Recipient Organization
TEXAS A&M UNIVERSITY
750 AGRONOMY RD STE 2701
COLLEGE STATION,TX 77843-0001
Performing Department
Veterinary Large Animal Clinical Sciences
Non Technical Summary
Regenerative medicine, such as autologous stem cells, hasbecome popular to treat a variety of orthopedic conditions. Most commonly, autologous (from the recipient) stem cells are utilized because use of allogeneic (from a donor) stem cells have been shown to cause inflammation and injection site reactions. This is despite the common thought that stem cells are immune privileged, meaning that the immune system will not recognize them. The purpose of this study is to evaluate differences between donors and recipients of stem cells in an attempt to narrow down the cause of the apparent immune response. This will be done by matching horses based on genetic markers and comparing the response after stem cell injection to horses that are genetically mis-matched. This matching and mis-matching will be based on MHC (major histocompatibility complex) compatibility, which is the major determinate of graft rejection in organ transplantation. Shedding light on this issue could have the potential to translate to human medicine and eventually lead to standardization of allogeneic stem cell usage.
Animal Health Component
50%
Research Effort Categories
Basic
50%
Applied
50%
Developmental
(N/A)
Goals / Objectives
GOALS AND OBJECTIVES: Our short-term goal is to determine if MHCI incompatibility is responsible for the adverse reaction to allogeneic equine bone marrow derived mesenchymal stem cell (MSC) therapy. We will compare synovial inflammation and immune responses after repeated intra-articular injection of MHC matched, MHC mismatched, and MHC null allogeneic MSCs. Our hypothesis is that joint inflammation will be significantly greater in the MHC mismatched group compared to MHC matched and MHC null groups and this will correspond with innate and adaptive immunity.OBJECTIVE 1: To evaluate recipient compatibility of MHC matched compared to MHC mismatched allogeneic MSCsSpecific Aim 1A: We will evaluate synovial inflammation following a naïve and primed intra-articular injection of MHC matched or mismatched MSCs by synovial cytology and synovial interferon-γ. We hypothesize that synovial inflammation will not be different after naive injection between the groups. After primed injection, we expect a significant increase in inflammation in the MHC mismatched group but not the MHC matched group.Specific Aim 1B: We will assess the immune response of horses in Aim 1A by measuring antibody and complement-dependent cytotoxicity and T-cell proliferation in vitro. We hypothesize there will be enhanced lymphocyte proliferation prior to intra-articular injection in the MHC mismatched group compared to MHC matched due to innate incompatibility, and a significant increase following the primed injection in the mismatched group because of an adaptive immune response. We expect development of donor-specific antibodies after the naïve injection in the MHC mismatched group, with a significant increase after primed injection.OBJECTIVE 2: To produce and evaluate recipient compatibility to MHC null MSCsSpecific Aim 2A: We will use CRISPR/Cas9 to eliminate MHCI expression on MSCs. We hypothesize that we will be able to produce a monoclonal population of MSCs lacking MHCI expression, which will be verified by PCR and flow cytometry.Specific Aim 2B: We will evaluate synovial inflammation following a naive and primed intra-articular injection of MHC null mismatched MSCs by synovial cytology and synovial interferon-γ. We hypothesize there will be no difference in inflammation compared to the control joint and significantly lower inflammation compared to MHC mismatched in Aim 1A.Specific Aim 2C: We will assess the primed immune response of horses in Aim 2B by measuring antibody and complement-dependent cytotoxicity and T-cell proliferation in vitro. We hypothesize there will be enhanced lymphocyte proliferation prior to intra-articular injection due to MHC haplotype incompatibility but it will not change after primed MHC null MSC injection. Additionally, there will be no change in donor-specific antibodies after naïve and primed MSC injection.Our long-term goal is to understand the mechanism of MSC allo-recognition so that allogeneic MSCs can be utilized to improve the welfare of horses with chronic pain and lameness.
Project Methods
We have screened 75 horses owned by Texas A&M University and identified four MHC homozygotes for well-characterized MHC haplotypes; two A5a and two A3b. There were nine and six eligible matched recipients for each donor type, respectively. The remaining screened horses (n=54) were dissimilar to A5a and A3b and are able to serve as mismatched recipients.Objective 1: To avoid xeno-contamination we will use platelet releasate instead of fetal bovine serum to expand MSCs. MSCs cultured in xenogen free media from the bone marrow of a single homozygote donor (n=4) will be injected into the metacarpophalangeal joint of MHC matched (n=3) and mismatched (n=3) recipients (Figure 3) on Day 0 and 29 (Figure 4). The contralateral metacarpophalangeal joint will be injected with MSC media as a control. Synovial total nucleated cell count (TNCC) and interferon-γ will be measured serially from the treated and control joints following naïve and primed injections (Figure 4). Antibody responses indicating immune recognition will be assessed using microcytotoxicity assays. Recipient serum separated from peripheral blood will be collected every other day for sixty days after intra-articular injection. Peripheral blood lymphocytes (PBLs) isolated from the homozygote donor will be tested against serum from the recipient. Serial dilutions of recipient serum will be added to PBLs under oil in Terasaki plates with rabbit complement added after 30 minutes. Wells will then be fixed with formalin and stained with eosin dye. Wells with ≥80% of PBL death is considered a positive response with the highest dilution being the cytotoxic antibody titer. As a measure of compatibility and primed cell mediated response ex vivo, mixed lymphocyte reactions (MLRs) will be performed using donor and recipient peripheral blood lymphocytes (PBLs) in co-culture before and after first injection. Whole blood will be collected from recipient horses on days -1 and 60 and PBLs isolated. Prior to the start of co-culture, recipient PBLs will be stained using a cytoplasmic dye and proliferation will be measured by flow cytometry after 5 days in co-culture.Objective 2: To produce and evaluate recipient compatibility to MHC null MSCs Non-homologous end joining, as part of the CRISPR/Cas9 system, will be used to target β2-microglobulin to prevent presentation of MHCI on the cell surface. The coding region of exon 1 of the β2-mincroglobulin coding region will be targeted, using a commercially available guide RNA (5' TCAGGGAGAGTAGCCCGAGC 3'). PCR will be used to determine success of gene editing, and modified MSCs will be expanded. Lack of MHCI expression will be confirmed using flow cytometry with a fluorescent monoclonal antibody. MHC null MSCs from a single homozygote donor will be injected into the metacarpophalangeal joint of an MHC mismatched (n=6) recipient on Day 0 and 29. The contralateral metacarpophalangeal joint will be injected with MSC media as a negative control. Synovial fluid collection and analysis will be performed at the same time points as in Objective 1. Microcytotoxicity assays and MLRs will be performed as in Objective 1.