Sowmya Viswanathan

Same or Not the Same? Comparison of Adipose Tissue-Derived Versus Bone Marrow-Derived Mesenchymal Stem and Stromal Cells

Mesenchymal stem/stromal cells (MSCs) comprise a heterogeneous population of cells with multilineage differentiation potential, the ability to modulate oxidative stress, and secrete various cytokines and growth factors that can have immunomodulatory, angiogenic, anti-inflammatory and anti-apoptotic effects. Recent data indicate that these paracrine factors may play a key role in MSC-mediated effects in modulating various acute and chronic pathological conditions. MSCs are found in virtually all organs of the body. Bone marrow-derived MSCs (BM-MSCs) were discovered first, and the bone marrow was considered the main source of MSCs for clinical application. Subsequently, MSCs have been isolated from various other sources with the adipose tissue, serving as one of the alternatives to bone marrow. Adipose tissue-derived MSCs (ASCs) can be more easily isolated; this approach is safer, and also, considerably larger amounts of ASCs can be obtained compared with the bone marrow. ASCs and BM-MSCs share many biological characteristics; however, there are some differences in their immunophenotype, differentiation potential, transcriptome, proteome, and immunomodulatory activity. Some of these differences may represent specific features of BM-MSCs and ASCs, while others are suggestive of the inherent heterogeneity of both BM-MSC and ASC populations. Still other differences may simply be related to different isolation and culture protocols. Most importantly, despite the minor differences between these MSC populations, ASCs seem to be as effective as BM-MSCs in clinical application, and, in some cases, may be better suited than BM-MSCs. In this review, we will examine in detail the ontology, biology, preclinical, and clinical application of BM-MSCs versus ASCs.

Ligand/Receptor Signaling Threshold (LIST) Model Accounts for gp130-Mediated Embryonic Stem Cell Self-Renewal Responses to LIF and HIL-6

We previously demonstrated that embryonic stem (ES) cell self‐renewal required sustained signaling by leukemia inhibitory factor (LIF) in a concentration‐dependent manner, allowing us to hypothesize that thresholds in ligand‐receptor signaling modulate stem cell differentiation control. To test this hypothesis, we have experimentally and computationally compared the abilities of two gp130‐signaling cytokines (LIF and Hyper‐interleukin‐6 [HIL‐6]) to sustain ES cell self‐renewal. Quantitative measurements of ES cell phenotypic markers (stage‐specific embryonic antigen‐1 and E‐cadherin), functional assays (alkaline phosphatase activity and embryoid body formation efficiency), and transcription factor (Oct‐4) expression over a range of LIF and HIL‐6 concentrations demonstrated a superior ability of LIF to maintain ES cell pluripotentiality at higher concentrations (≥500 pM). Additionally, we observed distinct qualitative differences in the ES cell self‐renewal dose response profiles between the two cytokines. A computational model permitted calculation of the number of signaling complexes as a function of receptor expression, ligand concentration, and ligand/receptor‐binding properties, generating predictions for the degree of self‐renewal as a function of cytokine concentration by comparison of these calculated complex numbers to experimentally determined threshold cytokine concentrations. Model predictions, consistent with experimental data, indicated that differences in the potencies of these two cytokines were based primarily on differences in receptor‐binding stoichiometries and properties. These results support a ligand/receptor signaling threshold model of ES cell fate modulation through appropriate types and levels of cytokine stimulation. Insights from these results may be more generally applicable to tissue‐specific stem cells and could aid in the development of stem cell‐based technologies.

Towards predictive models of stem cell fate

Quantitative approaches are essential for the advancement of strategies to manipulate stem cells or their derivatives for therapeutic applications. Predictive models of stem cell systems would provide the means to pose and validate non-intuitive hypotheses and could thus serve as an important tool for discerning underlying regulatory mechanisms governing stem cell fate decisions. In this paper we review the development of computational models that attempt to describe mammalian adult and embryonic stem (ES) cell responses. Early stochastic models that relied exclusively on statistical distributions to describe the in vitro or in vivo output of stem cells are being revised to incorporate the contributions of exogenous and endogenous parameters on specific stem cell fate processes. Recent models utilize cell specific data (for example, cell-surface receptor distributions, transcription factor half-lives, cell-cycle status, etc.) to provide mechanistic descriptions that are consistent with biologically observed phenomena. Ultimately, the goal of these computational models is to, a priori, predict stem cell output given an initial set of conditions. Our efforts to develop a predictive model of ES cell fate are discussed. The quantitative studies presented in this review represent an important step in developing bioengineering approaches to characterize and predict stem cell behavior. Ongoing efforts to incorporate genetic and signaling network data into computational models should accelerate our understanding of fundamental principles governing stem cell fate decisions.

