Introduction: Studies have proposed that mesenchymal stem cells (MSCs) improve the hematopoietic engraftment in allogeneic or xenogeneic transplants and this is probably due to the MSCs’ immunosuppressive properties. Our study aimed to discern, for the first time, whether MSC infusion could facilitate the engraftment of hematopoietic stem cells (HSCs) in autologous transplantations models, where no immune rejection of donor HSCs is expected. Methods: Recipient mice (CD45.2) mice, conditioned with moderate doses of radiation (5-7 Gy), were transplanted with low numbers of HSCs (CD45.1/CD45.2) either as a sole population or co-infused with increasing numbers of adipose-derived-MSCs (Ad-MSCs). The influence of Ad-MSC infusion on the short-term and long-term engraftment of donor HSCs was investigated. Additionally, homing assays and studies related with the administration route and with the Ad-MSC/HSC interaction were conducted. Results: Our data show that the co-infusion of Ad-MSCs with low numbers of purified HSCs significantly improves the short-term and long-term hematopoietic reconstitution of recipients conditioned with moderate irradiation doses. This effect was Ad-MSC dose-dependent and associated with an increased homing of transplanted HSCs in recipients’ bone marrow. In vivo and in vitro experiments also indicate that the Ad-MSC effects observed in this autologous transplant model are not due to paracrine effects but rather are related to Ad-MSC and HSC interactions, allowing us to propose that Ad-MSCs may act as HSC carriers, facilitating the migration and homing of the HSCs to recipient bone marrow niches. Conclusion: Our results demonstrate that Ad-MSCs facilitate the engraftment of purified HSCs in an autologous mouse transplantation model, opening new perspectives in the application of Ad-MSCs in autologous transplants, including HSC gene therapy.

Background and Aims: Mesenchymal stem cells [MSCs] are used in preclinical and clinical studies for treatment of immune-mediated disorders, thanks to their immunomodulatory properties. Cell therapy with MSCs induces multiple effects in the immune system which ultimately lead to increase in the number of immune cells with regulatory phenotype. In this study, we investigated whether the beneficial effects of MSC therapy are maintained in the long term in a clinically relevant mouse model of colitis. Methods: A single dose of adipose-derived MSCs [aMSCs] was infused into dextran sulphate sodium [DSS]-induced colitic mice during the induction phase of the disease. Following a latency period of 12 weeks, mice were re-challenged with a second 7-day cycle of DSS. Results: DSS-induced colitic mice treated with aMSCs showed significant reduction in their colitic disease activity index during the second DSS challenge when compared with non-aMSC treated DSS-induced colitic mice. Strikingly, the long-term protection induced by aMSC therapy was also observed in Rag-1-/- mice where no adaptive immune memory cell responses take place. Increased percentages of Ly6G+CD11b+ myeloid cells were observed 12 weeks after the first inflammatory challenge in the peritoneal cavity, spleen, and bone marrow of DSS-induced colitic mice that were infused with aMSCs. Interestingly, upon re-challenge with DSS, these animals showed a concomitant increase in the regulatory/inflammatory macrophage ratio in the colon lamina propria. Conclusions: Our findings demonstrate for the first time that MSC therapy can imprint an innate immune memory-like response in mice which confers sustained protection against acute inflammation in the long term.

Bone marrow transplantation in mice is performed by intravenous administration of haematopoietic repopulating cells, usually via the lateral tail vein. This technique can be technically challenging to carry out and may cause distress to the mice. The retro-orbital sinus is a large area where there is a confluence of several vessels that provides an alternative route for intravenous access. Retro-orbital injection, although aesthetically unpleasant, can be performed rapidly without requiring mechanical restriction or heat-induced vasodilation. In addition, this technique can be easily learned by novice manipulators. This route of administration has been reported for use in bone marrow transplantation but there is no comparison of retro-orbital and tail vein injections reported for this specific purpose, although both routes have been compared for many other applications. Here, we provide for the first time a comprehensive comparison between tail vein and retro-orbital injections for two different bone marrow transplant scenarios in P3B and B6D2F1 mice. In both cases, no significant differences regarding donor engraftment were observed between mice transplanted using each of the techniques. Haematological counts and leukocyte subpopulation distribution were practically identical between both animal groups. Moreover, donor engraftment levels were less homogenous when cells were transplanted by tail vein injection, probably due to a higher risk of failure associated with this technique. All these data suggest that retro-orbital injection is a compelling alternative to conventional tail vein injection for bone marrow transplant in mice, providing similar and more homogenous haematopoietic reconstitution.

In this study we propose a novel approach based on the use of mesenchymal stromal cells (MSCs), aiming at limiting risks of graft failure in gene therapy protocols associated with low conditioning regimens. Because the engraftment of corrected hematopoietic stem cells (HSCs) is particularly challenging in Fanconi anemia (FA), we have investigated the relevance of MSCs in an experimental model of FA gene therapy. Our results showed, first, that risks of graft failure in recipients conditioned with a moderate dose of 5 Gy and infused with limited numbers of wild-type HSCs are significantly higher in Fanca^−/− recipients as compared with wild-type recipients. However, when wild-type HSC numbers inducing 30–50% of graft failures in Fanca^−/− recipients were coinfused with MSCs, no graft failures were observed. Moreover, graft failures associated with the infusion of low numbers of gene-corrected Fanca^−/− HSCs were also significantly overcome by MSC coinfusion. Our study shows for the first time that MSC coinfusion constitutes a simple and nontoxic approach to minimize risks of graft failure in gene therapy applications associated with low conditioning regimens and infusion of limited numbers of corrected HSCs.

The in vivo fate of mesenchymal stromal cells (MSCs), including their clearance, interaction with host tissues, and persistence, remains incompletely understood following systemic or local clinical administration to patients. Although immune-mediated clearance mechanisms, such as triggering of the instant blood-mediated inflammatory reaction, activation of coagulation and complement pathways, apoptosis and efferocytosis have been identified, their contributions to MSC function and efficacy are still under investigation. To address these knowledge gaps, an international panel of experts in MSC biology and clinical regenerative medicine convened to assess current evidence and define key unanswered questions. Discussions were structured around three thematic domains: (i) biodistribution and mechanisms of action following systemic delivery; (ii) biological implications of local or depot-based administration and (iii) the dynamics of MSC persistence and clearance in vivo. A major focus was on the role of MSC apoptosis and its immunological consequences, particularly interactions between apoptotic MSCs, phagocytes and endothelial barriers. This perspective highlights the most urgent research questions identified during the meeting and in follow-up discussions and proposes experimental strategies to move beyond traditional cell tracking toward interrogating functional persistence, immune modulation and delivery context. Addressing these gaps will deepen our understanding of MSC behavior in vivo and guide the development of safer, more predictable and more effective MSC-based interventions.