Progress 06/15/18 to 06/14/23
Outputs
Target Audience:This project serves the interests of diverse audiences invested in equine wound management and antimicrobial stewardship. We delivered science-based knowledge to these audiences by publishing a review article on the use of Biologics and Stem Cells for Wound Healing in the Horse. This was upon invitation and was published in the Veterinary Clinics of North America: Equine Practice; Use of Biologic and Regenerative Therapies in Equine Practice. We also presented these data at several conferences; most notably, the PD Dr. Van de Walle was invited as a keynote speaker at the 2023 International Conference of the Veterinary Wound Healing Association in Milan, which is held in conjunction with the European Wound Management Association meeting. Moreover, we continued to deliver our findings via outreach to a broader audience, including horse owners, veterinarians, and people from the equine industry, where and when applicable. Also, women who have been working on this project are the PD, Dr. Gerlinde Van de Walle, her Research Support Specialist, Rebecca Harman, her lab technician Kelly Oxford, her post-doc Aarthi Rajesh, and her undergraduate student Esther Ju. This research provides a platform for them to gain skills and status in animal agriculture and outreach. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Post-doctoral Associate Dr. Aarthi Rajesh: Postdoctoral training under Dr. Van de Walle Dr. Rajesh has obtained her PhD on wound healing and since joining the lab in the spring of 2022, has been doing all in vitro experimental work with the equine immune cells and worked on the in vivo murine model as well. Dr. Rajesh was trained in various techniques, including flow cytometry, confocal microscopy, and image analysis. Working on the project, greatly strengthens her competences in optimizing protocols as well as her independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allows her to develop new analytical skills to interpret data. The work broadens her skills related to immunology and molecular bacteriology. How have the results been disseminated to communities of interest?The results from our work have been presented at various meetings and conferences with audiences from various scientific field of stem cell research, immunology research, microbiology research and veterinary research, as well as clinical veterinarians. Further, we published a review article on the use of Biologics and Stem Cells for Wound Healing in the Horse. This was upon invitation and was published in the Veterinary Clinics of North America: Equine Practice; Use of Biologic and Regenerative Therapies in Equine Practice. In addition, the results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?This is the final reporting period. No additional studies are planned. Data analysis and manuscript preparations are in final stages.
Impacts
What was accomplished under these goals?
Defining the antimicrobial effects of factors secreted by equine mesenchymal stromal cells (MSC) will help to understand the underlying antibacterial defense mechanisms of MSC. This could lead to alternative therapies of wound infections, reducing the medical use of antibiotics and hence, the hazard of increasing antibiotic resistance. Our group showed that conditioned medium (CM) of MSC inhibits a variety of wound pathogenic bacteria, importantly also biofilms of these bacteria that often cause therapy resistant infections. We found that the MSC CM affects both bacteria that are susceptible to antibiotics and those that are antibiotic-resistant. MSC CM further helped to increase antibiotics efficacy against bacteria in biofilms. In this fourth year, we focused on evaluating the indirect effects of the MSC CM on equine innate immune cells important during wound infections and we also initiated a pilot in vivo study of an equine wound model. These results will help to understand how the MSC secreted factors impact the infected wound milieu. Accomplishments for this final year include: Aim 1: All data to complete aim 1 were collected and analyzed in year 1 and these outcomes were reported in the first-year progress report, as well as in a paper published in a peer-reviewed journal. Accomplishment of aim 1 led to a change in knowledge. We showed for the first time that the antimicrobial properties of MSC not only consist of AMP activity, but that MSC CM destroy proteins in the extracellular matrix due to cysteine protease activity. Treatments with MSC CM inhibits a variety of biofilm-forming wound pathogens, importantly also the antibiotic-resistant strain MRSA. Bacteria in MRSA biofilms are more effectively killed by antibiotics if biofilms are pre-treated with MSC CM, indicating that the MSC CM is a valuable approach in cutaneous wound management. By showing that the MSC CM reduces live bacteria of S. aureus and MRSA biofilms in a physiological relevant ex vivo wound model, we confirmed a high clinical relevance of the MSC CM as a future therapeutic approach. Aim 2: The goal of this aim was to evaluate the effect of the MSC CM on resident skin cells and non-resident immune cells. The part that focused on the resident skin cells has been completed and has been published in a peer-reviewed journal. The part that focused on the non-resident immune cells was initiated in year 2, where we isolated equine neutrophils from equine blood and found that MSC CM stimulated neutrophil migration, a process that we found was mediated by the protein CXCL6. These findings have been reported in a paper published in a peer-reviewed journal. To complete this objective, we investigated the effects of MSC secreted factors on additional neutrophil functions. To this end, we conducted a series of neutrophil functional assays, including neutrophil chemotaxis, phagocytosis, and production of reactive oxygen species (ROS), in the presence and absence of CM from MSC that have been isolated from 3 different tissue sources (i.e., bone marrow, fat, and peripheral blood). Our findings are that equine MSC secreted factors (i) stimulate neutrophil chemotaxis (with bone marrow-derived MSC having the greatest effects), (ii) affects neutrophil maturation by increasing the percentage of banded neutrophils, reported to have a potential higher antimicrobial efficacy, at least in humans (with bone marrow-derived MSC having the greatest effects), (iii) dampen neutrophil phagocytosis (with all 3 MSC sources having an equal effect), and (iv) do not impact reactive oxygen species (ROS) production. We further expanded our in vitro studies to include macrophage functional assays as well. Equine monocytes were isolated from peripheral blood and differentiated into M0 macrophages. Macrophage polarization assays, phagocytosis and reactive oxygen species assays were carried out to evaluate the efficacy of the three different sources of MSC CM on macrophage function. These findings revealed that M0 macrophages cultured with PB-MSC CM exhibited a slight skew towards the M2 phenotype, while the treatment with other two sources of MSC CM increased polarization of macrophages to both M1 and M2 phenotypes. The macrophage phagocytic abilities were unaltered with MSC CM treatment. ROS production showed an increased trend, albeit not statistically significant, when treated with MSC CM. These data are currently put together in a manuscript in preparation that we intend to submit to Journal of Immunology. Working on aim 2 led to a change in knowledge. For the first time, we showed that the MSC secrete factors that stimulate keratinocytes to increase their antimicrobial immune