Guiding the osteogenic fate of mouse and human mesenchymal stem cells through feedback system control.

Stem cell-based disease modeling presents unique opportunities for mechanistic elucidation and therapeutic targeting. The stable induction of fate-specific differentiation is an essential prerequisite for stem cell-based strategy. Bone morphogenetic protein 2 (BMP-2) initiates receptor-regulated Smad phosphorylation, leading to the osteogenic differentiation of mesenchymal stromal/stem cells (MSC) in vitro; however, it requires supra-physiological concentrations, presenting a bottleneck problem for large-scale drug screening. Here, we report the use of a double-objective feedback system control (FSC) with a differential evolution (DE) algorithm to identify osteogenic cocktails of extrinsic factors. Cocktails containing significantly reduced doses of BMP-2 in combination with physiologically relevant doses of dexamethasone, ascorbic acid, beta-glycerophosphate, heparin, retinoic acid and vitamin D achieved accelerated in vitro mineralization of mouse and human MSC. These results provide insight into constructive approaches of FSC to determine the applicable functional and physiological environment for MSC in disease modeling, drug screening and tissue engineering

Development of a three-dimensional haematopoietic stem cell-permissive bone marrow niche model using magnetic levitation

Haematopoietic stem cells (HSCs) have a huge clinical relevance as they are regularly used in bone marrow transplants worldwide. This therapy has profound potential to alleviate diseases of the blood and immune system, where others are ineffective. However, HSCs cannot currently be cultured long term ex vivo, as they rapidly differentiate or senesce. Hence, genetically matched donors must often be found for transplant and studies of HSCs require costly animal models. Mimicking the microenvironment of the bone marrow, in which they reside, by incorporating supportive stromal cells including mesenchymal stem cells (MSCs), has the potential to overcome these limitations. This project aimed to create an HSC-permissive MSC spheroid culture system using magnetic nanoparticles and a collagen gel. An existing spheroid system was optimised for HSC-MSC co-culture and then characterised to assess the potential for HSC support. MSC spheroids were more quiescent, and expressed higher levels of HSC-supportive genes such as nestin and CXCL12. Subsequently, additional bone marrow cell types were introduced to the model to mimic vascular and endosteal areas of the bone marrow niche. HSC behaviour within these models was investigated. MSCs, endothelial cells, and osteoblasts exhibited gene expression changes in line with those predicted from examination of the physiology of endosteal and vascular regions of the bone marrow: i.e. higher activity at the vascular niche (model including endothelial cells), and lower activity at the endosteum (model including osteoblasts). However, gene transcription and phenotypic analyses of HSCs following culture within the bone marrow models produced more inconclusive results. Hence, further optimisation of the conditions of the model and repetition of results presented here are required to develop the system so that it truly mimics physiological bone marrow. The development of such a model has many applications, including in drug discovery, modelling disease states, and probing haematopoietic functions

Cells and Materials for Disease Modeling and Regenerative Medicine

Materials science and engineering are strongly developing tools with increasing impact in the biotechnological and biomedical areas. Interestingly, research in molecular and cellular biology is often at the core of the design and development of materials-based approaches, providing biological rationale. Focused on research relying on biology–materials interaction, IJMS launched a Special Issue named “Cells and Materials for Disease Modeling and Regenerative Medicine”. The aim of the Special Issue was to generate a compilation of in vitro and in vivo strategies based on cell–material interactions. This book compiles the papers published in that Special Issue and includes a selection of six original scientific experimental articles and six comprehensive reviews. We are convinced that this collection of articles shows representative examples of the state of the art in the field, unveiling the relevance of materials research in generating new regenerative medicine and disease modeling approaches

Guidelines for the use of flow cytometry and cell sorting in immunological studies (third edition)

The third edition of Flow Cytometry Guidelines provides the key aspects to consider when performing flow cytometry experiments and includes comprehensive sections describing phenotypes and functional assays of all major human and murine immune cell subsets. Notably, the Guidelines contain helpful tables highlighting phenotypes and key differences between human and murine cells. Another useful feature of this edition is the flow cytometry analysis of clinical samples with examples of flow cytometry applications in the context of autoimmune diseases, cancers as well as acute and chronic infectious diseases. Furthermore, there are sections detailing tips, tricks and pitfalls to avoid. All sections are written and peer-reviewed by leading flow cytometry experts and immunologists, making this edition an essential and state-of-the-art handbook for basic and clinical researchers

Comparability & reimbursement for the translation of scalable, automated stem cell cultures

