Development of a human umbilical cord-derived mesenchymal stromal cell-based advanced therapy medicinal product to treat immune and/or inflammatory diseases

Abstract

Background: Umbilical cord-derived mesenchymal stromal cells (UC-MSCs) revealed their key role in immune regulation, offering promising therapeutic perspectives for immune and inflammatory diseases. We aimed to develop a production process of an UC-MSC-based product and then to characterize UC-MSC properties and immunomodulatory activities in vitro, related to their clinical use and finally, to transfer this technology to a good manufacturing practice (GMP) compliant facility, to manufacture an advanced therapy medicinal product (ATMP).

Methods: Fifteen human umbilical cords (UCs) were collected to develop the production process. Three batches of UC-MSCs from a single donor were characterized at basal state and after in vitro pro-inflammatory stimulation by interferon-γ (IFNγ) and tumor necrosis factor-α (TNFα). Proliferation, immunophenotype, activation markers' expression and the inhibition of T cell proliferation were assessed. Finally, this technology was transferred to a GMP-compliant facility to manufacture an UC-MSC-based ATMP, from a single donor, using the explant method followed by the establishment of master and work cell stocks.

Results: Twelve UCs were processed successfully allowing to isolate UC-MSCs with doubling time and population doubling remaining stable until passage 4. CD90, CD105, CD73, CD44, CD29, CD166 expression was positive; CD14, CD45, CD31, HLA-DR, CD40, CD80 and CD86 expression was negative, while CD146 and HLA-ABC expression was heterogeneous. Cell morphology, proliferation and immunophenotype were not modified by inflammatory treatment. Indoleamine 2,3-dioxygenase (IDO) expression was significantly induced by IFNγ and IFNγ + TNFα versus non-treated cells. Intercellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) expression was induced significantly after priming. T cell proliferation was significantly decreased in the presence of UC-MSCs in a dose-dependent manner. This inhibitory effect was improved by IFNγ or IFNγ + TNFα, at UC-MSCs:PBMC ratio 1:10 and 1:30, whereas only IFNγ allowed to decrease significantly T cell proliferation at ratio 1:100. The manufacturing process of the UC-MSC-based ATMP was qualified and authorized by the French regulatory agency for clinical use (NCT04333368).

Conclusion: This work allowed to develop an investigational UC-MSC-based ATMP authorized for clinical use. Our results showed that an inflammatory environment preserves the biological properties of UC-MSCs with an improvement of their immunomodulatory functions.

Keywords: Advanced therapy medicinal product; Good manufacturing practice; Human umbilical cord; Immunomodulation; Inflammation; Mesenchymal stromal cells.

Conflict of interest statement

The authors declare that they have no competing interests.

© 2021. The Author(s).

Figures

Fig. 1

Development of UC-MSCs’ production process. A Characteristics of collected and processed UCs (n = 15 UCs). B UC-MSCs morphology at passage 1 (P1) at 4X (left) and 10X (right) magnifications. C UC-MSCs quantity at P0 and P1 (left) and DT (h) and PD calculated from P0 to P1 (right) for each UC (n = 12 UCs). D DT (h) and PD from P1 to P7 (n = 4 UCs) (left) and UC-MSCs morphology at P7 (right) representing senescent morphology (head arrows). E Comparison of DT (h) and PD between culture media composed of Nutristem® + PL5% and MEM-α + PL 5% at P3 and P4 (n = 4 UCs)

Fig. 2

UC-MSCs immunophenotype at basal state. A Expression of mesenchymal markers (CD90, CD105, CD73), adhesion molecules (CD44, CD29, CD166, CD146), hematopoietic (CD14, CD45) and endothelial (CD31) markers. B Expression of immunogenic (HLA-ABC, HLA-DR) and co-stimulatory (CD40, CD80, CD86) markers. C Summary of markers’ expression represented as mean ± standard deviation (%) (n = 3 batches from 1 UC)

Fig. 3

UC-MSCs’ characteristics after pro-inflammatory priming. A UC-MSCs’ morphology; B UC-MSCs’ size (µm); C DT (h) (left) and PD (right); and D UC-MSCs’ immunophenotype at basal state (NT) and after pro-inflammatory treatment by IFNγ, TNFα, IFNγ + TNFα, IL6, IL1β, GM-CSF and Mix. NA: Not applicable. n = 3 batches from 1 UC

Fig. 4

UC-MSCs’ biological activity in vitro after pro-inflammatory priming. A IDO expression (%) at basal state (NT) and after pro-inflammatory priming by IFNγ, TNFα, IFNγ + TNFα, IL1β, IL6, GM-CSF and Mix. B ICAM-1/CD54, PD-L1/CD274, VCAM-1/CD106, CD200, IFNγ-R/CD119, TNFα-RII/CD120b expression (%) at basal state (NT) and after pro-inflammatory priming by IFNγ, TNFα and IFNγ + TNFα. C T-lymphocyte proliferation (%) at ratio UC-MSCs:PBMC 0:1, 1:10, 1:30, 1:100, 1:300 and 1:1000 at basal state (NT) and after pro-inflammatory priming by IFNγ, TNFα and IFNγ + TNFα. */$p < 0.05, **/$$p < 0.01, ***/$$$p < 0.001, ****/$$$$p < 0.0001. n = 3 batches from 1 UC

Fig. 5

Qualification of the UC-MSC-based ATMP manufacturing process. A Quantities and proliferation data of UC-MSCs from P0 to P3. B Quality controls performed during MCS (n = 2 batches) and WCS steps (n = 3 batches)