Human artificial chromosome carrying R-spondin1 and IL-22 expression cassettes in rejuvenated MSCs enhances therapeutic efficacy in ulcerative colitis model mice

Ulcerative colitis (UC) is an incurable intestinal disease, with current treatments mainly focused on inflammation control and, in severe cases, surgical resection. Recent studies have highlighted the need for new therapies that promote tissue regeneration. R-spondin-1 (RSPO1) and interleukin-22 (IL-22) have shown anti-inflammatory and regenerative effects in UC models, but have short half-lives and poor targeting abilities. Another therapeutic tool, mesenchymal stem cells (MSCs), offer promising migratory and homing capabilities; however, the preparation of homogeneous therapeutic MSCs at sufficient levels in vitro is challenging. Therefore, we developed a novel line of MSCs (HAC-MSC) with significant therapeutic effects in dextran sodium sulfate (DSS)-induced colitis mice. Construction of HAC-MSC involved a two-part strategy: 1) establishment of a non-integrating human artificial chromosome (HAC) vector carrying therapeutic genes encoding IL22 and RSPO1; and 2) transfer of the HAC to previously characterized rejuvenated MSCs (rej-MSCs) prepared using Sendai virus technology for prolonged proliferation capacity in vitro. HAC-MSC stably and efficiently produced therapeutic factors in vitro and, following intraperitoneal administration to DSS-induced colitis mice, showed continuous expression of the therapeutic factors over 5 days. Additionally, HAC-MSC-treated mice showed alleviation of the disease activity index score, reduced depth of injury, and promotion of intestinal growth compared with MSC-treated mice. Furthermore, effective treatment with HAC-MSC required only a fraction (1 %-10 %) of the number of cells needed for conventional MSC therapy. These findings highlight the outstanding potential of rej-MSCs carrying therapeutic factor-loaded HACs as a cell therapy tool with prospective applications in the treatment of UC and other diseases.