Exploring the critical therapeutic window: Dose-frequency optimization of human umbilical cord mesenchymal stem cells for preclinical asthma treatment

Abstract

Background: Current drugs primarily target inflammation control but do not reverse tissue remodeling changes for asthma. Human mesenchymal stem cells are known for their anti-inflammatory and tissue remodeling capabilities. However, limited research has explored the therapeutic impact of varying doses and frequencies of human umbilical cord blood-derived mesenchymal stem cells (HUC-MSCs) on established airway remodeling in experimental asthma.

Aim: To explore and optimize the dosage and administration frequency of HUC-MSCs in experimental models of ovalbumin (OVA)-induced asthma.

Methods: BALB/c mice underwent sensitization and were challenged using OVA. Control animals were administered a saline solution following the same protocol. HUC-MSCs were identified using flow cytometry. HUC-MSCs at incremental dosages (1 × 10⁵, 2 × 10⁵, 4 × 10⁵) were injected via tail veins on day 30 (the second after the final stimulation). After comparing each group and determining the optimal dose, supplement the optimal dose twice on day 30 and day 33 (the second and fifth day after the final stimulation). Bronchoalveolar lavage fluid (BALF) and serum were harvested for analysis of concentrations of interleukin-4 (IL-4), IL-13, immunoglobulin E and interferon-gamma (IFN-γ) by enzyme-linked immunosorbent assay. Pharmacology of airways and lung functions were also evaluated to identify the optimal group.

Results: The study shows that HUC-MSC transplantation ameliorates OVA-induced asthma by significantly reducing airway inflammation and obstruction in preclinical models. This effect is associated with decreased Th2 cytokines IL-4 and IL-13, and increased Th1 cytokine IFN-γ. The optimal dose of 2 × 10⁵ cells/mouse was identified as the most effective in reducing local asthmatic airway inflammation and changing levels of IL-4, IL-13, and IFN-γ in serum and BALF compared to other single doses of HUC-MSC. Multiple treatments with the medium dose (2 × 10⁵ cells) of HUC-MSCs on days 30 and 33 yield the best pathological and lung function outcomes. However, double treatments do not reduce IL-4 and IL-13 expression or enhance IFN-γ production in serum or BALF more effectively than a single medium dose.

Conclusion: HUC-MSCs effectively regulate pro-inflammatory mediators in serum and BALF, modulating airway remodeling and lung function. In this acute mouse asthma model, a single dosage of 2 × 10⁵ is optimal, with more significant effects of decreasing airway obstruction requiring repeated administration.

Keywords: Asthma; Dose; Frequency; Human umbilical cord mesenchymal stem cells; Mouse.

Figure 1

Schematic flow diagram of anti-asthma experiment with different dosages of human umbilical cord blood-derived mesenchymal stem cell treatment. OVA: Ovalbumin; HUC-MSCs: Human umbilical cord blood-derived mesenchymal stem cells.

Figure 2

Immunophenotypic analysis of human umbilical cord blood-derived mesenchymal stem cell. A and B: Human umbilical cord blood-derived mesenchymal stem cell cells were incubated with specific antibodies against the cell surface antigens CD29, CD73, CD34, and CD45 by flow cytometry; C: Representative images of human umbilical cord blood-derived mesenchymal stem cells. HUC-MSC: Human umbilical cord blood-derived mesenchymal stem cell.

Figure 3

Gross lung photography of ovalbumin-induced asthmatic mice treated with different dosages of human umbilical cord blood-derived mesenchymal stem cell. A-E: Gross lung images of a group of control, model, and three different dosages of human umbilical cord blood-derived mesenchymal stem cells treatment. L-D HUC-MSC: Low dosage (1 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell; M-D HUC-MSC: Medium dosage (2 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell; H-D HUC-MSC: High dosage (4 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell.

Figure 4

Effect of human umbilical cord blood-derived mesenchymal stem cell on the levels of inflammatory cytokines. A-D: Interleukin-4 (IL-4) (A); IL-13 (B); interferon-gamma (C), and immunoglobulin E (D) in serum of mice with ovalbumin-induced asthma; E-G: IL-4 (E), IL-13 (F), interferon-gamma (G) in bronchoalveolar lavage fluid of mice with ovalbumin-induced asthma. ^aP < 0.05 indicate a significant difference from the control group; ^bP < 0.05 indicate a significant difference from the model group. Bonferroni correction was applied to evaluate cytokines in multiple treatment groups. IL: Interleukin; IFN: Interferon; IgE: Immunoglobulin E; BALF: Bronchoalveolar lavage fluid; L-D HUC-MSC: Low dosage (1 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell; M-D HUC-MSC: Medium dosage (2 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell; H-D HUC-MSC: High dosage (4 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell; M-2D HUC-MSC: Medium dosage (2 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell twice.

Figure 5

Pathological features of human umbilical cord blood-derived mesenchymal stem cell in attenuating airway inflammation in ovalbumin-induced asthmatic mice. A-F: Hematoxylin and eosin staining images of control, model, low dosage, medium dosage, high dosage, and two medium dosage human umbilical cord blood-derived mesenchymal stem cell treated lung tissue of mice. Hematoxylin and eosin staining showed that the control group displayed normal lung tissue architecture and cellular morphology, with no inflammatory cell infiltration. Conversely, the model group demonstrated pronounced inflammatory cell infiltration (indicated by black arrows), thickening of the alveolar walls (indicated by red arrows), sparse inflammatory cells (black arrows), desquamation of bronchial epithelial cells (indicated by blue arrows), and vascular congestion (indicated by green arrows) across the various dose groups. Notably, the group receiving a double medium dose of human umbilical cord blood-derived mesenchymal stem cell exhibited significant improvement in the condition of the alveolar walls compared to the model group (red arrows). L-D HUC-MSC: Low dosage (1 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell; M-D HUC-MSC: Medium dosage (2 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell; H-D HUC-MSC: High dosage (4 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell; M-2D HUC-MSC: Medium dosage (2 × 10⁵) of human umbilical cord blood-derived mesenchymal stem cell twice.

Figure 6

Effects of human umbilical cord blood-derived mesenchymal stem cells on the lung function of acute ovalbumin-induced asthmatic mice. A: Forced expiratory volume 0.1/forced vital capacity; B: Dynamic lung compliance; C: Airway resistance; D: Respiratory system elastic resistance; E: Forced vital capacity. ^aP < 0.05, ^bP < 0.01, ^cP < 0.001, ^dP < 0.0001. Bonferroni correction was applied to evaluate lung function metrics in multiple treatment groups. FEV: Forced expiratory volume; FVC: Forced vital capacity; Cdyn: Dynamic lung compliance; RL: Airway resistance; Re: Respiratory system elastic resistance.