Background and ObjectivesThere is an unmet need for neuroregenerative therapies in multiple sclerosis (MS). Mesenchymal stem cells (MSCs) have shown immunomodulatory and regenerative effects in preclinical models and early clinical studies. Intrathecal administration may enhance therapeutic potential by direct delivery to the CNS. However, randomized, placebo-controlled trials are needed to establish safety and efficacy. The objective of this trial was to assess whether a single intrathecal administration of autologous MSCs could provide evidence of a neuroregenerative effect in progressive MS.MethodsIn this randomized, double-blind, placebo-controlled phase I/II trial (NCT04749667), patients with progressive MS enrolled at 4 Norwegian tertiary hospitals received a single intrathecal injection of autologous bone marrow-derived MSCs (1 & times; 106 cells/kg) in a crossover design. The primary end point was the change in latency of combined evoked potentials at 6 months. Secondary end points included safety, brain MRI measures, functional and ophthalmologic assessments, and serum biomarkers at 6 and 12 months. Exploratory analyses comprised proteomic profiling of CSF. Outcomes were analyzed using baseline-adjusted regression models.ResultsA total of 18 patients were included (mean age 46.7 years; 55.6% female). No significant between-group difference was observed for the primary end point (beta = -0.31, 95% CI -1.84 to 1.22, p = 0.668). At 6 months, patients in the MSC group showed reduced cerebral atrophy on MRI (beta = 9.37, 95% CI 0.29 to 18.45, p = 0.044) and lower serum glial fibrillary acidic protein levels (beta = -16.3 pg/mL, 95% CI -33.0 to 0.3, p = 0.054), but neither were sustained at 12 months. Exploratory CSF proteomics revealed reductions in multiple inflammation-related proteins at 6 months. One serious adverse event was deemed probably related to MSC treatment. Common adverse events included fever (n = 9) and low back pain (n = 10) after MSC administration, and spinal MRI abnormalities with fluid loculations and nerve root clumping (n = 7) at 6 months. One patient developed chronic coccygeal pain attributed to arachnoiditis.DiscussionA neuroregenerative effect was not detected, although interpretation may be limited by the small sample size. Adverse events suggest an acute localized inflammatory reaction after MSC administration. Our findings suggest that intrathecal administration of MSCs in progressive MS should be approached with caution in future studies.Classification of EvidenceThis study provides Class III evidence that, in patients with progressive multiple sclerosis, treatment with a single intrathecal administration of autologous mesenchymal stem cells does not provide neuroregenerative effect, as assessed by a composite evoked potential score.Trial registrationClinicalTrials.gov (NCT04749667); registered February 8, 2021; first patient enrolled August 9, 2021.

BACKGROUND AND OBJECTIVES:There is an unmet need for neuroregenerative therapies in multiple sclerosis (MS). Mesenchymal stem cells (MSCs) have shown immunomodulatory and regenerative effects in preclinical models and early clinical studies. Intrathecal administration may enhance therapeutic potential by direct delivery to the CNS. However, randomized, placebo-controlled trials are needed to establish safety and efficacy. The objective of this trial was to assess whether a single intrathecal administration of autologous MSCs could provide evidence of a neuroregenerative effect in progressive MS. METHODS:In this randomized, double-blind, placebo-controlled phase I/II trial (NCT04749667), patients with progressive MS enrolled at 4 Norwegian tertiary hospitals received a single intrathecal injection of autologous bone marrow-derived MSCs (1 × 106 cells/kg) in a crossover design. The primary end point was the change in latency of combined evoked potentials at 6 months. Secondary end points included safety, brain MRI measures, functional and ophthalmologic assessments, and serum biomarkers at 6 and 12 months. Exploratory analyses comprised proteomic profiling of CSF. Outcomes were analyzed using baseline-adjusted regression models. RESULTS:A total of 18 patients were included (mean age 46.7 years; 55.6% female). No significant between-group difference was observed for the primary end point (β = -0.31, 95% CI -1.84 to 1.22, p = 0.668). At 6 months, patients in the MSC group showed reduced cerebral atrophy on MRI (β = 9.37, 95% CI 0.29 to 18.45, p = 0.044) and lower serum glial fibrillary acidic protein levels (β = -16.3 pg/mL, 95% CI -33.0 to 0.3, p = 0.054), but neither were sustained at 12 months. Exploratory CSF proteomics revealed reductions in multiple inflammation-related proteins at 6 months. One serious adverse event was deemed probably related to MSC treatment. Common adverse events included fever (n = 9) and low back pain (n = 10) after MSC administration, and spinal MRI abnormalities with fluid loculations and nerve root clumping (n = 7) at 6 months. One patient developed chronic coccygeal pain attributed to arachnoiditis. DISCUSSION:A neuroregenerative effect was not detected, although interpretation may be limited by the small sample size. Adverse events suggest an acute localized inflammatory reaction after MSC administration. Our findings suggest that intrathecal administration of MSCs in progressive MS should be approached with caution in future studies. CLASSIFICATION OF EVIDENCE:This study provides Class III evidence that, in patients with progressive multiple sclerosis, treatment with a single intrathecal administration of autologous mesenchymal stem cells does not provide neuroregenerative effect, as assessed by a composite evoked potential score. TRIAL REGISTRATION:ClinicalTrials.gov (NCT04749667); registered February 8, 2021; first patient enrolled August 9, 2021.

