Immunohistochemistry indicated that DLK1 was expressed exclusively in the intestinal stroma at E14.5 at the top of emerging villi, decreased after birth, and shifted to the intestinal epithelium in adulthood. In coculture experiments, addition of rDLK1 to adult IMCs inhibited organoid differentiation, whereas Dlk1 knockdown in embryonic IMCs increased epithelial differentiation to secretory lineage cells. Dlk1-/- mice had restricted Ki67+ cells in the villi base and increased secretory lineage cells compared with Dlk1+/+ embryos. Mesenchyme-derived DLK1 plays an important role in the promotion of epithelial stem/precursor expansion and prevention of differentiation to secretory lineages in the developing intestine.NEW & NOTEWORTHY Using a novel coculture system, transcriptomics, and transgenic mice, we investigated differential molecular signaling between the intestinal epithelium and mesenchyme during development and in the adult. We show that the Notch pathway factor delta-like protein-1 (DLK1) is stromally produced during development and uncover a new role for DLK1 in the regulation of intestinal epithelial stem/precursor expansion and differentiation to secretory lineages.

Morphological profiling using machine learning reveals emergent subpopulations of interferon-γ-stimulated mesenchymal stromal cells that predict immunosuppression.

BACKGROUND: Although a preponderance of pre-clinical data demonstrates the immunosuppressive potential of mesenchymal stromal cells (MSCs), significant heterogeneity and lack of critical quality attributes (CQAs) based on immunosuppressive capacity likely have contributed to inconsistent clinical outcomes. This heterogeneity exists not only between MSC lots derived from different donors, tissues and manufacturing conditions, but also within a given MSC lot in the form of functional subpopulations. We therefore explored the potential of functionally relevant morphological profiling (FRMP) to identify morphological subpopulations predictive of the immunosuppressive capacity of MSCs derived from multiple donors, manufacturers and passages. METHODS: We profiled the single-cell morphological response of MSCs from different donors and passages to the functionally relevant inflammatory cytokine interferon (IFN)-γ. We used the machine learning approach visual stochastic neighbor embedding (viSNE) to identify distinct morphological subpopulations that could predict suppression of activated CD4+ and CD8+ T cells in a multiplexed quantitative assay. RESULTS: Multiple IFN-γ-stimulated subpopulations significantly correlated with the ability of MSCs to inhibit CD4+ and CD8+ T-cell activation and served as effective CQAs to predict the immunosuppressive capacity of additional manufactured MSC lots. We further characterized the emergence of morphological heterogeneity following IFN-γ stimulation, which provides a strategy for identifying functional subpopulations for future single-cell characterization and enrichment techniques. DISCUSSION: This work provides a generalizable analytical platform for assessing functional heterogeneity based on single-cell morphological responses that could be used to identify novel CQAs and inform cell manufacturing decisions.

Functional Profiling of Chondrogenically Induced Multipotent Stromal Cell Aggregates Reveals Transcriptomic and Emergent Morphological Phenotypes Predictive of Differentiation Capacity.

Multipotent stromal cells (MSCs) are an attractive cell source for bone and cartilage tissue repair strategies. However, the functional heterogeneity of MSCs derived from different donors and manufacturing conditions has limited clinical translation, emphasizing the need for improved methods to assess MSC chondrogenic capacity. We used functionally relevant morphological profiling to dynamically monitor emergent morphological phenotypes of chondrogenically induced MSC aggregates to identify morphological features indicative of MSC chondrogenesis. Toward this goal, we characterized the morphology of chondrogenically stimulated MSC aggregates from eight different human cell-lines at multiple passages and demonstrated that MSC aggregates exhibited unique morphological dynamics that were both cell line- and passage-dependent. This variation in 3D morphology was shown to be informative of long-term MSC chondrogenesis based on multiple quantitative functional assays. We found that the specific morphological features of spheroid area, radius, minimum feret diameter, and minor axis length to be strongly correlated with MSC chondrogenic synthetic activity but not gene expression as early as day 4 in 3D culture. Our high-throughput, nondestructive approach could potentially serve as a tool to identify MSC lines with desired chondrogenic capacity toward improving manufacturing strategies for MSC-based cellular products for cartilage tissue repair. Stem Cells Translational Medicine 2018;1-12.