response, and we also showed that MSC secreted factors promote migration of equine neutrophils, an important first line defense mechanism to combat wound infections. In addition, MSC secreted factors contribute to both M1 and M2 macrophage polarization, which is essential for both anti-bacterial and pro-healing components during wound healing. We further showed that MSC CM alters additional neutrophil and macrophage functions in vitro, suggesting that the equine MSC secretome might have the potential to restore impaired immune cell functions in infected wounds. Aim 3: In addition to the collection of preliminary data that have been reported in earlier annual reports, we have initiated a pilot study of an experimental skin wound infection model in horses to evaluate the efficacy of equine MSC-derived secreted factors in vivo. To this end, two horses were enrolled in the study, with each horse receiving 12 full thickness skin wounds on the torso, 6 per side. The wounds on one side of each horse were inoculated with S. aureus bacteria. On each side, following treatments were administered topically, in duplicate; (i) positive control (mupirocin), (ii) negative control (base culture medium) and (iii) MSC CM. The procedures of creating and inoculating the wounds went smoothly, and the administration of treatments was successful. The read outs for the study were (i) counts of bacteria collected from wounds on days 1, 2, 3, 4, 7, 14, 21 and 28 post wounding, (ii) wound scores based on photographs taken on days 1, 2, 3, 4, 5, 6, 7, 9, 11, 14, 16, 18, 21, 23, 25, 28 and 30 post wounding, (iii) biopsies taken from wound margins on days 7 and 28 post wounding that will be submitted for histology and/or additional immunohistochemistry staining, and (iv) days to wound closure. The bacterial data from the first couple of days indicate that the MSC CM reduced bacterial wound loads to levels comparable to the positive antibiotic control, when compared to the negative control DMEM treated wounds. The remaining data analysis is ongoing, and once finalized, will be put together in a manuscript in preparation that we intend to submit to either a wound-focused or a stem cell-focused veterinary journal.
Publications
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Rajesh A, Harman RM, Van de Walle GR. Impact of equine mesenchymal stromal cell secretome on innate immune response during wound healing. Annual Meeting of CRWAD, Chicago.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Van de Walle GR. The potential of the stem cell secretome in wound healing. International Conference of the Veterinary Wound Healing Association, Italy.
- Type:
Journal Articles
Status:
Published
Year Published:
2023
Citation:
Harman RM, Rajesh A, Van de Walle GR. Use of Biologics and Stem Cells for Wound Healing in the Horse. Veterinary Clinics of North America: Equine Practice; Use of Biologic and Regenerative Therapies in Equine Practice. Epub ahead of print.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2023
Citation:
Rajesh A, Oxford K, Harman RM, Van de Walle GR. Evaluating the efficacy of equine mesenchymal stromal cell secretome on innate immune responses in vitro and in vivo. North American Veterinary Regenerative Medicine Association, Hawaii.
- Type:
Conference Papers and Presentations
Status:
Submitted
Year Published:
2023
Citation:
Rajesh A, Oxford K, Harman RM, Van de Walle GR. Evaluating the efficacy of equine mesenchymal stromal cell secretome on innate immune responses in vitro and in vivo. Upstate New York Immunology Conference, NY.
|
Progress 06/15/21 to 06/14/22
Outputs
Target Audience:This project serves the interests of diverse audiences invested in equine wound management and antimicrobial stewardship. We delivered science-based knowledge to these audiences by publishing one research paper in a peer-reviewed journal, which was selected by the Editor-of-Chief for a press release. We also presented these data at several conferences; most notably, the PD Dr. Van de Walle was invited as a keynote speaker at the virtual 2021 TERMIS meeting. Moreover, we have been delivering our findings via outreach to a broader audience, including horse owners, veterinarians and people from the equine industry, where and when applicable. Also, women who have been working on this project are the PD, Dr. Gerlinde Van de Walle, her Research Support Specialist, Rebecca Harman, and her post-doc Aarthi Rajesh. The ongoing in vivo preliminary experiment of a horse wound healing model involved the board-certified equine surgeon Dr. Michelle Delco, her graduate student Megan Fahey, and 3 female veterinary students helping with horse care. This research provides a platform for them to gain skills and status in animal agriculture and outreach. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?* Post-doctoral Associate Dr. Aarthi Rajesh: Postdoctoral training under Dr. Van de Walle Dr. Rajesh has obtained her PhD on wound healing and since joining the lab in the spring of 2022, has been doing all in vitro experimental work with the equine immune cells. Dr. Rajesh was trained in various techniques, including flow cytometry, confocal microscopy, and image analysis. Working on the project, greatly strengthens her competences in optimizing protocols as well as her independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allows her to develop new analytical skills to interpret data. The work broadens her skills related to immunology and molecular bacteriology. * Graduate Student Megan Fahey: Training under Dr. Delco Megan Fahey is a combined DVM-PhD student at Cornell university, working in the lab of her mentor Dr. Delco. Megan has been involved in the in vivo pilot study of the experimental skin wound infection model in horses and she will take an important lead on analyzing the collected data. This will help Megan to develop important lab skills including bacteriology, histology and immunohistochemistry. Further, she gained first-hand experiences in developing, establishing, and executing experimental in vivo protocols. How have the results been disseminated to communities of interest?The results from our work have been presented at various virtual meetings and conferences with audiences from various scientific field of stem cell research, immunology research, microbiology research and veterinary research, as well as clinical veterinarians. Further, we published a research article in the peer-reviewed journal Stem Cell Translational Medicine. This article was selected by the Editor-of-Chief for a press release. In addition, the results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?We will continue with our experiments to complete aim 2. Specifically, we will evaluate macrophage polarization, phagocytosis, and ROS production. The most effective MSC source will then be used to investigate the effects of the equine MSC secretome on immune cells in acute and infected wounds in vivo. Masson's trichrome staining will be used to determine the impact of MSC CM on different aspects of healing, including re-epithelialization, wound contraction, and granulation tissue formation. Immunofluorescence staining for different immune cell markers will be carried out to investigate the changes in immune cell populations at the different phases of healing in the presence and absence of MSC CM treatment. For aim 3, we are currently analyzing the samples collected during our pilot study, which will help us guide planning our next in vivo experiment. Based on what we have learned thus far from this preliminary study, we plan to implement slight variations to the protocol.