Natural killer cell lines preferentially kill clonogenic multiple myeloma cells and decrease myeloma engraftment in a bioluminescent xenograft mouse model

Background Novel therapies capable of targeting drug resistant clonogenic MM cells are required for more effective treatment of multiple myeloma. This study investigates the cytotoxicity of natural killer cell lines against bulk and clonogenic multiple myeloma and evaluates the tumor burden after NK cell therapy in a bioluminescent xenograft mouse model. Design and Methods The cytotoxicity of natural killer cell lines was evaluated against bulk multiple myeloma cell lines using chromium release and flow cytometry cytotoxicity assays. Selected activating receptors on natural killer cells were blocked to determine their role in multiple myeloma recognition. Growth inhibition of clonogenic multiple myeloma cells was assessed in a methylcellulose clonogenic assay in combination with secondary replating to evaluate the self-renewal of residual progenitors after natural killer cell treatment. A bioluminescent mouse model was developed using the human U266 cell line transduced to express green fluorescent protein and luciferase (U266eGFPluc) to monitor disease progression in vivo and assess bone marrow engraftment after intravenous NK-92 cell therapy. Results Three multiple myeloma cell lines were sensitive to NK-92 and KHYG-1 cytotoxicity mediated by NKp30, NKp46, NKG2D and DNAM-1 activating receptors. NK-92 and KHYG-1 demonstrated 2- to 3-fold greater inhibition of clonogenic multiple myeloma growth, compared with killing of the bulk tumor population. In addition, the residual colonies after treatment formed significantly fewer colonies compared to the control in a secondary replating for a cumulative clonogenic inhibition of 89–99% at the 20:1 effector to target ratio. Multiple myeloma tumor burden was reduced by NK-92 in a xenograft mouse model as measured by bioluminescence imaging and reduction in bone marrow engraftment of U266eGFPluc cells by flow cytometry. Conclusions This study demonstrates that NK-92 and KHYG-1 are capable of killing clonogenic and bulk multiple myeloma cells. In addition, multiple myeloma tumor burden in a xenograft mouse model was reduced by intravenous NK-92 cell therapy. Since multiple myeloma colony frequency correlates with survival, our observations have important clinical implications and suggest that clinical studies of NK cell lines to treat MM are warranted.

Current Concepts: The Role of Mesenchymal Stem Cells in the Management of Knee Osteoarthritis

Context: The number of adults with osteoarthritis in the United States is expected to nearly double from 21.4 million in 2005 to 41.1 million by 2030. As a result, medical costs and associated comorbidity will exponentially increase in the coming decades. In the past decade, mesenchymal stem cells (MSCs) have emerged as a novel treatment for degenerative joint disease. Evidence Acquisition: PubMed (from 1990 to 2013) was searched to identify relevant studies. Reference lists of included studies were also reviewed. Study Design: Clinical review. Level of Evidence: Level 3. Results: We identified 9 animal and 7 human studies investigating the use of MSCs in the treatment of osteoarthritis, with varying levels of support for this therapy. Conclusion: While MSCs have shown potential for improving function and decreasing inflammation in animal studies, translation to patients is still in question. There is a great deal of heterogeneity in treatment methods. Standardizing the manufacturing and characterization of MSCs will allow for better comparisons.

Clonal evolution of stem and differentiated cells can be predicted by integrating cell-intrinsic and -extrinsic parameters.

Stem cells and their derivatives represent a renewable source of cells for therapeutic applications. However, the inability to quantitatively integrate and exploit the effects of multiple parameters on the fate of stem cells limits their use in clinical applications. To address this, we developed a computational model that combines probabilistic, individual‐cell and deterministic cell‐population parameters to simultaneously calculate the specific effects of exogenous and endogenous factors on the overall population‐dynamics behaviour. The model tracks the progeny trajectory of individual cells over several generations as a threshold function of ligand–receptor signalling interactions. Simulations in silico were validated against an Oct4‐promoter‐driven green‐fluorescent‐protein‐expressing murine embryonic stem cell line, and used to understand the effects of key parameters on the clonal evolution of stem versus differentiated cells in this system. Our approach demonstrated the ability to distinguish between individual‐cell and population‐averaged parameters with respect to their effects on governing dynamic behaviour. Moreover, we could discriminate between digital versus graded regulation of the Oct4 transcription factor in accounting for experimental observations. Finally, we showed that our approach could be generalized to other stem‐cell systems, in particular the previously characterized intestinal crypt system, in elucidating relative contributions of stem and progenitor cells to population output. On the basis of all these results, we believe that our iterative experimental and computational approach has been found to be useful for the study of various stem‐cell systems.

