“Dr. Vorp’s quest,
Stem cells dance, hope is expressed,
Future health’s behest.”

– ChatGPT, using GPT-3.5 architecture by OpenAI, prompted to compose a haiku that summarizes a RoosterBio webinar via input of a real-time speech-to-text transcript.

Much pioneering work to clinically translate mesenchymal stem/stromal cells (MSCs) and their exosomes (MSC-EVs) comes from academic labs dedicated to what RoosterBio calls “MSC 2.0” concepts, where MSCs leapfrog “traditional” uses when MSCs were once the sole ingredient in a regenerative therapy. One exemplar is the Lab of Professor David Vorp at the University of Pittsburgh’s Vascular Bioengineering Laboratory. Dr. Vorp seeks collaborative, interdisciplinary solutions for diseases and injuries of tubular organs such as blood vessels, urethra, and esophagus, combining expertise in biomechanics, cell and molecular biology, and tissue engineering. Engaging with the UPMC, the McGowan Institute for Regenerative Medicine, and notably, RoosterBio, the Vorp Lab is accelerating such MSC 2.0 solutions as shown in a 2023 webinar hosted by RoosterBio.

Crystal Cruz (Bioprocessing Consultant for Europe of RoosterBio) kicked off the presentations with a brief summary of RoosterBio, a leading institution for driving innovative MSC and MSC-EV concepts towards more seamless translation into clinical programs that are easier to scale in both product and process development. Before introducing the four speakers, Crystal highlighted RoosterBio’s long and fruitful collaboration with the University of Pittsburgh—and especially with Dr. David Vorp’s lab—who have worked together for over five years. The topics included:

1. David A. Vorp, Ph.D., John A. Swanson Professor of Bioengineering, Surgery, and Cardiothoracic Surgery​, Sr. Associate Dean for Research & Facilities, Swanson School of Engineering, University of Pittsburgh. — Ongoing Applications of Vascular Tissue Engineering & Regenerative Medicine (3:10)

2. Ande X. Marini, NIH F31 (NHLBI) Graduate Research Fellow, University of Pittsburgh. — Delivery system for local EV treatment of small AAA (24:54)

3. Amanda R. Pellegrino, National Science Foundation (NSF) Graduate Research Fellow, University of Pittsburgh. — EV-based TEVG (30:37)

4. Kamali Charles, Cardiovascular Bioengineering Training Fellow (CBTP) Graduate Research Fellow, University of Pittsburgh. — EV-based regenerative  dressings for chronic wound care (34:20)

5. Conclusion / Q&A (39:03)

Professor Vorp first provided some background on two projects aimed at two significant health challenges— aortic aneurysms and bypass grafts for surgical repair of damaged hearts. Abdominal aortic aneurysms (AAAs) ¹ are defined when a region of the aorta balloons up to 150% of its original diameter. While many remain undetected until they grow larger, they nevertheless pose a high risk of rupture. Once an aneurysm reaches a size of 5.5 centimeters, its risk of rupture exceeds the risks of surgical repair. Since rapid death from AAA rupture exceeds 90%, this condition with 200,000 new diagnoses per year in the USA alone is sometimes called a ticking time bomb that kills 15,000 annually. Nevertheless, a significant fraction of aneurysms will in fact rupture prior to attaining widths of 5.5 cm, inspiring Vorp’s and colleagues’ work to intervene in this unmet medical need with adipose-derived MSCs (AD-MSCs). ² It was hypothesized that AD-MSCs’ immunomodulatory activity could block the AAA pathobiology “Cycle of Destruction” that weakens the aorta and causes it to bulge.

A collaboration with Dr. John Curci (Division of Vascular Surgery, Vanderbilt University) enabled assistance via an induced AAA mouse model ³ that combined highly elastase perfusion and temporary ligatures of the aorta. Initial published studies ⁴ showed that local application of the AAA with AD-MSCs could halt progression of the model aneurysms as measured by both aorta width as well as restoration of normal elastin layering. This work would be the foundation for new, exciting studies led by 5^th year Ph.D. candidate and webinar speaker, Ande Marini, BS.

Professor Vorp’s prior tissue engineering studies also involved AD-MSCs—but here, in a different modality, for the generation of improved arterial bypass grafts. Autologous vein and arterial graft options are limited for any given patient, making their short supply an unmet medical need. While artificial grafts could supply a much larger number of quality-controlled tissue products, no fully synthetic grafts are yet successful. Vorp accordingly described his group’s prior efforts to generate tissue engineered vascular grafts (TEVGs) ⁵ from bilayered, tubular, biodegradable PEUU scaffolds seeded with AD-MSCs. ⁶ AD-MSCs from healthy human donors dramatically improve patency of the TEVGs in live rat models from 0% (non-seeded) to 100% (MSC-seeded). Such efforts blazed a trail for new work to be performed by Amanda R. Pellegrino, BSN and RN, a 3rd year Bioengineering PhD student.