Impacts
What was accomplished under these goals?
Defining the antimicrobial effects of factors secreted by equine mesenchymal stromal cells (MSC) will help to understand the underlying antibacterial defense mechanisms of MSC. This could lead to alternative therapies of wound infections, reducing the medical use of antibiotics and hence, the hazard of increasing antibiotic resistance. Our group showed that conditioned medium (CM) of MSC inhibits a variety of wound pathogenic bacteria, importantly also biofilms of these bacteria that often cause therapy resistant infections. We found that the MSC CM affects both bacteria that are susceptible to antibiotics and those that are antibiotic-resistant. MSC CM further helped to increase antibiotics efficacy against bacteria in biofilms. In this fourth year, we focused on evaluating the indirect effects of the MSC CM on equine innate immune cells important during wound infections and we also initiated a pilot in vivo study of an equine wound model. These results will help to understand how the MSC secreted factors impact the infected wound milieu. Accomplishments for this fourth year include: Aim 1: All data to complete aim 1 were collected and analyzed in year 1 and these outcomes were reported in the first-year progress report, as well as in a paper published in a peer-reviewed journal. Accomplishment of aim 1 led to a change in knowledge. We showed for the first time that the antimicrobial properties of MSC not only consist of AMP activity, but that MSC CM destroy proteins in the extracellular matrix due to cysteine protease activity. Treatments with MSC CM inhibits a variety of biofilm-forming wound pathogens, importantly also the antibiotic-resistant strain MRSA. Bacteria in MRSA biofilms are more effectively killed by antibiotics if biofilms are pre-treated with MSC CM, indicating that the MSC CM is a valuable approach in cutaneous wound management. By showing that the MSC CM reduces live bacteria of S. aureus and MRSA biofilms in a physiological relevant ex vivo wound model, we confirmed a high clinical relevance of the MSC CM as a future therapeutic approach. Aim 2: The goal of this aim was to evaluate the effect of the MSC CM on resident skin cells and non-resident immune cells. The part that focused on the resident skin cells has been completed and has been published in a peer-reviewed journal. The part that focused on the non-resident immune cells was initiated in year 2, where we isolated equine neutrophils from equine blood and found that MSC CM stimulated neutrophil migration, a process that we found was mediated by the protein CXCL6. These findings have been reported in a paper published in a peer-reviewed journal. To complete this objective, we are currently investigating the effects of MSC secreted factors on additional neutrophil functions. Briefly, we are conducting a series of neutrophil functional assays, including neutrophil chemotaxis, phagocytosis, and production of reactive oxygen species (ROS), in the presence and absence of CM from MSC that have been isolated from 3 different tissue sources (i.e., bone marrow, fat, and peripheral blood). Our findings thus far are that equine MSC secreted factors (i) stimulate neutrophil chemotaxis (with bone marrow-derived MSC having the greatest effects), (ii) affects neutrophil maturation by increasing the percentage of banded neutrophils, reported to have a potential higher antimicrobial efficacy, at least in humans (with bone marrow-derived MSC having the greatest effects), (iii) dampen neutrophil phagocytosis (with all 3 MSC sources having an equal effect), and (iv) do not impact reactive oxygen species (ROS) production. Working on aim 2 led to a change in knowledge. For the first time, we showed that the MSC secrete factors that stimulate keratinocytes to increase their antimicrobial immune response, and we also showed that MSC secreted factors promote migration of equine neutrophils, an important first line defense mechanism to combat wound infections. We further showed that MSC CM alters additional neutrophil functions in vitro, suggesting that the equine MSC secretome might have the potential to restore impaired immune cell functions in infected wounds. Aim 3: In addition to the collection of preliminary data that have been reported in earlier annual reports, we have initiated a pilot study of an experimental skin wound infection model in horses to evaluate the efficacy of equine MSC-derived secreted factors in vivo. To this end, two horses were enrolled in the study, with each horse receiving 12 full thickness skin wounds on the torso, 6 per side. The wounds on one side of each horse were inoculated with S. aureus bacteria. On each side, following treatments were administered topically, in duplicate; (i) positive control (mupirocin), (ii) negative control (base culture medium) and (iii) MSC CM. The procedures of creating and inoculating the wounds went smoothly, and the administration of treatments was successful. The read outs for the study were (i) counts of bacteria collected from wounds on days 1, 2, 3, 4, 7, 14, 21 and 28 post wounding, (ii) wound scores based on photographs taken on days 1, 2, 3, 4, 5, 6, 7, 9, 11, 14, 16, 18, 21, 23, 25, 28 and 30 post wounding, (iii) biopsies taken from wound margins on days 7 and 28 post wounding that will be submitted for histology and/or additional immunohistochemistry staining, and (iv) days to wound closure. The bacterial data from the first couple of days indicate that the MSC CM reduced bacterial wound loads to levels comparable to the positive antibiotic control, when compared to the negative control DMEM treated wounds. The remaining data analysis is ongoing.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Marx C, Gardner S, Harman RM, Wagner B., Van de Walle GR. Mesenchymal stromal cell secreted CCL2 promotes antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes. Stem Cell Translational Medicine. 10: 1666-1679.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Harman RM, Patel RS, Park JE, Rosenberg BR, Van de Walle GR. Single cell RNA sequencing of equine mesenchymal stromal cells from donor-matched tissues reveals functional heterogeneity. 102th Annual Meeting of CRWAD, virtual.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Van de Walle GR. The potential of the mesenchymal stromal cell secretome in equine regenerative medicine. Tissue Engineering and Regenerative Medicine International Society (TERMIS), virtual.
