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.

Purpose: To assess the safety and feasibility of intravitreal injection of autologous CD34+ bone marrow stem cells (BMSCs) in eyes with vision loss from central retinal vein occlusion (CRVO). Design: Phase I/II single-center, prospective, randomized, sham-controlled, double-masked study. Participants: Participants with CRVO of 6 to 42 months duration, best-corrected visual acuity (VA) of 20/40 to 20/400, and no concurrent retinopathy or optic neuropathy contributing to vision loss in the study eye. The exclusion criteria include any concurrent systemic condition that would alter bone marrow components. Methods: Participants were randomized to immediate cell injection followed by sham injection at month 6 or immediate sham injection followed by cell injection at month 6. Cell injection consisted of a bone marrow aspiration and intravitreal injection of autologous CD34+ BMSCs. CD34+ BMSCs were isolated from the mononuclear cell fraction of bone marrow using Miltenyi CliniMACS system under current good manufacturing practices. Isolated cells were released for intravitreal injection if they passed the release criteria for quantity, sterility, and viability accepted by the US Food and Drug Administration. Sham injection consisted of a sham bone marrow aspiration and intravitreal injection without entering bone or eye. Eye examination, microperimetry, fundus photography, fluorescein angiography, electroretinography, OCT, and OCT angiography were performed at baseline and during study follow-up of 12 months. Main Outcome Measures: Adverse events (AEs) associated with study treatment, number of CD34+ BMSCs injected intravitreally. Results: Sixteen participants (16 eyes) were randomized to 1 of 2 study groups. All received intravitreal injection of autologous CD34+ BMSCs (mean 4.3 million cells) and completed the study follow-up. Rubeosis with vitreous hemorrhage occurred in 1 study eye, <1 month after sham injection and 7 months after cell injection, attributed to normal progression of CRVO. There were no other serious ocular AEs. The most common AE related to the study cell injection was new floaters (15/16, 93%). Other ocular AEs were similarly noted after sham injection. No eye had persistent VA loss of ≥15 letters after cell injection. Conclusions: Intravitreal injection of autologous CD34+ BMSCs appears well-tolerated and feasible in eyes with vision loss from CRVO. Financial Disclosure(s): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.

Growth factor proteins are essential reagents for cell culture and tissue engineering but require quality control for bioactivity. Currently, growth factor bioactivity measurements made by immunoblot or ELISA are limited in evaluating the kinetics and heterogeneity of cellular responses, and there is a need for convenient methods with higher temporal and spatial resolution. In this study, we evaluate the advantages of using genetically encoded biosensors to quantify growth factor bioactivity in living cells. Using fibroblasts expressing a FRET-based biosensor of ERK activity, we compare methods for quantifying the cellular response across several doses and sources of recombinant basic fibroblast growth factor (bFGF). In contrast to immunological methods, the biosensor-based approach provides single-cell ERK activity kinetics and robust dose-response curves with minimal experimental processing. We additionally demonstrate that this method can assess bFGF activity in induced pluripotent stem cells and resolve spatial activity patterns. We conclude that biosensors represent a rapid, high-quality bioactivity assay extendable to other growth factors and signaling pathways.

Background aims: Despite promising results in pre-clinical studies, mesenchymal stromal cells (MSCs) face significant challenges in clinical translation. A scoping review by our group highlighted two key issues contributing to this gap: (i) lack of a clear and consensus definition for MSCs and (ii) under-reporting of critical parameters in MSC clinical studies. To address these issues, we conducted a modified Delphi study to establish and implement a consensus definition for MSCs and develop reporting guidelines for MSC clinical studies. Methods: A steering committee of 22 international experts, including stakeholders from different MSC research fields, participated in the three Delphi rounds. For the first round, to obtain a broad perspective, additional investigators recommended by the steering committee were invited to participate. The first two rounds consisted of online surveys, whereas the third round took the form of a virtual meeting. Participants were asked to rate a series of potential defining characteristics of MSCs and items for reporting guidelines. Consensus was defined as at least 80% of the participants rating the item in the same category of importance. Results: Eighty-seven international participants participated in the first round survey (spring 2023), 17 participants participated in the second online survey (fall 2023) and 15 participants participated in the final virtual consensus meeting (January 2024). For the MSC definition, 20 items were considered and nine reached consensus. Items included terminology (one item), cell marker expression (five items), tissue origin (one item), stemness (one item) and description of critical quality attributes (one item). For the reporting guidelines, with the 28 initial items and the additional items suggested during round 1, a total of 33 items to report were included. This included items on MSC intervention group and control (e.g., MSC product, dose and administration), MSC characteristics (e.g., MSC provenance, "fitness," viability and immune compatibility) and MSC culture conditions (e.g., oxygen environment, culture medium and use of serum). Conclusions: By applying a Delphi method to establish a consensus definition for MSCs and reporting guidelines for MSC-based clinical trials, this work represents a significant advance in improving transparency and reproducibility in the conduct and reporting of MSC research. (c) 2024 International Society for Cell & Gene Therapy. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/)

