Lab Research Projects

                                      

Mesenchymal Stromal Cells (MSCs)
1) Enhanced MSCs
We are exploring transient culture priming strategies to augment the therapeutic potency of MSCs for osteoarthritis. Specifically, we have engineered a proprietary 3D culture method and are comparing the 3D MSCs to MSCs cultured in 2D under hypoxic or normoxic conditions. We are applying advanced statistical tools to rank the MSC enhancement methods in terms of their immunomodulatory, angiogenic, and anti-fibrotic properties. Our analysis will enable the selection of a disease-specific approach to osteoarthritis treatment based on analysis of enhanced MSC mechanism of action.
2) Using miRs to identify potent vs. less potent MSCs
MicroRNAs (miRs) are non-coding RNAs that play important roles in regulating gene expression. MSCs extracellular vesicles contain a variety of cargo including miRs that could be transferred to target cells thereby inducing genetic and epigenetic changes. We are identifying novel miRs defining MSCs potency via linking miR profile of MSCs from our OA clinical trail participants to patients reported outcome post MSCs administration. The role of novel miRs in anti-inflammatory/anti-fibrotic action of MSCs will be validated through MSCs co-culture with macrophages/ synovial fibroblasts.
3) Understanding role of IL6 in MSC potency
IL6 is known as a cytokine with dual role. Binding IL6 to membrane bound IL6 receptor will initiate the IL6 classical pathway with anti-inflammatory action, whereas Il6 - soluble IL6 receptor (sIL6R) complex promotes the pro-inflammatory trans-signalling pathway. We identified IL6 as one of top MSC secreted factors which appeared to be positively linked to MSC potency in OA. Controversially, OA microenvironment contains high level of sIL6R potentially enhancing the pro-inflammatory action of IL6. We are investigating the role of IL6 in MSCs potency in OA focusing on distinguishing the role of the two opposing signaling pathways.
4) Using iPSCs to generate MSC
We are optimizing and evaluating iPSC-derived MSCs that are enhanced by proprietary methods
using iPSCs to generate MSCs. 
5) Using mesenchymal stromal cells on mineralized collagen mimics the osteogenic differentiation
The extracellular matrix of bone comprises primarily mineralized collagen fibrils, which have a uniquely intricate ultrastructural arrangement of apatite mineral and collagen. Researchers have developed in vitro approaches to recreating mineralized collagen ultrastructure in bone. However, our understanding of how MSCs, which are used to study osteogenic differentiation in vitro, respond to ultrastructurally biomimetic mineralized collagen fibrils is an area of emerging interest.
6) Using MSC-based bone tissue engineering for mandibular reconstruction using bioengineered composite scaffolds
This is a project in collaboration with University of Brescia, Italy, for developing the entire workflow of primary reconstruction of mandibular defects with bioengineered composite scaffolds in an animal model. This multi-institutional study focuses on the evaluation of bone formation, osteointegration, periosteal formation, vascularization, and mucosal lining in novel bioscaffolds with MSCs. The in vitro characterization of these bioscaffolds are done in Viswanathan lab, where the scaffolds are evaluated for viability, proliferation and osteogenic differentiation of MSCs. The in vivo evaluation of new bone, periosteal, and mucosal formation from these scaffolds seeded with MSCs in rabbit models are done in Dr. Jonathan Irish’s lab at UHN. Once this model to simulate complex head and neck defects bioengineering reconstruction will be developed and optimized, a potential translation towards the surgical application is possible.