|
Progress 06/15/20 to 06/14/21
Outputs
Target Audience:This project serves the interests of diverse audiences invested in equine wound management and antimicrobial stewardship. We delivered science-based knowledge to these audiences by publishing one research paper and one review paper in peer-reviewed journals. The research paper was selected as one of the Cornell Center for Vertebrate Genomics (CVG) Director's Highlights in Spring of 2021. We also presented these data at several conferences and meetings and have been delivering our findings via outreach to a broader audience, including horse owners, veterinarians and people from the equine industry, where and when applicable. Also, women who have been working on this project are the PD, Dr. Gerlinde Van de Walle, her Research Support Specialist, Rebecca Harman, her post-doc Charlotte Marx, and her undergraduate student Sophia Gardner. This research provides a platform for them to gain skills and status in animal agriculture and outreach. We also collaborated with our co-PD, Dr. Bettina Wagner, on the identification of equine chemokines and cytokines in the secretome of equine MSC. Changes/Problems:Due to the Covid pandemic, our lab was fully shut down from March till August 2020, which had an overall serious impact on the research progress of all our projects. This pandemic also resulted in our post-doctoral Associate to return to her home country due to family reasons. Therefore, we requested a 1-year no-cost extension through our university, which was approved. We are also actively interviewing candidates to replace our post-doc, so that we can finalize the experiments proposed in the next reporting period to accomplish our goals. What opportunities for training and professional development has the project provided?* Post-doctoral Associate Dr. Charlotte Marx: Postdoctoral training under Dr. Van de Walle Dr Marx has been doing all in vitro experimental molecular work as well as the preliminary in vivo experiments, including treating the wounds and samplings. Dr Marx was trained in various techniques, including flow cytometry, confocal microscopy and image analysis. Working on the project, greatly strengthens her competences in optimizing protocols as well as her independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allowed to develop new analytical skills to interpret data. The work broadens her skills related to molecular bacteriology and cell biology. Importantly, she was successful in securing a Morris Animal Foundation Fellowship, building on the work she has been doing on this grant. * Undergraduate Student Sophia Gardner: Training under Dr Van de Walle Sophia Gardner is an undergraduate student at Cornell university in the field of bioengineering. The work on the project helped Sophia to develop important lab skills and she learnt various commonly used techniques in cell culture, histology and immunofluorescence. Further, she gained first-hand experiences in developing, establishing, and executing experimental protocols by working on the equine skin explant model. How have the results been disseminated to communities of interest?The results from our work have been presented at various virtual meetings and conferences with audiences from various scientific field of stem cell research, immunology research, microbiology research and veterinary research as well as clinical veterinarians. Further, we published a research article in the peer-reviewed journal Stem Cells Research and Therapy. This article was selected as one of the Cornell Center for Vertebrate Genomics (CVG) Director's Highlights in Spring of 2021. We also published a review article in the peer-reviewed journal Frontiers in Cell and Developmental Biology. In addition, the results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?We will continue with our experiments to complete aim 2. Specifically, we will further determine the effects of equine MSC-derived cytokines on AMP expression in neutrophils and/or neutrophil function. To position ourselves well for executing aim 3, we will first determine the dosage of MSC CM to be used in vivo, and will then initiate the experimental wound infections in horses.
Impacts
What was accomplished under these goals?
Defining the antimicrobial effect of factors secreted by equine mesenchymal stromal cells (MSC) will help to understand the underlying antibacterial defense mechanisms of MSC. This could lead to alternative therapies of wound infections, reducing the medical use of antibiotics and hence, the hazard of increasing antibiotic resistance. Our group showed that conditioned medium (CM) of MSC inhibits a variety of wound pathogenic bacteria, importantly also biofilms of these bacteria that often cause therapy resistant infections. We found that the MSC CM affects both bacteria that are susceptible to antibiotics and those that are antibiotic-resistant. MSC CM further helped to increase antibiotics efficacy against bacteria in biofilms. In the third year, we focused on identifying the underlying mechanisms that cause biofilm inhibition and finalized our studies to evaluate the effects of the MSC on resident skin cells. These results will help to understand how the MSC secreted factors impact the infected wound milieu. Accomplishments for this third year include: Aim 1: All data to complete aim 1 were collected and analyzed in year 1 and these outcomes were reported in the first-year progress report, as well as in a paper published in a peer-reviewed journal. Accomplishment of aim 1 led to a change in knowledge. We showed for the first time that the antimicrobial properties of MSC not only consist of AMP activity, but that MSC CM destroy proteins in the extracellular matrix due to cysteine protease activity. Treatments with MSC CM inhibits a variety of biofilm-forming wound pathogens, importantly also the antibiotic-resistant strain MRSA. Bacteria in MRSA biofilms are more effectively killed by antibiotics if biofilms are pre-treated with MSC CM, indicating that the MSC CM is a valuable approach in cutaneous wound management. By showing that the MSC CM reduces live bacteria of S. aureus and MRSA biofilms in a physiological relevant ex vivo wound model, we confirmed a high clinical relevance of the MSC CM as a future therapeutic approach. Aim 2: The main goals of aim 2 were initiated in the second year period and most data to complete aim 2 have been collected and analyzed in year 3. The goal of this aim was to evaluate the effect of the MSC CM on resident skin cells and non-resident immune cells. The part that focused on the resident skin cells has been completed and we recently submitted a manuscript to a peer-reviewed journal to report these results. This manuscript will be resubmitted soon to address the reviewers' comments before publication. The part that focused on the non-resident immune cells was initiated in year 2, where we isolated equine neutrophils from equine blood and found that MSC CM stimulated neutrophil migration, a process that we found was mediated by the protein CXCL6. These findings have been reported in a paper published in a peer-reviewed journal. To complete this objective, we need to investigate if the MSC secreted factors, besides promoting neutrophil migration, also promotes the direct antimicrobial response of neutrophils. Working on aim 2 led to a change in knowledge. For the first time, we showed that the MSC secrete factors that stimulate keratinocytes to increase their antimicrobial immune response, and we also showed that MSC secreted factors promote migrations of equine neutrophils, an important first line defense mechanism to combat wound infections. Aim 3: In addition to the collection of preliminary data that have been reported in earlier annual reports, we have not yet determined the efficacy of equine MSC-derived secreted factors in an experimental skin wound infection model in horses, but are planning this experiment in the last year of this grant proposal.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Harman RM, Patel RS, Fan JC, Park JE, Rosenberg BR, Van de Walle GR. Single-cell RNA sequencing of mesenchymal stromal cells from primary donor-matched tissue sources reveals functional heterogeneity in immune modulation and cell motility. Stem Cell Research & Therapy, 11, 524.
