Mesenchymal Stromal Cell-Based Therapies as Promising Treatments for Muscle Regeneration After Snakebite Envenoming

Abstract

Snakebite envenoming is a global neglected disease with an incidence of up to 2.7 million new cases every year. Although antivenoms are so-far the most effective treatment to reverse the acute systemic effects induced by snakebite envenoming, they have a limited therapeutic potential, being unable to completely neutralize the local venom effects. Local damage, such as dermonecrosis and myonecrosis, can lead to permanent sequelae with physical, social, and psychological implications. The strong inflammatory process induced by snake venoms is associated with poor tissue regeneration, in particular the lack of or reduced skeletal muscle regeneration. Mesenchymal stromal cells (MSCs)-based therapies have shown both anti-inflammatory and pro-regenerative properties. We postulate that using allogeneic MSCs or their cell-free products can induce skeletal muscle regeneration in snakebite victims, improving all the three steps of the skeletal muscle regeneration process, mainly by anti-inflammatory activity, paracrine effects, neovascularization induction, and inhibition of tissue damage, instrumental for microenvironment remodeling and regeneration. Since snakebite envenoming occurs mainly in areas with poor healthcare, we enlist the principles and potential of MSCs-based therapies and discuss regulatory issues, good manufacturing practices, transportation, storage, and related-procedures that could allow the administration of these therapies, looking forward to a safe and cost-effective treatment for a so far unsolved and neglected health problem.

Keywords: advanced therapy medicinal products; envenoming; mesenchymal stromal cells; muscle regeneration; snakebite.

Figure 1

Summary of the main hypothetical factors that determine the poor outcome in skeletal muscle regeneration after myonecrosis induced by viperid venoms, associated to viperid toxins and the steps of the SMR which they affect. Different venom components induce hemorrhage, myotoxicity, or/and neurotoxicity. These deleterious effects impair the normal SMR acting on all their three steps. IR, inflammatory reaction; RP, regenerative/phase; RrP, remodeling-repair phase; CRISP, cysteine-rich secretory protein [Protein Data Bank accession ID (PDB ID): 3MZ8]; CTL, C-type lectin-like protein (PDB ID: 1IXX); LAO, l-amino acid oxidase (PDB ID: 2IID); Myo, low molecular mass myotoxin (PDB ID: 4GV5); PLA2, phospholipase A2 (PDB ID:1TGM for the monomer and PDB ID:3R0L for the dimer); SVMP, snake venom metalloproteinase (PDB ID:3DSL for class Plll and PDB ID: 1ND1 fo class PI); SVSP, snake venom serine proteinase (PDB 1D:1OP0).

Figure 2

Illustration of the three phases of skeletal muscle tissue regeneration. (A) In normal conditions, satellite cells are located between the sarcolemma and basement membrane of terminally-differentiated muscle fibers. (B) Soon after damage, the inflammatory reaction starts. First, the cells of the immune system (neutrophils and macrophages) infiltrate the damaged tissue produce a proinflammatory stage. Over time, an anti-inflammatory stage starts to replace the proinflammatory one, in this transition, proinflammatory M1 macrophages switch to anti-inflammatory M2 macrophages. (C) Once the proinflammatory stage starts to decay, the regenerative phase starts with the satellite cells activation. Then, activated satellite cells proliferate and differentiate into myoblast and finally myoblasts fuse into myotubes. (D) The remodeling/repair phase consists in the extra-cellular microenvironment (ECM) remodeling and maturation of the new myofibers. If an inadequate remodeling/repair phase occurs, the myotubes grow unorganized generating dysfunctional muscle tissue.

Figure 3

Four main properties of mesenchymal stem cells (MSC). MSCs have a potential for differentiation into ectoderm cells (neurons, epithelial cells), mesoderm (osteocyte, endothelial cell, chondrocyte, and adipocyte), and endoderm (muscle cells, gut epithelial cells and lung cells). Secretion of factors such as proteins, miRNAs, mitochondria, and exosomes can promote repair of damaged tissue and immunomodulatory potential. The immunomodulatory effect is mainly immunosuppressive, several secreted cytokines inhibit the activity of natural killer cells (NK cells), T cells and B cells; other cytokines activate the proliferation of regulatory T cells (T reg) and the switch from macrophage M1 (pro-inflammatory) to macrophage M2 (anti-inflammatory). The property of migration and homing is possible by the expression of specific ligands and receptors in the site of injury. Dashed arrows: Controversial transdifferentiation in vivo.

Figure 4

Relationship on main properties of MSC-based therapies regarding the recovery of the impaired SMR after myonecrosis induced by snakebite envenomation. It is detailed the direct and indirect relationship between properties of MSC-based therapies and which step or steps of SMR would be benefited by each property. Treg, regulatory T cells; NK cells, natural killer cells.

Figure 5

hMSCs derived from omentum adipose tissue, grown for long periods in chemically defined media have optimal morphological and proliferative properties. The hMSC were cultivated in control medium supplemented with fetal bovine serum (Control SFB medium) on adherent plates and XANADU chemically defined medium and commercial MQD on plates functionalized with vitronectin for eight consecutive passes. (A) Morphology and culture density of hMSC. (B) Duplication time. (C) Cumulative doubling of the population. The figures in (A) are representative of three independent experiments. The data of (A, B) are the mean plus the deviation of at least three independent experiments. (*) p ≤0.05 when compared to the control SFB medium.

Figure 6

Secretoma of hMSC cultured in chemically defined medium. The hMSCs derived from omentum adipose tissue were isolated and cultured in XANADU chemically defined medium (hMSC- line Sev5). The obtained secretome was used for the assay of cytokine array. Both the fresh non-secretoma medium and the secretion-enriched medium of hMSC-line Sev5 were incubated on membranes of the Human Cytokine Antibody Array (Abcam) kit, following the manufacturers’ recommended protocol. The red circles indicate the cytokines that are significantly increased in the secretion-enriched XANADU after culture of the MSCs for 5 days compared to the fresh medium. The figure is representative of three independent experiments.

Figure 7

Vasculogenesis after Intrarterial adipose-derived MSC administration (intrarterial 1 x 106 cell/kg). Left: basal, middle: 6 months after and right: 12 months after intrarterial administration in a type 2 diabetic patient with a Grade 6 Rutherford (from Soria B. 2016. La Nueva Biología y sus Aplicaciones Médicas, with permission) (124).

Figure 8

Preliminary assay. Creatine kinase levels of mice sera 3 h, 72 h, and 1 week after inoculation. Group PBS+PBS was injected with 50 µl of PBS by both intramuscular (i.m) and intravenous (i.v) routes. Group BaV+PBS received 50 µg of Bothrops atrox venom diluted in 50 µl of PBS and only PBS by i.v route. Group BaV+SEC was inoculated with 50 µg of B. atrox venom diluted in 50 µl of PBS i.m. and 50 µl of MSC secretome injected in the animal’s tail vein. Group PBS+SEC received PBS i.m and secretome i.v. * = p ≤0.05 when compared to the control PBS-PBS.

Figure 9

Summarizes the physiological and pathological response in the inflammation-regeneration crossroad. Under physiological circumstances the response to an injury activate platelets, N1 neutrophils and M1 macrophages. Platelets not only promote coagulation to stop hemorrhage but release growth factors that, in cooperation with Angiogenic neutrophils and M2 macrophages, contribute t tissue regeneration through the mobilization of local progenitors, angiogenesis and ECM remodeling.