The research in this thesis focuses primarily on two critical challenges that inhibit the late stage translation of cell-based therapies and Regenerative Medicines (RMs). These include product comparability after a change in manufacturing process or site; and the reimbursement of RMs, in particular those which target multiple simultaneous indications, or Multimorbidity . The automation and standardisation of stem cell cultures also represent key themes of this thesis, which may facilitate the development of scalable, reproducible manufacturing processes for cell-based therapies. Furthermore, given the current uncertainty regarding the characterisation and potency of Human Mesenchymal Stromal (or Stem) Cells (hMSCs) that has inhibited the successful clinical translation of hMSC-based products, understanding the characterisation and putative modes of action of these cells was also a priority throughout this research. Also, due to the increasing number of Human Embryonic Stem Cell (hESC) derived therapies progressing towards market, and the industry-wide shift towards Human Induced Pluripotent Stem Cells (hiPSC) as an alternative to hESCs, the measurement of the growth and characterisation of these cells types represents an important method of demonstrating product comparability after alternative manufacturing process steps in the present thesis. Finally, due to the potential of hiPSCs as a source of large numbers of hMSCs, the culture conditions required to direct the differentiation of hiPSCs to hMSCs are explored

Mesenchymal stem cells as trophic mediators of neural differentiation

Intense excitement and optimism surrounds the rapidly-expanding field of stem cell research, owing to their high capacity for self-renewal and intrinsic ability to differentiate into mature cell lineages. Although it may be envisioned that embryonic stem cells will be of significantly greater therapeutic value than their adult stem cell counterparts, the use of embryonic stem cells is fraught with both technical and ethical challenges and, as such, significant impetus has been placed on adult stem cell-based research. In particular, mesenchymal stem cells (MSCs) present as exciting candidates for potential use in cellular therapies and tissue engineering strategies. MSCs are defined at the functional level in terms of their ability to differentiate into mesodermal derivatives such as bone and fat. However, this functional definition is evolving, and there is considerable evidence to suggest that MSCs have a key role within their niche involving the release and/or uptake of soluble factors and cytokines, significantly influencing the behaviour of other cell types within the niche. Both facets of MSC behaviour are valuable from a clinical perspective, and have been examined in the present thesis. The most obvious and realistically-achievable clinical application of MSCs at present is in the treatment of osseous and adipose tissue defects. However, before the use of MSCs in the clinic becomes more commonplace, it is crucial to gain a more comprehensive understanding of the complex molecular and cellular mechanism(s) by which MSCs commit to a given fate and undergo differentiation to produce mature, fully-functional derivatives. Much of our present knowledge is derived from studies performed on the highly unnatural, 2D environment of tissue culture plastic. The present study assessed the behaviour of MSCs cultured on AlvetexTM, a novel, 3D scaffold manufactured by ReInnervate, with particular emphasis on the ability of MSCs to undergo osteogenic and adipogenic differentiation. Results obtained suggest that AlvetexTM may provide a more realistic and physiologically-relevant system in which to study osteogenesis and adipogenesis, in a manner more pertinent to that which occurs in vivo. Furthermore, the ability of MSCs to influence the behaviour of other cell types via the release of trophic factors and cytokines was examined, with particular emphasis on the nervous system. An in vitro conditioned media model was developed in order to investigate the influence(s) of MSC-derived soluble factors/cytokines on neural development and plasticity, using the adult rat hippocampal progenitor cell (AHPC) line as a model system. Results obtained suggest that, under defined conditions, MSCs secreted a complement of soluble factors/cytokines that induce AHPCs to commit to and undergo astrogenesis. This effect was characterised at both the cellular and molecular level. The specific complement of bioactive factors secreted by MSCs has been investigated using a combination of targeted transcriptional profiling and shotgun proteomics, and several putative candidate factors have been identified for further investigation

CHEMICAL SCREEN FOR EPIGENETIC BARRIERS TO SINGLE ALLELE ACTIVATION OF OCT4

Induced pluripotent stem cells (iPSCs) have applications in many research fields. However, current methods to produce iPSCs have been inefficient and largely carcinogenic. The current study uses the chromatin in vivo assay (CiA) mouse platform explore barriers to small molecule facilitated activation of a single allele of Oct4. The CiA:Oct4 allele contains an engineered EGFP reporter that replaces one allele of the Oct4 gene combined with an upstream Gal4 array in the promoter at this engineered locus. A high throughput small molecule screen was performed with and without recruitment of a transcriptional activator, VP16. From this screen, we identified that Azacytidine and multiple different HDAC inhibitors facilitated CiA:Oct4 gene activation. Furthermore, an HDAC inhibitor, Mocetinostat, was found to increase reprogramming efficiency during transcription factor reprogramming approximately 20 fold. Our results identified more recently discovered HDAC inhibitors which could be generally useful for future reprogramming studies. Furthermore, our results highlight the differences between testing small molecules using single allele activation versus whole cell reprogramming.Doctor of Philosoph

3D Culture Strategies for the Dynamic Expansion and Preconditioning of Adipose-derived Stromal Cells on Decellularized Adipose Tissue Bioscaffolds