What if possible translational challenges related to MSCs and other therapeutic cells could be minimized by utilizing their stockpiling-friendly and quality-controllable secreted products instead of live injected cells? One compelling application of “MSC 2.0” that is generating recent buzz around regenerative medicine clinical trials is the use of MSCs as bioproduction cells for extracellular vesicles (EVs), sometimes loosely referred to as “exosomes.” Dr. Vorp stated, “EVs are neatly packaged signals and protein secreted in lipid membranes from their parent cells and we believe would have the same effect in our regenerative medicine and tissue engineering applications as we have shown for MSCs.” He noted RoosterBio’s role as an accelerator partner to help secure a Catalyze (R61) Grant in 2020, which is providing the bridge for a new application for an R33 grant that is to involve scaleup studies in large animals. His ongoing studies with MSC-EVs ^{7, 8} have also extended into work in cutaneous wounds via a NIH UO1 grant that involves silk-based biomaterial wound dressings containing “clickable” MSC-EVs, to be pursued by his graduate student, Kamali Charles, DPT.

At this point in the webinar, Ande Marini took the reins to describe recent work on a Delivery system for local EV treatment of small abdominal aortic aneurysm. Building on the previous efforts via Professor Vorp and Vanderbilt’s Curci Lab, Marini explored cell-free regenerative treatment of EVs on the AAA model, with an objective being the localization of EVs to the aneurysm site. Marini first characterized the prep of tangential flow filtration (TFF)-isolated EVs from RoosterBio AD-MSCs by Western blotting for CD63 and nanoparticle tracking analysis (NTA). Next, she used fluorescence microscopy techniques to visualize uptake of these EVs into healthy vascular smooth muscle epithelial cells (VSMCs) seeded in fibrin gel, a metric of their bioactivity that might be extrapolated to ex vivo utility. VSMCs do indeed internalize the dye-labeled EVs whether in 2D or when embedded (3D) in fibrin. Presently, Marini is perfecting assay conditions to determine ex vivo bioactivity of AD-MSC-EVs that are delivered via microparticle (MP) carriers, examining their effect on VSMC secretion of elastin and collagen, which are putative biomarkers that are disrupted in AAA pathology.

After Ande’s presentation, Amanda Pellegrino described previous work with EV-functionalized TEVGs, which showed improved patency in vivo in rats (100%), above and beyond AD-MSC seeded controls, as well as more elastin and collagen production and infiltration of smooth muscle cells and fewer (pro-inflammatory) macrophages. Pellegrino’s recent studies involves transition from ultracentrifugation of EVs to the use of a TFF system from Repligen, which can generate much larger yields. Pellegrino has been observing more than 4-fold faster proliferation of smooth muscle cells (SMCs) in the presence of EVs, and 2- 3-fold greater migration as measured by a transwell assay system. Moving forward, Amanda and her colleagues are looking into enhancing EV retention into EV-seeded TEVGs that would enable a more extended release time, as well as diving into effects on how EVs affect the formation of an endothelial cell monolayer. These experiments are paving the way to perform studies with the TEVGs in scaled-up, clinically relevant large animal models.

Pellegrino then turned over the presentation, introducing Kamali Charles as the next student speaker. Charles began by outlining the difference between acute and chronic wounds. Acute wounds have a clear healing trajectory, while chronic wounds stall in the inflammatory phase, leading to complications like infections, biofilm formation, and impaired wound closure. He described wound healing as relatively new for the Vorp Lab but noted existing literature that exhibits potential benefits towards various wound models, including chronic, diabetic, and burn wounds. Many of these studies have reported enhanced wound closure, increased neovascularization, and reduced inflammation. However, one of the main challenges with EVs may be their effective delivery and retention in the wound site. Systemic delivery is often hindered by premature clearance, while topical methods may not ensure sustained EV presence for optimal healing. The proposed solution by Charles and Professor Vorp is to engineer silk fibroin wound mesh that are embedded with AD-MSC-EVs. After completing preliminary in vitro tests, the team plans to evaluate their dressings using a validated chronic wound model in mice. The hypothesis is that the dressings with immobilized EVs will facilitate faster wound closure compared to other dressing types.

After Kamali Charles’ webinar segment, Professor Vorp gave some concluding remarks and opened the floor for some brief questions and answers. Here were some of the questions (paraphrased), but be sure to “tune in” to hear their speakers’ astute answers:

• Why use adipose-derived MSCs when there are MSCs that can be obtained from other tissues?
• How was RoosterCollect™-EV useful for gathering EVs into AD-MSC conditioned media?
• Are there noticeable differences between EVs obtained via ultracentrifugation and EVs obtained via TFF?
• What is the benefit of using silk fibroins as a wound dressing versus other biomaterials?