Therapeutic options for progressive multiple sclerosis (MS) remain limited, and the biological mechanisms engaged by intrathecal mesenchymal stem cell (MSC) therapy are incompletely understood. MSCs are proposed to exert immunomodulatory and trophic effects, yet most studies rely on targeted biomarkers and lack systems-level analysis across immune compartments. Here, we applied an integrated multi-omics framework to characterize immune and cerebrospinal fluid (CSF) responses to MSC therapy in patients with progressive MS enrolled in the SMART-MS trial, a randomized, placebo-controlled crossover study of a single intrathecal MSC injection. Although the clinical trial did not demonstrate a clear neuro-regenerative signal as the primary endpoint, exploratory MRI findings and adverse events (e.g., fever, back pain) suggested localized biological responses following intrathecal administration. Longitudinal peripheral blood mass cytometry and matched CSF proteomics were analysed from 18 participants sampled at baseline, 6 months, and 12 months. Immune phenotypes and composite functional program scores were quantified using mixed-effects modelling. To synthesize CSF proteomic changes into biologically interpretable patterns, we summarized protein-level responses into composite CSF functional programs reflecting extracellular matrix and CNS interface remodelling, innate and vascular inflammatory stress, metabolic and cytoskeletal adaptation, and biological reactivity. These program-level scores enabled structured cross-compartment integration with circulating immune programs. MSC exposure did not broadly alter peripheral immune composition or functional programs across circulating lymphocyte and monocyte populations. Instead, the dominant signal emerged within the CSF proteome, where treatment was associated with coordinated extracellular matrix and CNS interface remodelling alongside attenuation of acute-phase inflammatory pathways in many individuals. Elevated inflammatory and metabolic signatures were largely confined to patients with clinical adverse events or spinal MRI reactivity, consistent with amplified biological responsiveness rather than distinct MSC-specific mechanisms. Cross-compartment analyses revealed weak and heterogeneous coupling between circulating immune programs and CSF remodelling, supporting predominantly compartmentalized intrathecal effects. Together, these findings suggest that intrathecal MSC therapy in progressive MS is associated with selective remodelling at the immune–CNS interface rather than broad systemic immunosuppression and demonstrate the value of integrated multi-omics approaches for dissecting treatment-associated biology in neuroinflammatory disease.