TLR3 or TLR4 Activation Enhances Mesenchymal Stromal Cell‐Mediated Treg Induction via Notch Signaling

Mesenchymal stromal cells (MSCs) are the subject of numerous clinical trials, largely due to their immunomodulatory and tissue regenerative properties. Toll‐like receptors (TLRs), especially TLR3 and TLR4, are highly expressed on MSCs and their activation can significantly modulate the immunosuppressive and anti‐inflammatory functions of MSCs. While MSCs can recruit and promote the generation of regulatory T cells (Tregs), the effect of TLR activation on MSC‐mediated Treg induction is unknown. In this study, we investigated the effect of ligand‐mediated activation of TLR3 and TLR4 on Treg induction by human MSCs. We found that generation of Tregs in human CD4(+) lymphocyte and MSC cocultures was enhanced by either TLR3 or TLR4 activation of MSCs and that the increase was abolished by TLR3 and TLR4 gene‐silencing. Augmented Treg induction by TLR‐activated MSCs was cell contact‐dependent and associated with increased gene expression of the Notch ligand, Delta‐like 1. Moreover, inhibition of Notch signaling abrogated the augmented Treg levels in the MSC cocultures. Our data show that TLR3 or TLR4 activation of MSCs increases Treg induction via the Notch pathway and suggest new means to enhance the potency of MSCs for treating disorders with an underlying immune dysfunction, including steroid resistant acute graft‐versus‐host disease. Stem Cells 2017;35:265–275

Bringing regenerative medicines to the clinic: the future for regulation and reimbursement.

Significant investments in regenerative medicine necessitate discussion to align evidentiary requirements and decision-making considerations from regulatory, health system payer and developer perspectives. Only with coordinated efforts will the potential of regenerative medicine be realized. We report on discussions from two workshops sponsored by NICE, University of Alberta, Cell Therapy Catapult and Centre for Commercialization of Regenerative Medicine. We discuss methods to support the assessment of value for regenerative medicine products and services and the synergies that exist between market authorization and reimbursement regulations and practices. We discuss the convergence in novel adaptive licensing practices that may promote the development and adoption of novel therapeutics that meet the needs of healthcare payers.

TLR3 or TLR4 Activation Enhances MSC‐Mediated Treg Induction via Notch Signaling

Mesenchymal stromal cells (MSCs) are the subject of numerous clinical trials, largely due to their immunomodulatory and tissue regenerative properties. Toll‐like receptors (TLRs), especially TLR3 and TLR4, are highly expressed on MSCs and their activation can significantly modulate the immunosuppressive and anti‐inflammatory functions of MSCs. While MSCs can recruit and promote the generation of regulatory T cells (Tregs), the effect of TLR activation on MSC‐mediated Treg induction is unknown. In this study, we investigated the effect of ligand‐mediated activation of TLR3 and TLR4 on Treg induction by human MSCs. We found that generation of Tregs in human CD4(+) lymphocyte and MSC cocultures was enhanced by either TLR3 or TLR4 activation of MSCs and that the increase was abolished by TLR3 and TLR4 gene‐silencing. Augmented Treg induction by TLR‐activated MSCs was cell contact‐dependent and associated with increased gene expression of the Notch ligand, Delta‐like 1. Moreover, inhibition of Notch signaling abrogated the augmented Treg levels in the MSC cocultures. Our data show that TLR3 or TLR4 activation of MSCs increases Treg induction via the Notch pathway and suggest new means to enhance the potency of MSCs for treating disorders with an underlying immune dysfunction, including steroid resistant acute graft‐versus‐host disease. Stem Cells 2017;35:265–275

Proceedings: International Regulatory Considerations on Development Pathways for Cell Therapies

Regenerative medicine is a rapidly evolving field that faces novel scientific and regulatory challenges. In September 2013, the International Workshop on Regulatory Pathways for Cell Therapies was convened to discuss the nature of these challenges and potential solutions and to highlight opportunities for potential convergence between different regulatory bodies that might assist the fields development. The workshop discussions generated potentially actionable steps in five main areas that could mitigate cell therapy development pathway risk and accelerate moving promising therapies to patients. These included the need for convergence of regulatory guidelines on donor eligibility and suitability of lines for use in clinical trials and subsequent commercialization for cell therapies to move forward on a global basis; the need to challenge and encourage investigators in the regenerative medicine field to share information and provide examples of comparability studies related to master cell banks; the need for convergence of guidelines across regulatory jurisdictions on requirements for tumorigenicity studies, based on particular cell types and on biodistribution studies; the need to increase transparency in sharing clinical trial information more broadly and disseminating results more rapidly; and the need to establish a forum for sharing the experiences of various approaches being developed to expedite regulatory approvals and access for patients to innovative cell and regenerative therapies in the different regulatory jurisdictions and to assess their potential strengths and weaknesses.