- Type:
Journal Articles
Status:
Published
Year Published:
2021
Citation:
Harman RM, Marx C, Van de Walle GR (2021). Translational animal models provide insight into mesenchymal stromal cell (MSC) secretome therapy. Frontiers in Cell and Developmental Biology, 9: 654885.
- Type:
Journal Articles
Status:
Under Review
Year Published:
2021
Citation:
Marx C, Gardner S, Harman RM, Wagner B., Van de Walle GR. Mesenchymal stromal cell secreted CCL2 promotes antibacterial defense mechanisms through increased antimicrobial peptide expression in keratinocytes. Stem Cell Trans Med.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
101th Annual Meeting of CRWAD, virtual.
Charlotte Marx, Sophie Gardner, Gerlinde R. Van de Walle. Equine mesenchymal stromal cells display antimicrobial properties in an ex vivo skin biofilm explant model
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2021
Citation:
Havemeyer Horse Genome workshop, virtual.
Harman RM, Patel RS, Fan JC, Park JE, Rosenberg BR, Van de Walle GR. Single cell RNA sequencing of equine mesenchymal stromal cells from different tissue sources reveals genotypic heterogeneity with phenotypic consequences.
|
Progress 06/15/19 to 06/14/20
Outputs
Target Audience:This project serves the interests of diverse audiences invested in equine wound management and antimicrobial stewardship. We delivered science-based knowledge to these audiences by publishing a research paper in a peer-reviewed journal and presenting data at several conferences and meetings. A broader audience, e.g. horse owners, veterinarians and people from the equine industry were reached by a press release of the research article in various non-scientific journals. We will further deliver our findings by outreach to the audiences where and when applicable. Also, women who have been working on this project are the PD, Dr. Gerlinde Van de Walle, her Research Support Specialist, Rebecca Harman, her post-doc Charlotte Marx, and her undergraduate student Sophia Gardner. This research provides a platform for them to gain skills and status in animal agriculture and outreach. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?Post-doctoral Associate Dr. Charlotte Marx: Postdoctoral training under Dr. Van de Walle Dr Marx is doing all in vitro experimental molecular work and the in vivo experiments, including treating the wounds and samplings. Dr Marx was trained in various techniques, including flow cytometry, confocal microscopy and image analysis. Working on the project, greatly strengthens her competences in optimizing protocols as well as her independence to establishing new protocols, realizing innovative ideas and presenting data to the scientific community. Further, the project allowed to develop new analytical skills to interpret data. The work broadens her skills related to molecular bacteriology and cell biology. Undergraduate Student Sophia Gardner: Training under Dr Van de Walle Sophia Gardner is an undergraduate student at Cornell university in the field of bioengineering. The work on the project helped Sophia to develop important lab skills and she learnt various commonly used techniques in cell culture, histology and immunofluorescence. Further, she gained first-hand experiences in developing, establishing, and executing experimental protocols by working on the equine skin explant model. How have the results been disseminated to communities of interest?The results from our work have been presented at various meetings and conferences with audiences from various scientific field of stem cell research, immunology research, microbiology research and veterinary research as well as clinical veterinarians. Further, we published a research article in the peer-reviewed journal Stem Cells Translational Medicine. This article was selected for a press release by the Editor-ibn-Chief of the journal, and was featured in a variety of non-scientific journals reaching a broad non-scientific community as well. In addition, the results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?Aim 1 has been completed, and we will continue with our experiments to complete aim 2. Specifically, we will confirm which MSC-derived cytokines are responsible for AMP upregulation in resident equine skin cells, and we will further determine the effects of equine MSC-derived cytokines on AMP expression in neutrophils and/or neutrophil function. To position ourselves well for executing aim 3, we will determine the dosage of MSC CM to be used in vivo, and will initiate experimental wound infections in horses and start with evaluations and sample collections.
Impacts
What was accomplished under these goals?