Sepsis is a life-threatening condition with cardiac complications being an independent predictor of poor outcome. Although their mechanisms have been widely investigated, therapeutic options remain limited. One promising therapeutic tool are mesenchymal stromal cells (MSCs). The aim of this study is to investigate the immunomodulatory effects of human MSCs from two different sources (bone marrow/BMMSC and adipose tissue/ASC) and to evaluate their cardioprotective potential. 60 adult male C57BL/6 mice were divided into sham, sepsis (cecal ligation puncture (CLP)) and two i.v. treatment groups CLP + human BMMSC and CLP + human ASC with 5 animals in each group. The observation periods were 8, 24 and 72 h. Left ventricular tissue was analyzed histologically, by qPCR (C3ar, C5ar1, Il-1b, Il-6, Il-10, Tlr2, Tlr4, Tnfa, and Nlrp3) and western blot. Cardiac damage markers troponin I and heart fatty acid binding protein (HFABP) were detected in serum by ELISA. Troponin I and HFABP were significantly increased in CLP group after 8 h compared to sham. In cardiac tissue the expression of C3ar, C5ar1, Il-1b, Il-6, Il-10, Tlr2, Tlr4, Tnfa and Nlrp3 inflammasome was upregulated up to 24h after CLP compared to sham. After BMMSC treatment, C3ar as well as C5ar, Tlr2 and Il-10 mRNA expression in left ventricle was downregulated compared to CLP, whereas ASC treatment was associated with the downregulation of Il-6 and Nlrp3. CLP-induced polymicrobial sepsis in mice was associated with cardiac damage and increased inflammation in left ventricular tissue. Therapeutic systemic application of human BMMSC and ASC ameliorated damage and inflammation in the heart.

Objectives To assess the efficacy and safety of a cell-based therapy for 3D bone augmentation of severe alveolar bone defects prior to dental implant placement.Materials and Methods A Phase 2 randomized controlled clinical trial evaluated the safety and efficacy of a cell therapy using expanded autologous iliac crest-derived mesenchymal cells seeded on a synthetic bioabsorbable bone substitute covered with a non-resorbable membrane. The control group received an autogenous bone block graft. After 5 months, CBCT scans were compared to measure the bone volume changes achieved after the regenerative surgery. Subsequently, dental implants were placed in the regenerated areas.Results A total of 48 patients were included and randomized (36 patients in the test group and 12 in the control group). However, seven patients did not reach the minimum required number of expanded MSCs and were therefore unable to be treated. The tested intervention demonstrated significantly greater gains in bone volume, with a mean difference of 480.01 mm3 (p = 0.032). Similarly, the mean change in bone crest volume from baseline to 5 months was notably higher in the test group (1066.91 mm3) compared to the control group (586.9 mm3). Adverse reactions and patient morbidity were minor in both groups. Implants were placed on the regenerated bone, and all were integrated successfully in both groups.Conclusions The cell-based therapy resulted in significant changes in bone volume compared to the control treatment, enabling dental implants in all patients. The procedure was associated with minimal adverse effects and patient morbidity.Trial Registration ClinicalTrials.gov: NCT03373052, NCT04297813

OBJECTIVES:This study aims to evaluate the efficacy of intra-articular autologous adipose-derived ex vivo expanded mesenchymal stromal cells (ADSC) on patient-reported outcome (pain and function) in symptomatic mild-to-moderate tibiofemoral knee osteoarthritis (OA). METHODS:Participants were randomised in a prospective, double-blind, controlled phase 2b study to receive 1 single intra-articular injection in 3 separate groups: ADSC low dose 2 × 106 cells; ADSC high dose 10 × 106 cells; or saline placebo. The primary outcome was the rate of OsteoArthritis Research Society International (OARSI)/Outcome MEasures in Rheumatology (OMERACT) 'strict responders' defined by improvements from baseline reported to 0 to 100 mm scale in Western Ontario and McMaster University Osteoarthritis index (WOMAC) pain or physical function subscores ≥50% with minimal absolute changes ≥20 mm at 6 months. Secondary outcomes included an evaluation of WOMAC pain, WOMAC function, quality of life (36-Item Short Form Health Survey), WOMAC and Knee injury and OA Outcome Score global change, magnetic resonance imaging cartilage thickness changes and safety from baseline to 12-month follow-up. RESULTS:One hundred thirty-five patients were randomised from September 2016 to March 2022 (mean age, 58.3 [SD, 6.5] years; 58 [43%] women), 99 (73%) received allocated intervention, and 97 were analysed. After 6 months, treatment with ADSC versus placebo injection, 26 patients (47.3%) versus 23 patients (54.8%) were strict OARSI/OMERACT responders (relative risk 0.86 [95% CI: 0.58-1.28]; P = .46). Individuals showed different patterns in cartilage thickness change between timepoints across tibiofemoral articular surfaces, but overall secondary outcomes showed no significant-group differences. CONCLUSIONS:Among patients with symptomatic mild-to-moderate knee OA, a single intra-articular injection of 2 × 106 or 10 × 106 autologous ADSC compared with injection of saline placebo did not significantly improve pain and function. TRIAL REGISTRATION NUMBER:ClinicalTrials.gov Identifier: NCT02838069, https://clinicaltrials.gov/study/NCT02838069/EudraCT number: 2015-002125-19, https://www.clinicaltrialsregister.eu/ctr-search/search?query=2015-002125-19.

