Enrichment and expansion of human umbilical cord mesenchymal stem cells as an angiogenic therapy for critical limb ischemia

Critical Limb ischemia (CLI) or which is otherwise known as Chronic limb threatening ischemia is the advanced form of PAD, which can lead to ischemic rest pain, non healing wounds, ulcers gangrene, and in its worst form to limb amputation and consequent death. CLI occurs due to atherosclerosis in the arteries supplying the lower extremities. This causes the tissues below the plaque to be deficient in oxygen and nutrients; and leads to micro and macro vascular complications. Among the CLI patients, the non-revascularizable percentage of 10%-50 % or the “no-option” segment has poor prognosis with high mortality rates and reduced quality of life. The no-option patients have to undergo limb amputation; even upon amputation in addition to loss of quality of life, 50% die in 5 years and 70% die in 10 years of amputation. There is no effective treatment for CLI; hence a therapy for CLI is an unmet medical need. In response, a scalable, reproducible sterile system using 3D microbioprinting methodology was developed, to allow high-throughput bioprinting of GMP compliant, sorted and Quantum closed bioreactor expanded 3D UC-362- MSC-gel platforms. Innovative devices were developed and prototyped in this approach, and their proof of concept was sought. An invention disclosure form (IDF) for the novel innovative device was prepared and filed at the University of Galway Technology Transfer Office. The system received an Enterprise Ireland feasibility grant for obtaining IP rights and a startup development. The IP application process was initiated in this regard. The novel innovation will benefit numerous 3D bioprinting applications in different fields by enhancing their capacities. An in vivo randomized study was carried out in a nude BALB/c HLI mouse model for evaluation of the angiogenic capacity of the umbilical cord derived CD 362-MSC-bioprinted-cell gel platform and consisted of four groups with 12 animals in each. The treatment groups included saline/ sham, UC-362-MSC-IDO cells alone, UC-362-MSC-IDO-gel platforms, and acellular-gel platforms. Although the perfusion imaging methods of laser Doppler imaging and the novel HLI-MSOT used for the first time ever in HLI perfusion imaging did not detect increases in perfusion among treatment groups, the highest gains in capillary density with greatest significance (p<0.0001) were observed in the calf muscle of UC-362-MSC-IDO-gel platform recipients (368 BV /mm2 ± 197 s.d.). Muscle analysis of the ischaemic posterior calf muscles revealed high capillary densities in UC-362-MSC-IDO-gel treated animals (368 BV /mm2 ± 197 s.d.), and in UC-362-MSC-IDO cell treated animals (341 BV /mm2 ± 293 s.d.) compared to the saline treated group (p<0.0001). On average, the UC-362-MSC-IDO-gel treated animals developed 2.6 times more blood vessels per unit area compared to that of the saline recipients. The muscle fiber diameter analysis revealed the average of the median Feret fiber diameter was significantly higher in UC-CD-362-MSC-IDO cell treated (p<0.0001) and in UC-CD-362-MSC-IDO-gel platform treated (p<0.05) mice compared to the saline treated mice. The nature of added benefits the UC-362-MSC-IDO-gel platform can confer over UC-362-MSC-IDO cells alone warrants disease-specific application analysis. Therefore, based on the present study, the high-throughput 3D microbioprinted UC-362-MSC-IDO-gel platform with potent in vitro and in vivo proangiogenic capacity may require further dose-based fine-tuning through HLI in vivo studies for more amplified therapeutic benefit, before its use in critical limb ischemia. Additionally, a novel three-dimensional imaging modality for mouse hind limb ischemia based on multi-spectral optoacoustic tomography (MSOT) was developed and compared against the standard laser Doppler imaging modality. A comprehensive description of methodology, process, benchmark values for ischemia HLI-MSOT, and dos and don’ts were introduced for conducting MSOT in mouse HLI studies based on the learning experience and results. In the proof of concept studies, the novel mouse HLI-MSOT method was successful in detecting all ischemic events with 100% specificity and sensitivity relative to the standard LDI system in the ischemic mouse foot pads. The novel HLI-MSOT imaging modality would enhance the accurate determination capabilities of small animal-based HLI perfusion studies particularly in the thicker denser tissue 3D space which is not sufficiently addressed by the LDI based imaging. Thus, the novel MSOT based HLI imaging system could therefore be utilized successfully as either a stand-alone three-dimensional perfusion data acquisition modality instead of the LDI system, for more accurate perfusion information acquisition; or as a complementary system to the standard LDI already in use in a conjunctive manner in mouse HLI studies. Abbreviations-: Indoleamine-2,3-dioxygenase (IDO); umbilical cord derived CD-362-MSC and IDO secreting cells: UC-362-MSC-IDO;3D bioprinted umbilical cord derived CD-362-MSC and IDO secreting cell-hydrogel platforms: UC-362-MSC-IDO-gel; blood vessels: BV; Standard deviation: s.d

Funder

Irish Research Council (IRC)

Publisher

University of Galway

Collections

University of Galway Theses (PhD Theses)