Defining the antimicrobial effect of factors secreted by equine mesenchymal stromal cells (MSC) will help to understand the underlying antibacterial defense mechanisms of MSC. This could lead to alternative therapies of wound infections, reducing the medical use of antibiotics and hence, the hazard of increasing antibiotic resistance. Our group showed that conditioned medium (CM) of MSC inhibits a variety of wound pathogenic bacteria, importantly also biofilms of these bacteria that often cause therapy resistant infections. We found that the MSC CM affects both bacteria that are susceptible to antibiotics and those that are antibiotic-resistant. MSC CM further helped to increase antibiotics efficacy against bacteria in biofilms. In the second year, we focused on identifying the underlying mechanisms that cause biofilm inhibition, and we started to investigate the effects of the MSC on resident skin cells. These results will help to understand how the MSC secreted factors impact the infected wound milieu. Accomplishments for this second year include: Aim 1: Majority of goals were completed in year 1 and outcomes were reported in the first-year progress report as well as in a paper published in a peer-reviewed journal. Additional investigations to complete aim 1 were as follows: The effect of MSC CM on MRSA biofilms, especially on the extracellular matrix, were studied by performing fluorescent staining and confocal imaging. Fluorescent live and dead staining were used to determine if MSC CM leads to bacterial killing by membrane depolarization, and further, if pre-treatments of the biofilms with MSC CM increase antibiotic efficacy against bacteria in biofilms. An ex vivo cutaneous wound model that we established in year 1 was used to test the effect of MSC CM on S. aureus and MRSA biofilms in a physiological relevant model. Moreover, single cell RNA sequencing data from MSC were used to identify a variety of genes encoding antimicrobial peptides (AMP). All data to complete aim 1 were collected and analyzed. We found that the biofilm inhibitory effect of the MSC CM is due to significant reduction of proteins in the biofilm´s extracellular matrix. This effect was mediated by cysteine protease activity which we confirmed with blocking antibodies. Cysteine proteases that were identified in high levels in the MSC CM were Cathepsin B and Cathepsin V. Previously, we found that MSC CM killed bacteria in the planktonic state by membrane depolarization. In the biofilm phenotype, a significantly reduction in live bacteria could be observed after MSC CM treatment. At the same time, a reduction of total bacteria in the biofilm was found while no significant increase in dead bacteria could be observed. Additional experiments revealed that biofilms that were treated with MSC CM had significantly less bacteria that were able to from CFU, pointing out that the MSC CM reduces viability of bacteria in biofilm. Additionally, pretreatments with MSC CM led to significantly increased antibiotic efficacy against bacteria in MRSA biofilms. We used our skin explant wound model to test the effect of MSC CM on biofilms in a more physiological relevant model. Treatments with MSC CM reduced live bacteria in S. aureus and MRSA biofilm to a level comparable with the antibiotic control. In case of MRSA biofilms, the reduction of live bacteria in the biofilms was significant compared to medium control. Accomplishment of aim 1 led to a change in knowledge. We showed for the first time that the antimicrobial properties of MSC not only consist of AMP activity, but that MSC CM destroy proteins in the extracellular matrix due to cysteine protease activity. Treatments with MSC CM inhibits a variety of biofilm-forming wound pathogens, importantly also the antibiotic-resistant strain MRSA. Bacteria in MRSA biofilms are more effectively killed by antibiotics if biofilms are pre-treated with MSC CM, indicating that the MSC CM is a valuable approach in cutaneous wound management. By showing that the MSC CM reduces live bacteria of S. aureus and MRSA biofilms in a physiological relevant ex vivo wound model, we confirmed a high clinical relevance of the MSC CM as a future therapeutic approach. Aim 2: One of the main goals of aim 2 has been initiated in the second year period, namely the effect of the MSC CM on resident skin cells and non-resident immune cells. We successfully isolated equine keratinocytes and tested the effect of MSC CM on the keratinocytes´ innate immune response. In order to evaluate the indirect antimicrobial properties of MSC, keratinocytes were stimulated with MSC CM and the antimicrobial effect of the stimulated keratinocytes was evaluated. Stimulated keratinocytes CM significantly inhibited the growth of S. aureus and MRSA compared to unstimulated keratinocytes. Keratinocytes were stained for AMP and we are currently analyzing AMP expression by confocal imaging investigations. Preliminary results indicate that stimulated keratinocytes have an increased expression of the AMP cathelicidin, lipocalin, and elafin. We further established an ex vivo skin explant model to test the effect of MSC CM on resident skin cells innate immune response in a physiological relevant model. Sample collection is complete and data analyzation is ongoing. Preliminary results show that keratinocytes are the primary cells in the skin that express AMP and that cathelicidin expression is increased in the skin after stimulation with MSC CM. To preliminary test the effect of MSC CM on non-resident immune cells, we isolated equine neutrophils from equine blood and found that MSC CM stimulated neutrophil migration mediated by JAM-2. To complete this objective, we need to confirm the presence of the cytokines in the MSC CM on a protein level and confirm cytokine activity towards keratinocytes. We further need to investigate if the MSC secreted factors, beside promoting neutrophil migration, also promote the direct antimicrobial response of neutrophils. Working on aim 2 led to a change in knowledge. For the first time, we showed that the MSC secrete factors that stimulate keratinocytes to increase their antimicrobial immune response. Aim 3: We already collected some preliminary data during the first year period that were reported in the first-year report. Briefly summarized, we used the skin explant model to determine the effect of the MSC CM on cell viability to assess safety and treated natural occurring wounds of allergic horses with MSC CM to asses efficacy in vivo. In the second year, we further confirmed the MSC CM therapeutic safety using isolated keratinocytes and ex vivo skin explants, as follows. Isolated keratinocytes were treated with MSC CM for 24 h and cell viability was tested. Importantly, we found no significant difference in cell viability compared to culture medium control. The keratinocytes that were cocultured with the MSC CM even had a higher viability than keratinocyte that were maintained in culture medium. Although, the difference was not significant, it indicates that no negative effects from the MSC on keratinocytes are to be expected when applied in vivo. Moreover, MSC CM-treated skin explants were investigated histological and no changes in epidermal thickness as well as in overall histological appearance were observed. Our preliminary investigation shows a high therapeutic safety. To determine the best therapeutic concentration, we plan to test effects of various concentrations of the MSC CM on keratinocytes viability and cell survival, as well as histological changes. Effects on biofilm inhibition will be assessed using the clinically relevant wound pathogen MRSA before moving into in vivo experiments.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2020
Citation:
Marx C, Gardner S, Harman RM, Van de Walle GR. The mesenchymal stromal cell secretome impairs methicillin-resistant Staphylococcus aureus biofilms via cysteine protease activity in the equine model. Stem Cell Trans Med. 2020. DOI: 10.1002/sctm.19-0333.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Charlotte Marx, Sophie Gardner, Rebecca M. Harman, Gerlinde R. Van de Walle. The antimicrobial properties of equine mesenchymal stromal cells as a biological alternative to antibiotics.