FDA and NIST collaboration on standards development activities supporting innovation and translation of regenerative medicine products.

The development of standards for the field of regenerative medicine has been noted as a high priority by several road-mapping activities. Additionally, the U.S. Congress recognizes the importance of standards in the 21st Century Cure Act. Standards will help to accelerate and streamline cell and gene therapy product development, ensure the quality and consistency of processes and products, and facilitate their regulatory approval. Although there is general agreement for the need of additional standards for regenerative medicine products, a shared understanding of standards is required for real progress toward the development of standards to advance regenerative medicine. Here, we describe the roles of standards in regenerative medicine as well as the process for standards development and the interactions of different entities in the standards development process. Highlighted are recent coordinated efforts between the U.S. Food and Drug Administration and the National Institute of Standards and Technology to facilitate standards development and foster science that underpins standards development.

Functionally-Relevant Morphological Profiling: A Tool to Assess Cellular Heterogeneity.

Heterogeneity in cell function has presented a significant hurdle to the successful clinical translation of many cellular therapies. Current techniques for assessing cell quality and the effects of microenvironmental cues and manufacturing processes on cell behavior often inadequately address heterogeneity due to issues such as population versus single-cell measurements and the therapeutic relevance and throughput/robustness of the assay. Due to the well-established relationship between morphology and cellular function, morphological profiling has become increasingly utilized to better understand functional heterogeneity and its impact on therapeutic development. In this review, we introduce an emerging field we term functionally-relevant morphological profiling with great potential to improve our understanding of cellular heterogeneity through discovering novel quality attributes, optimizing manufacturing, and screening drugs/biomaterials.

Adaptation of a Simple Microfluidic Platform for High-Dimensional Quantitative Morphological Analysis of Human Mesenchymal Stromal Cells on Polystyrene-Based Substrates.

Multipotent stromal cells (MSCs, often called mesenchymal stem cells) have garnered significant attention within the field of regenerative medicine because of their purported ability to differentiate down musculoskeletal lineages. Given the inherent heterogeneity of MSC populations, recent studies have suggested that cell morphology may be indicative of MSC differentiation potential. Toward improving current methods and developing simple yet effective approaches for the morphological evaluation of MSCs, we combined passive pumping microfluidic technology with high-dimensional morphological characterization to produce robust tools for standardized high-throughput analysis. Using ultraviolet (UV) light as a modality for reproducible polystyrene substrate modification, we show that MSCs seeded on microfluidic straight channel devices incorporating UV-exposed substrates exhibited morphological changes that responded accordingly to the degree of substrate modification. Substrate modification also effected greater morphological changes in MSCs seeded at a lower rather than higher density within microfluidic channels. Despite largely comparable trends in morphology, MSCs seeded in microscale as opposed to traditional macroscale platforms displayed much higher sensitivity to changes in substrate properties. In summary, we adapted and qualified microfluidic cell culture platforms comprising simple straight channel arrays as a viable and robust tool for high-throughput quantitative morphological analysis to study cell-material interactions.

Morphological features of IFN-γ-stimulated mesenchymal stromal cells predict overall immunosuppressive capacity.

Human mesenchymal stromal cell (MSC) lines can vary significantly in their functional characteristics, and the effectiveness of MSC-based therapeutics may be realized by finding predictive features associated with MSC function. To identify features associated with immunosuppressive capacity in MSCs, we developed a robust in vitro assay that uses principal-component analysis to integrate multidimensional flow cytometry data into a single measurement of MSC-mediated inhibition of T-cell activation. We used this assay to correlate single-cell morphological data with overall immunosuppressive capacity in a cohort of MSC lines derived from different donors and manufacturing conditions. MSC morphology after IFN-γ stimulation significantly correlated with immunosuppressive capacity and accurately predicted the immunosuppressive capacity of MSC lines in a validation cohort. IFN-γ enhanced the immunosuppressive capacity of all MSC lines, and morphology predicted the magnitude of IFN-γ-enhanced immunosuppressive activity. Together, these data identify MSC morphology as a predictive feature of MSC immunosuppressive function.