The therapeutic efficacy of mesenchymal stromal cells (MSCs) has been shown to rely on their immunomodulatory and regenerative properties. In order to obtain sufficient numbers of cells for clinical applications, MSCs have to be expanded ex vivo. Expansion media with xenogeneic-free (XF) growth-promoting supplements like human platelet lysate (PL) or serum- and xenogeneic-free (SF/XF) formulations have been established as safe and efficient, and both groups provide different beneficial qualities. In this study, MSCs were expanded in XF or SF/XF media as well as in mixtures thereof. MSCs cultured in these media were analyzed for phenotypic and functional properties. MSC expansion was optimal with SF/XF conditions when PL was present. Metabolic patterns, consumption of growth factors, and secretome of MSCs differed depending on the type and concentration of supplement. The lactate per glucose yield increased along with a higher proportion of PL. Many factors in the supernatant of cultured MSCs showed distinct patterns depending on the supplement (e.g., FGF-2, TGFβ, and insulin only in PL-expanded MSC, and leptin, sCD40L PDGF-AA only in SF/XF-expanded MSC). This also resulted in changes in cell characteristics like migratory potential. These findings support current approaches where growth media may be utilized for priming MSCs for specific therapeutic applications.

Mesenchymal stromal cells (MSCs) are promising therapeutic candidates in a variety of diseases due to having immunomodulatory and pro-regenerative properties. In recent years, MSC-derived small extracellular vesicles (sEVs) have attracted increasing interest as a possible alternative to conventional cell therapy. However, translational processes of sEVs for clinical applications are still impeded by inconsistencies regarding isolation procedures and culture conditions. We systematically compared different methods for sEV isolation from conditioned media of ex vivo expanded bone marrow-derived MSCs and demonstrated considerable variability of quantity, purity, and characteristics of sEV preparations obtained by these methods. The combination of cross flow filtration with ultracentrifugation for sEV isolation resulted in sEVs with similar properties as compared to isolation by differential centrifugation combined with ultracentrifugation, the latter is still considered as gold standard for sEV isolation. In contrast, sEV isolation by a combination of precipitation with polyethylene glycol and ultracentrifugation as well as cross flow filtration and size exclusion chromatography resulted in sEVs with different characteristics, as shown by surface antigen expression patterns. The MSC culture requires a growth-promoting supplement, such as platelet lysate, which contains sEVs itself. We demonstrated that MSC culture with EV-depleted platelet lysate does not alter MSC characteristics, and conditioned media of such MSC cultures provide sEV preparations enriched for MSC-derived sEVs. The results from the systematic stepwise evaluation of various aspects were combined with culture of MSCs in a hollow fiber bioreactor. This resulted in a strategy using cross flow filtration with subsequent ultracentrifugation for sEV isolation. In conclusion, this workflow provides a semi-automated, efficient, large-scale-applicable, and good manufacturing practice (GMP)-grade approach for the generation of sEVs for clinical use. The use of EV-depleted platelet lysate is an option to further increase the purity of MSC-derived sEVs.

<p>Supplementary-Figure 2 Characterization of the affected stromal population after PTH/ZA treatment (A-B) Using flow cytometry, potential markers of mesenchymal stromal cells were evaluated in bone marrow of PTH/ZA-treated mice. Gating was applied after the exclusion of hematopoietic and erythroid cells (CD45- Ter119-). (B) PTH/ZA diminished the size of populations expressing the following surface markers: Sca1+, CD29+, CD31+, CD44-, αSMA+, leptin receptor+, and CD146+; n=15/15 in 3 experiments.</p>