100th Annual Meeting of CRWAD, Chicago, Illinois.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2019
Citation:
Charlotte Marx, Sophie Gardner, Rebecca M. Harman, Gerlinde R. Van de Walle. The antimicrobial properties of equine mesenchymal stromal cells as a biological alternative to antibiotics.
Baker seminar series, Baker Institute for Animal Health, Ithaca, New York.
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Charlotte Marx, Sophie Gardner, Rebecca M. Harman, Gerlinde R. Van de Walle. The antimicrobial properties of equine mesenchymal stromal cells as a biological alternative to antibiotics.
Postdoctoral Research Seminar, Department of Microbiology and Immunology, Cornell University, Ithaca, New York
- Type:
Conference Papers and Presentations
Status:
Published
Year Published:
2020
Citation:
Charlotte Marx, Sophie Gardner, Rebecca M. Harman, Gerlinde R. Van de Walle. The antimicrobial properties of equine mesenchymal stromal cells as a biological alternative to antibiotics.
Stem Cell Work-In-Progress Meeting, Cornell University, Ithaca, New York
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Progress 06/15/18 to 06/14/19
Outputs
Target Audience:This project serves the interests of diverse audiences invested in equine wound management and antimicrobial stewardship, and will result in expanded academic collaborative networks. We will deliver science-based knowledge to these audiences by outreach when and where applicable. Also, women who have been working on this project are the PD, Dr. Gerlinde Van de Walle and her Research Support Specialist, Dr. Rebecca Harman. Other female scientists that were involved in the first year of this project include the Post-doctoral Associate, Dr. Charlotte Marx, and the high school students, Sophie Gardner and Safiya Bashir, that she mentored. This research provides a platform for them to gain skills and status in animal agriculture and outreach. Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided?* Post-doctoral Associate Dr. Charlotte Marx: Postdoctoral training under Dr. Van de Walle. Dr. Marx is doing all the in vitro experimental molecular work and the in vivo experiments, including treating the wounds and sampling. Dr. Marx was trained in various techniques, including flow cytometry, confocal microscopy and image analysis. Working on the project greatly strengthens her competences in optimizing protocols as well as her independence to establishing new protocols and realizing innovative ideas. Further, the project allowed to develop new analytical skills to interpret data. The work broadens her skills related to molecular bacteriology and cell biology. * High School Student Sophia Gardener: Training under Dr. Van de Walle. Sophia Gardener gathered first research experiences as a High School Student of the "New Vision in Life Science" program. She characterized the horse skin explant model and determined the effects of MSC CM on S.aureus in skin wounds. She had the opportunity to present her results at the New Vision Research Symposium. Based on her excellent work, she will further participate in the project as an undergraduate student at Cornell university in the Field of Bioengineering. * High School Student Safiya Bashir: Training under Dr. Van de Walle. Safiya Bashir participated in the project as a Summer Student. She had the opportunity to learn cell culture techniques and basic microscopic skills. How have the results been disseminated to communities of interest?The results from our work have been presented at various conferences and symposia, both within Cornell University as well as outside Cornell (see under Publications). Our work has also been featured on social media (https://www2.vet.cornell.edu/news/20180118/equine-stem-cells-rein-bacteria#.WmXty9Fzlix.facebook) In addition, the results have been informally discussed with other scientists and equine clinicians and have been received with great enthusiasm by scientists in the field. What do you plan to do during the next reporting period to accomplish the goals?In the next year, we plan to write a manuscript describing the results we have generated thus far, and to submit this to a peer-reviewed journal. We also will further focus on Aim 1 and 2: * To complete aim 1, we plan to identify the full spectrum of AMPs and confirm their activity * To complete aim 2, we plan to confirm which MSC-derived cytokines are responsible for AMP upregulation in resident equine skin cells and to determine the effects of equine MSC-derived cytokines on AMP expression in neutrophils and/or neutrophil function.
Impacts
What was accomplished under these goals?