Monocytes/Macrophages
1) Adoptive Transfer of monocyte/macrophages 
a. This project involves development of rapid polarization protocols using cytokines or small molecules to generate pro-resolving monocytes/macrophage phenotypes.
b. Adaptation of polarization protocols into a closed-system platform technology to facilitate future clinical translation.
c. Proof-of-concept investigation of effect of pro-resolving versus pro-inflammatory monocytes/macrophages in in vitro and in vivo models of osteoarthritis.
2) Using small molecules to polarize monocytes/macrophages
Small molecules have a range of 50-1500 kD and can enter the cells easily or by cell surface transporters. These molecules are important in metabolic changes in cells by effect other molecules such as proteins (enzymes, transcription factors, etc.). They can change the fate and property of cells truly. These molecules have been used to polarize monocytes to macrophages. Small molecule therapy can be used for M2 like macrophage polarization as a treatment in pro-inflammatory diseases such as osteoarthritis.

Note: Monocytes are a subset of peripheral mononuclear cells in circulating blood which infiltrate the target tissues in response to chemokines. Monocytes are able to differentiate to a different type of macrophages in response to microenvironmental signals during polarization. These polarized macrophages are phenotypically and functionally different cells. For example, pro-inflammatory cytokines such as interferon ɣ or bacterial lipopolysaccharides polarize monocytes to M1 like macrophages with strong pro-inflammatory properties. However, IL10 and TGFβ cytokines polarize monocytes to M2 like macrophages that have anti-inflammatory, wound healing, and tissue repair properties.

Osteoarthritis (OA)
1) Clinical trial results
My lab, working with the Arthritis Program and orthopedic surgeons at UHN conducted Canada’s first trial using MSCs to treat OA patients. The results are published (Chahal et al, 2019, ranked #1 most-read paper of 2019 by SCTM) and show that single injection of bone marrow-derived MSCs resulted in significant improvements to patient-reported outcomes, relative to baseline. Important mechanistic insights showed that MSCs reduced inflammation in the joint and reduced pro-inflammatory monocyte subsets, 3 months after baseline. Importantly, donor heterogeneity was significant and MSCs with stronger anti-inflammatory properties results in more robust clinical outcomes.
2) Joint-in-a-dish model
a. Optimization and characterization of an in-house co-culture explants of late-stage OA cartilage and synovium as a multivariate tool to predict therapeutic outcomes.
b. Testing of cell-based therapies (MSCs, monocytes/macrophages) within the human explant model to complement in vivo animal model outcomes. 
c. Exploring next generation additions to this model including perfusion component, diffusion gradients to generate joint-on-a-chip prototype.
3) OA animal models
Our lab uses the well-established injury-induced osteoarthritis mouse model known as the destabilization of the medial meniscus (DMM) mouse model in order to test cell-based therapies that we are developing in the lab. More specifically, we are using the DMM model to evaluate the efficacy and mechanisms of action of ex vivo polarized monocyte/macrophages as well as MSCs that are enhanced through culture priming strategies.
We also used another model known as collagenase induced osteoarthritis (CIOA) mouse whereby collagenase type VII is injected to intra-articular in mice. After 4 weeks injection of 1 U collagenase into the knee joints twice on alternate days, osteoarthritis is developed by disruption of the ligaments and local instability of the joint.
We are also exploring pain, using monoiodoacetate (MIA) models to destroy cartilage and assess pain using standard paw withdrawal assays. 

MSC bioengineering and bioprocess
Using cellular manufacturing and bioengineering expertise in my lab along with design of experiment principles, we are investigating combinations of manufacturing process parameters and their dual effects on MSC potency and yield with Prof. Julie Audet.
Concurrently, we are optimizing scalability of growing MSCs in 3D configurations using vertical wheel bioreactor systems. Cryopreservation of enhanced 3D-configured MSCs are also being optimized (OIRM grant, Viswanathan PI, 2019-2020). We have a research collaboration with Miltenyi Biotec to explore developing scalable, closed-systems to process monocytes/macrophages for adoptive immunotherapies. Miltenyi is interested in modifying their T cell processing device (CliniMACS Prodigy®) to process monocytes/macrophages as a platform technology. The CliniMACs Prodigy® is currently being used to process differentiated macrophages for a clinical trial in liver cirrhosis.