<p>Supplementary-Figure 3 Correlations between MSCs, CFU-f and homing (A) The percentage of detected MSCs in vivo in control mice and after PTH/ZA treatment correlates negatively with the number of detected tumor cells in the bone marrow, n=40, Pearson''s correlation r=-0.59. (B) The percentage of detected MSCs in vivo in control mice and after PTH/ZA treatment correlates positively with the number of CFU-fs in vitro. (n=27; Pearson correlation r=0.57). (C) In the absence of tumor cells, the combination of PTH/ZA diminishes the MSC subpopulation. Please compare to Fig. 1G obtained in the presence of cancer cells, n=9/12/9/9 in three experiments. Apoptosis after PTH/ZA treatment (D) Apoptosis in CD45- Ter119- CD31+ cells increased after treatment with PTH/ZA and ZA alone, whereas apoptosis in CD45- Ter119- CD146+ cells only increased in the PTH/ZA-treated group, n=10/12/7/7 in 3 experiments.</p>

<p>Supplementary-Figure 2 Characterization of the affected stromal population after PTH/ZA treatment (A-B) Using flow cytometry, potential markers of mesenchymal stromal cells were evaluated in bone marrow of PTH/ZA-treated mice. Gating was applied after the exclusion of hematopoietic and erythroid cells (CD45- Ter119-). (B) PTH/ZA diminished the size of populations expressing the following surface markers: Sca1+, CD29+, CD31+, CD44-, αSMA+, leptin receptor+, and CD146+; n=15/15 in 3 experiments.</p>

Supplementary-Figure 5 Flow cytometry of MSC markers in human bone marrow biopsies Bone marrow aspirates from patients with prostate cancer (A) and breast cancer (B) who had an evaluation of their bone marrows for the presence of cytokeratin-stained cells to detect disseminated cancer cells (DTCs) were stained for the various MSC markers and analyzed by flow cytometry. The expression of the following markers was not different between cytokeratin negative or positive biopsies: MSCA1, Stro1, CD146, FAP, CD44, CD144, CD73, CD349, CD56 and CD140b in prostate cancer biopsies, n=15/21 (A) and MSCA1, Stro1, CD146, FAP, CD44, CD73 and CD349 in breast cancer biopsies, n=30/30 (B). (C) To exclude any effect of neoadjuvant chemotherapy on the MSC population, the breast cancer patient samples were divided into samples from patients with or without neoadjuvant chemotherapy. Both groups showed an increase in the MSC subpopulation in samples without evidence of cytokeratin-stained cells in bone marrow, n= 10/11/20/19.

Supplementary-Figure 1 Tumor cell infiltration of other organs after PTH/ZA treatment (A) The combined treatment with PTH/ZA does not affect the homing of MDA tumor cells to other organs; n=14/15 in 2 experiments. Evaluation of blood vessel numbers and binding of cancer cells to vascular cells after PTH/ZA treatment (B) PTH/ZA did not affect blood vessel numbers in the treated mice. Proximal tibiae from 4 mice/group were evaluated and the number of blood vessels adjusted to the evaluated area. (C) Binding of cancer cells to immune cells (CD45+) or vascular cells (CD31 or PLVAP) was not affected by treatment with PTH/ZA, n=13/11. Bone marrow from control and treated mice that received cancer cells 24 hours prior to bone marrow isolation was subjected to addition of magnetic beads precoated with CD45 antibody and carefully removed. Addition of either CD31 or PLVAP antibody coated beads followed and beads were used for DNA isolation and evaluation of cancer cell presence. The amount of cancer cell specific DNA was corrected to murine -actin. PTH/ZA treatment does not affect colony forming units of hematopoietic populations (D) There were no changes in various progenitor colonies when bone marrow from treated mice was cultured in vitro for 2 weeks in specialized media, stained with crystal violet and counted; n=6/6/6/6 in 3 experiments. CFU-GEMM is shown in Fig. 1C. Examples of the various colonies are shown below the graphs (light microscopy, 40x).