Fetus-derived DLK1 is required for maternal metabolic adaptations to pregnancy and is associated with fetal growth restriction.

Pregnancy is a state of high metabolic demand. Fasting diverts metabolism to fatty acid oxidation, and the fasted response occurs much more rapidly in pregnant women than in non-pregnant women. The product of the imprinted DLK1 gene (delta-like homolog 1) is an endocrine signaling molecule that reaches a high concentration in the maternal circulation during late pregnancy. By using mouse models with deleted Dlk1, we show that the fetus is the source of maternal circulating DLK1. In the absence of fetally derived DLK1, the maternal fasting response is impaired. Furthermore, we found that maternal circulating DLK1 levels predict embryonic mass in mice and can differentiate healthy small-for-gestational-age (SGA) infants from pathologically small infants in a human cohort. Therefore, measurement of DLK1 concentration in maternal blood may be a valuable method for diagnosing human disorders associated with impaired DLK1 expression and to predict poor intrauterine growth and complications of pregnancy.

Role of mir-15a/16-1 in early B cell development in a mouse model of chronic lymphocytic leukemia.

In both human chronic lymphocytic leukemia (CLL) and the New Zealand Black (NZB) murine model of CLL, decreased levels of microRNAs miR-15a/16 play an important role in the disease. Here we investigate the effects of this microRNA on early steps of B cell development and the capacity of miR-15a-deficient hematopoietic stem cells (HSC) and B1 progenitor cells (B1P) to reproduce CLL-like phenotype both in vitro and in vivo. Our results demonstrate that both miR-15a deficient HSC and B1P cells are capable of repopulating irradiated recipients and produce higher numbers of B1 cells than sources with normal miR-15a/16 levels. Furthermore, induced pluripotent stem (iPS) cells derived for the first time from NZB mice, provided insights into the B cell differentiation roadblock inherent in this strain. In addition, exogenously delivered miR-15a into the NZB derived B cell line provided valuable clues into novel targets such as Mmp10 and Mt2. Our data supports the hypothesis that miR-15a/16 deficient stem cells and B1Ps experience a maturation blockage, which contributes to B1 cells bias in development. This work will help understand the role of miR-15a in early events of CLL and points to B1P cells as potential cells of origin for this incurable disease.

Identification of Predictive Gene Markers for Multipotent Stromal Cell Proliferation.

Multipotent stromal cells (MSCs) are known for their distinctive ability to differentiate into different cell lineages, such as adipocytes, chondrocytes, and osteocytes. They can be isolated from numerous tissue sources, including bone marrow, adipose tissue, skeletal muscle, and others. Because of their differentiation potential and secretion of growth factors, MSCs are believed to have an inherent quality of regeneration and immune suppression. Cellular expansion is necessary to obtain sufficient numbers for use; however, MSCs exhibit a reduced capacity for proliferation and differentiation after several rounds of passaging. In this study, gene markers of MSC proliferation were identified and evaluated for their ability to predict proliferative quality. Microarray data of human bone marrow-derived MSCs were correlated with two proliferation assays. A collection of 24 genes were observed to significantly correlate with both proliferation assays (|r| >0.70) for eight MSC lines at multiple passages. These 24 identified genes were then confirmed using an additional set of MSCs from eight new donors using reverse transcription quantitative polymerase chain reaction (RT-qPCR). The proliferative potential of the second set of MSCs was measured for each donor/passage for confluency fraction, fraction of EdU+ cells, and population doubling time. The second set of MSCs exhibited a greater proliferative potential at passage 4 in comparison to passage 8, which was distinguishable by 15 genes; however, only seven of the genes (BIRC5, CCNA2, CDC20, CDK1, PBK, PLK1, and SPC25) demonstrated significant correlation with MSC proliferation regardless of passage. Our analyses revealed that correlation between gene expression and proliferation was consistently reduced with the inclusion of non-MSC cell lines; therefore, this set of seven genes may be more strongly associated with MSC proliferative quality. Our results pave the way to determine the quality of an MSC population for a particular cellular therapy in lieu of an extended in vitro or in vivo assay.