We aim to better define the antimicrobial effects of factors secreted by equine mesenchymal stromal cells (MSC), a.k.a. the stem cell secretome. Taking advantage of the antimicrobial properties of the secretome could lead to alternative and adjunct therapies for treating wound infections, thereby reducing the medical use of antibiotics and, hence, the potential treat of antibiotic resistance development. Our group already showed that conditioned medium of MSCs (MSC CM) inhibit the growth of the Gram+ bacterium S.aureus and the Gram- bacterium E.coli. In the first year of the project, we focused on determining the effect of MSC CM on additional bacteria that are commonly found in infected skin wounds. The results will increase our understanding of how MSC secreted factors impact the wound milieu that is populated with a variety of bacterial strains. Accomplishments for this first year include Aim 1: * We tested the effect of MSC CM on various wound-related bacteria in their planktonic form or in biofilms. For this we used field strains of P.aeruginosa, S.epidermidis, A.baumannii and A.viridans that were isolated from horse skin wounds. We also included an isolate of the Methicillin-resistant S.aureus (MRSA) USA 300 strain as well as a biofilm-negative MRSA Δsigβ strain as control, since antibiotic-resistant bacteria have a high clinical relevance. We found MSC CM to significantly inhibit the growth of planktonic P.aeruginosa, A.baumannii, A.viridans and MRSA. Although the growth of S.epidermidis was not inhibited by the secretome of naïve MSC, the secretome of MSC that were pre-exposed to living bacteria significantly inhibited the growth of S.epidermidis, indicating that the MSC secretome is altered after the MSCs were stimulated by bacteria. * We determined the effect of MSC CM on biofilm formation and on mature biofilms. A.baumannii, A.viridans and MRSA form significantly less biofilm mass after treatment with MSC CM. Importantly, MSC CM was found to be superior in reducing bacterial growth when compared to various antibiotics that were included as controls. S.epidermidis was not found to form significant biofilms in the assay and hence, was excluded from the study. P.aeruginosa biofilm formation was not impaired by MSC CM. * We tested the effect of MSC CM on mature biofilms. Supporting the fact that bacteria in mature biofilms are hard to treat, MSC CM did not affect the mature biofilms of A.viridans and A.baumannii. Surprisingly, the antibiotics also could not reduce the bacterial biofilms, pointing out the importance of investigating alternative therapies. However, we found that MSC CM significantly reduced the biofilm mass of P.aeruginosa, S.epidermidis and MRSA, when compared to the vehicle control. Importantly, MRSA biofilms were not affected by antibiotics, conforming that conventional therapies to treat antibiotic-resistant bacteria are ineffective. To further investigate the effects on MSC CM on bacteria in biofilms, we established immunofluorescence protocols to visualize and analyze the composition of the biofilm´s extracellular matrix and the viability of the bacteria in the biofilm. Our preliminary experiments show that MSC secrete bioactive factors that impact the composition of the bacterial extracellular matrix. * We established an ex vivo equine infected wound skin explant model, and evaluated the effects of MSC CM on bacteria. To this end, wounds were inoculated with S. aureus and the effect of MSC CM was tested on biofilm formation and mature biofilms in this more physiologically relevant assay. In line, with the in vitro results, we found that MSC CM significantly decrease the number of living bacteria in the biofilm. Interestingly, the negative effect of MSC CM on the survival of bacteria in mature biofilms was significantly higher compared to the effect observed with antibiotic treatment (i.e. much less bacteria survived in biofilms when exposed to MSC CM). To complete Aim 1, we need to identify the full spectrum of antimicrobial peptides (AMPs) produced by equine MSC and confirm their activity. We established a flow cytometry protocol, in order to investigate the effect of AMPs on the bacterial cells and further studies will elucidate how these AMPs work, e.g. by causing cell wall disruption. Aim 2: * We tested the effect of MSC CM on equine resident skin cells in an ex vivo skin explant model and determined the LIVE/DEAD ratio of the skin cell. Explant that were treated with MSC CM had significantly higher numbers of living eukaryotic cells when compared to culture medium control. This finding supports our hypothesis that MSC secrete factors that stimulate resident skin cells. The next steps will be to confirm that MSC secrete cytokines, or other bioactive factors, that affect resident skin cells. Aim 3: * We used our established equine skin explant models to collect ex vivo data about the toxicity of MSC CM on resident skin cells, by analyzing the numbers of dead cells after 3 subsequent treatments with MSC CM or culture medium control. Importantly, we did not detect any negative effects on the resident skin cells in the preliminary experiments. In further investigation, we will increase the concentration of the MSC CM and test the cytotoxicity on equine resident skin cells my measuring the viability from cells in culture and determine the LIVE/DEAD ratio of skin cells in the explant model. * Additionally, we established the procedure to inoculate wounds in the skin explant model with a dosage of S.aureus that forms a strong, mature biofilm within 3 days. * Finally, we already had the chance to perform a first preliminary in vivo study. Natural occurring skin wounds of allergic horsed were treated with MSC CM, and the bacterial load as well as the bacterial composition in the wound milieu were determined by CFU-forming assays on non-selective and selective agars. Interestingly, we detected a decrease of the total bacterial load in all MSC CM treated wounds after a 7 days trial. Additionally, we also found a shift in the bacterial composition in the wound milieu. After treatments with MSC CM, the ratio of Gam-/Gram+ bacteria changed to benefit Gram+ bacteria.
Publications
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Charlotte Marx, Sophie Gardner, Rebecca M. Harman, Gerlinde R. Van de Walle. The antimicrobial effect of equine mesenchymal stromal cells. 7th Mesenchymal Stem Cell Symposium, Alice Statler Auditorium, Ithaca, New York.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Charlotte Marx, Sophie Gardner, Rebecca M. Harman, Gerlinde R. Van de Walle. The Antimicrobial Properties Of Equine Mesenchymal Stromal Cells As A Biological Alternative To Antibiotics. North American Veterinary Regenerative Medicine Association meeting, Ontario-on-the-Lake, Ontario, Canada.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Charlotte Marx, Sophie Gardner, Rebecca M. Harman, Gerlinde R. Van de Walle. The antimicrobial effect of equine mesenchymal stromal cells. 10th annual Harry M. Zweig Memorial Fund for equine Research Faculty Presentation and Poster Session, College of Veterinary Medicine, Cornell University Ithaca, New York.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2018
Citation:
Rebecca M. Harman, Charlotte Marx, Gerlinde R. Van de Walle. The Antimicrobial Properties Of Equine Mesenchymal Stromal Cells As A Biological Alternative To Antibiotics.99th Conference of Research Workers in Animal Diseases (CRWAD), Chicago, Illinois.
- Type:
Conference Papers and Presentations
Status:
Other
Year Published:
2019
Citation:
Sophie Gardner, Charlotte Marx, Gerlinde R. Van de Walle. Antimicrobial properties of equine mesenchymal Stromal Cells. The 6th New Vision in Life Science Research Symposium, Weill Hall, Cornell University, Ithaca New York.
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