Human multipotent mesenchymal stromal cells (hMSCs) are of significant therapeutic interest due to their ability to deliver oncolytic adenoviruses to tumors. This approach is also investigated for targeting head and neck squamous cell carcinomas (HNSCCs). HAdV-5-HexPos3, a recently reported capsid-modified vector based on human adenovirus type 5 (HAdV-5), showed strongly improved infection of both hMSCs and the HNSCC cell line UM-SCC-11B. Given that, we generated life cycle-unmodified and -modified replication-competent HAdV-5-HexPos3 vector variants and analyzed their replication within bone marrow- and adipose tissue-derived hMSCs. Efficient replication was detected for both life cycle-unmodified and -modified vectors. Moreover, we analyzed the migration of vector-carrying hMSCs toward different HNSCCs. Although migration of hMSCs to HNSCC cell lines was confirmed in vitro, no homing of hMSCs to HNSCC xenografts was observed in vivo in mice and in ovo in a chorioallantoic membrane model. Taken together, our data suggest that HAdV-5-HexPos3 is a potent candidate for hMSC-based oncolytic therapy of HNSCCs. However, it also emphasizes the importance of generating optimized in vivo models for the evaluation of hMSC as carrier cells.

Supplementary-Figure 4 Absolute numbers of HSPCs and MSCs after G-CSF and G-CSF+PTH/ZA treatment and in Mx 1fl/fl knockout mice (A) The absolute number of HSPCs per femur was lower after treatment with G-CSF, whereas the number of MSCs was higher, n=12/14 in 3 experiments. (B) Addition of PTH/ZA to G-CSF did not affect HSPCs further, but reduced the absolute number of MSCs, n=14/14 in 3 experiments. (C) Conditional deletion of integrin 1 (Mx 1fl/fl) in the bone marrow had no effect on HSPCs, but led to increased absolute numbers of MSCs, n=6/9 in 2 experiments. Expression of MSC markers on low-passage human MSCs (D) In order to evaluate potential MSC markers, adherent human bone marrow cells (passages 0 or 1) were dissociated from the plates and stained for flow cytometry analysis. Shown is a graph summarizing the findings. In addition, in each panel the unstained control is shown on the left and the bone marrow staining on the right (E). The cells were 79% positive for CD31, 91% positive for CD44, 22% positive for CD56, 95% positive for CD73, 59% positive for CD140b, 61% positive for CD144, 72% positive for CD146, 87% positive for CD271, 87% positive for CD349, 90% positive for FAP, 90% positive for MSCA1, and 92% positive for Stro1; n=4 different aspirates.

<p>Supplementary-Table 1A. The markers for murine mesenchymal stromal cells were selected based on literature. Supplementary-Table 1B. Model summary of stepwise multiple-regression analysis of the relation of the selected murine MSC markers and homing of tumor cells; *p<0.05; ***p<0.001 (n=48). Supplementary-Table 2A. The markers for human mesenchymal stromal cells were selected based on literature. Supplementary-Table 2B. Model summary of stepwise or enter multiple-regression analysis of proposed human MSC markers and cytokeratin staining in bone marrow aspirates from patients with prostate (PC: n=36) and breast cancer (BC: n=60); *p<0.05, **p<0.01, ***p<0.001. Supplementary-Table 3A. Clinical characteristics of prostate cancer patients (n=36) at the time of diagnosis. Numbers (percentages) of affected patients are provided. Supplementary-Table 3B. Clinical characteristics of breast cancer patients (n=60) at the time of surgery. The cohort was followed for a median of 21.4 months (Range 1.9-40.0 months). T: tumor size; N: nodal status; HR: hazard ratio; CI: confidence interval; ref.: reference. Significant p-values are bolded.</p>

Supplementary-Figure 1. Tumor cell infiltration of other organs after PTH/ZA treatment. Evaluation of blood vessel numbers and binding of cancer cells to vascular cells after PTH/ZA treatment. PTH/ZA treatment does not affect colony forming units of hematopoietic populations. Supplementary-Figure 2. Characterization of the affected stromal population after PTH/ZA treatment. Supplementary-Figure 3. Correlations between MSCs, CFU-f and homing. Apoptosis after PTH/ZA treatment. Supplementary-Figure 4. Absolute numbers of HSPCs and MSCs after G-CSF and G-CSF PTH/ZA treatment and in Mx 1fl/fl knockout mice. Expression of MSC markers on low-passage human MSCs. Supplementary-Figure 5. Flow cytometry of MSC markers in human bone marrow biopsies.