Adipose tissue engineering holds promise for the development of therapeutic strategies for subcutaneous adipose tissue regeneration to treat defects resulting from congenital birth defects, invasive surgical procedures and traumatic injuries. Decellularized adipose tissue (DAT) scaffolds represent a potential off-the-shelf tissue substitute for volume augmentation. Seeding the DAT with adipose-derived stromal cells (ASCs) has been shown to enhance adipose tissue regeneration in immunocompetent animals in vivo. Although promising, this strategy is limited by low cell attachment on the DAT. As such, this thesis focused on the development of bioreactor strategies to enhance the capacity of human ASCs to stimulate angiogenesis and adipogenesis within the DAT. Culturing human ASCs on the DAT scaffolds within a perfusion bioreactor under hypoxia (2% O2) promoted ASC expansion, and altered cell phenotype, upregulating the expression of hypoxia inducible factor 1-alpha (HIF-1a), inducible nitric oxide synthase (iNOS) and tumour necrosis factor-alpha (TNF-a) in the ASCs in the peripheral regions of the DAT. Further, bioreactor culture modulated the expression of pro-angiogenic and immunomodulatory paracrine factors, suggesting that the capacity of the ASCs to stimulate regeneration may have been altered by shear stress stimulation. In vivo testing in athymic nude mice demonstrated that angiogenesis and adipogenesis within the DAT were markedly enhanced when the ASCs were cultured for 14 days within the perfusion bioreactor under 2% O2 prior to implantation as compared to bioreactor culture under 20% O2, as well as static-cultured, freshly-seeded and unseeded controls. Analysis of host cell infiltration indicated that there was increased CD31+ endothelial cell recruitment and potentially adipogenic progenitor cell recruitment into the DAT implants, as well as a shift towards a more pro-regenerative macrophage response, in the 2% O2 bioreactor group relative to static cultured controls. Building from this work, a novel scalable rocking bioreactor platform was explored as an expansion and preconditioning system for ASCs. Preliminary studies indicated that culturing ASCs on DAT coatings within the rocking bioreactor enhanced the expression of the pro-angiogenic and immunomodulatory markers CD146 and iNOS. Collectively, this work supports that dynamic culture systems can be applied to enhance the pro-regenerative potential of ASC-seeded DAT bioscaffolds

Monitoring the Systemic Immune System to Understand and Improve the Efficacy of Immunotherapy for Metastatic Osteosarcoma

Osteosarcoma (OS) is a complex tumor with no effective targeted therapies due to its genomic heterogeneity and pleomorphism. The immune response it creates, particularly against metastatic lesions, is considerable; however, various suppressive mechanisms induced by the tumor prohibit its effectiveness. The presence of infiltrating lymphocytes suggests that therapeutic disinhibition through checkpoint blockade could increase antitumor immunity, though none have been successful in clinical trials. The complexities of the immune response to OS tumors have yet to be unraveled; however, there is evidence to suggest that cell-mediated immunity (CMI, specifically T cells, Natural Killer [NK] cells, and myeloid-lineage cells [MLCs]) plays an important role. New technologies have made it possilble to assess large numbers of cell antigens simultaneously, producing detailed information on the immune status of an individual. A snapshot of the immunological status of an organ or tumor can be conveyed by means of an immunophenotype (IPT), which uses cytometry, microarray, or gene set enrichment analysis to determine the relative percent populations and activation states of immune cells. The majority of data collected on the OS IPT are from primary tumors. Unlike metastatic tumros, primary tumors are easily removed by surgeons and do not account for aossicated high mortality rates. As such, we have investigated the systemic immune response to disease progression with and without immunotherapy. Using a highly metastatic luciferase(+) K7M2 (luc-K7M2) orthotopic OS model in an immunocompetent host, it was determined that the introduction of luciferase into tumor cells had little to no effect on the overall antitumor immune response and clinical disease progression. A systems-wide comparison of tumor-bearing and sham (surgery only) mice showed that there are significant changes in the IPTs of tumor-bearing mice at various time points in disease progression, and that these changes are consistent across different tissues. As disease progresses, T and NK cells are gradually depleted, and macrophages (Mφs) adopt intermediate phenotypes through increased expression of both M1 and M2 polarization markers. Moreover, NK cell depletion in blood is specific to disease progression. T cells, under constant exposure to OS tumor antigens, become overstimulated and upregulate exhaustion markers like programmed cell death protein 1 (PD-1) and T cell immunoglobulin and mucin domain-containing protein 3 (TIM-3). Further, a notable systemic immune biomarker was uncovered using a co-expression ratio of immunosuppressive programmed death ligand 1 (PD-L1) to immunostimulatory major histocompatibility complex class II (MHC-II) on monocytic-myeloid-derived suppressor cells (M-MDSCs), a specific marker of OS disease progression in blood. Monotherapy with monoclocal antibody anti(α)-PD-L1 reversed the malignancy-induced immunological disturbances but did coindice with increased survival. Conversely, treatment of OS tumor-bearing mice with poly(lactic-co-glycolic) acid (PLGA)-encapsulated interleukin(IL)-12 decreased metastatic rate and increased cure rate in a immunophenotype-dependent manner that coincided with treatment-induced NK cell proliferation. These results demonstrate that systemic immunophenotyping can be used to non-invasively monitor OS patient disease progression and response to immunotherapeutics, potentially offering clinicians the opportunity to modify treatment regimens in real-time