RoosterBio is extremely grateful for the opportunity to have been collaborating with Professor Vorp and his talented group for more than five years as new discoveries and engineered constructs develop into viable clinical opportunities with “MSC 2.0.” We look forward to what the next years will bring as MSCs and their exosomes/EVs integrate into future cures for dire unmet medical needs.

In closing, we returned to ChatGPT via OpenAI to compose a brief ode regarding its unique take on the research described in this webinar. The results aren’t exactly Shakespeare, but… possibly earnest enough. It’s almost as if ChatGPT could catch the pulse of the state of the art on vascular biomedical engineering. Or hopefully(?) it’s at least good enough for a jolly chuckle or smile:

“In labs where bioengineers toil and strive,
Professor Vorp’s wisdom helps ideas thrive.
From aortic aneurysms to wounds that stall,

His guidance leads, ensuring progress for all.
Andy, Amanda, and Kamali spoke clear,
Of EVs and graphs, innovations near.

From vascular cures to healing’s hopeful grace,
Their research charts the future’s earnest pace.
Yet, questions linger, answers sought and found,

In this symposium, knowledge does abound.
With every query and each insight shared,
The quest for health’s advancements is declared.”

References

1. Vorp, D. A., Introduction to the JVS-VS Special Issue, “Aneurysms”. JVS Vasc Sci 2023, 4, 100110. 10.1016/j.jvssci.2023.100110
2. Krawiec, J. T.; Liao, H. T.;  Kwan, L. L.;  D’Amore, A.;  Weinbaum, J. S.;  Rubin, J. P.;  Wagner, W. R.; Vorp, D. A., Evaluation of the stromal vascular fraction of adipose tissue as the basis for a stem cell-based tissue-engineered vascular graft. J Vasc Surg 2017, 66 (3), 883-890 e1. 10.1016/j.jvs.2016.09.034
3. Gueldner, P. H.; Marini, A. X.;  Li, B.;  Darvish, C. J.;  Chung, T. K.;  Weinbaum, J. S.;  Curci, J. A.; Vorp, D. A., Mechanical and matrix effects of short and long-duration exposure to beta-aminopropionitrile in elastase-induced model abdominal aortic aneurysm in mice. JVS Vasc Sci 2023, 4, 100098. 10.1016/j.jvssci.2023.100098
4. Blose, K. J.; Ennis, T. L.;  Arif, B.;  Weinbaum, J. S.;  Curci, J. A.; Vorp, D. A., Periadventitial adipose-derived stem cell treatment halts elastase-induced abdominal aortic aneurysm progression. Regen Med 2014, 9 (6), 733-41. 10.2217/rme.14.61
5. Haskett, D. G.; Saleh, K. S.;  Lorentz, K. L.;  Josowitz, A. D.;  Luketich, S. K.;  Weinbaum, J. S.;  Kokai, L. E.;  D’Amore, A.;  Marra, K. G.;  Rubin, J. P.;  Wagner, W. R.; Vorp, D. A., An exploratory study on the preparation and evaluation of a “same-day” adipose stem cell-based tissue-engineered vascular graft. J Thorac Cardiovasc Surg 2018, 156 (5), 1814-1822 e3. 10.1016/j.jtcvs.2018.05.120
6. Krawiec, J. T.; Weinbaum, J. S.;  Liao, H. T.;  Ramaswamy, A. K.;  Pezzone, D. J.;  Josowitz, A. D.;  D’Amore, A.;  Rubin, J. P.;  Wagner, W. R.; Vorp, D. A., In Vivo Functional Evaluation of Tissue-Engineered Vascular Grafts Fabricated Using Human Adipose-Derived Stem Cells from High Cardiovascular Risk Populations. Tissue Eng Part A 2016, 22 (9-10), 765-75. 10.1089/ten.TEA.2015.0379
7. Cunnane, E. M.; Weinbaum, J. S.;  O’Brien, F. J.; Vorp, D. A., Future Perspectives on the Role of Stem Cells and Extracellular Vesicles in Vascular Tissue Regeneration. Front Cardiovasc Med 2018, 5, 86. 10.3389/fcvm.2018.00086
8. Cunnane, E. M.; Ramaswamy, A. K.;  Lorentz, K. L.;  Vorp, D. A.; Weinbaum, J. S., Extracellular Vesicles Derived from Primary Adipose Stromal Cells Induce Elastin and Collagen Deposition by Smooth Muscle Cells within 3D Fibrin Gel Culture. Bioengineering (Basel) 2021, 8 (5). 10.3390/bioengineering8050051