The transplantation of human neurons into the central nervous system (CNS) offers transformative opportunities for modeling neurodegenerative diseases in vivo. This study evaluated the survival, integration, and functional properties of cryopreserved forebrain GABAergic neurons (iGABAs) derived from human induced pluripotent stem cells (iPSCs) across three species used in translational research. iGABAs were stereotactically injected into the striatum of Sprague-Dawley rats, immunodeficient RNU rats, R6/2 Huntington's disease (HD) mice, wild-type controls, and Cynomolgus monkeys. Post-transplantation, long-term assessments revealed robust neuronal survival, extensive neurite outgrowth, and integration with host CNS environments. In immunodeficient rats, iGABAs innervated the neuraxis, extending from the prefrontal cortex to the midbrain, while maintaining mature neuronal phenotypes without uncontrolled proliferation. Similarly, grafts in nonhuman primates showed localized survival and stable phenotype at one month. In the neurodegenerative milieu of HD mice, iGABAs survived up to six months, projecting into the host striatum and white matter, with evidence of mutant huntingtin aggregates localized within the graft, indicating pathological protein transfer. These findings underscore the utility of cryopreserved iGABAs as a reproducible, scalable model for disease-specific CNS investigations and mechanistic studies of proteinopathic propagation. This work establishes a critical platform for studying neurodegenerative diseases and developing therapeutic interventions.

Purpose To evaluate the feasibility and safety of intravitreal injection of autologous CD34+ stem cells from bone marrow (BMSCs) in eyes with vision loss from retinitis pigmentosa (RP). Design Phase I prospective open-labeled single center study. Participants Seven eyes (7 patients) with RP with best corrected visual acuity (BCVA) of 20/100 to 20/400 or visual field constriction to within 10 degrees. Methods A comprehensive exam with ETDRS BCVA, macular optical coherence tomography, perimetry, fluorescein angiography was performed at baseline, 1 to 3 months, and 6 months after study treatment. Bone marrow aspiration, isolation of CD34+ BMSCs under Good Manufacturing Practice (GMP) conditions, and intravitreal cell injection were performed on the same day. The CD34+ cells were isolated from bone marrow using a Ficoll gradient and the Miltenyi CliniMACs system. Isolated CD34+ cells were released for clinical use if viability, sterility and purity met the release criteria accepted by the Food and Drug Administration for this clinical study. Main Outcome Measures Number of CD34+ cells isolated for injection and adverse events associated with study treatment during follow-up. Secondary outcome measures are changes in BCVA and perimetry. Results All isolated CD34+ cells passed the release criteria. A mean of 3.26 + 0.66 million viable CD34+ cells (range 1.6 to 7.05 million) were injected intravitreally per eye. No adverse event was noted during the study follow-up except for one participant who was noted with transient cells in the anterior chamber with mild elevation in intraocular pressure at 18 hours after study injection which normalized by 24 hours. BCVA remained within 2 lines of baseline or improved in all participants at 6 months follow-up. Perimetry was stable or improved in all eyes during study follow-up except one eye with transient improvement at 1 month and worsening of both eyes at 6 months. Conclusion Intravitreal injection of autologous CD34+ BMSCs is feasible and appears to be well tolerated in eyes with vision loss from RP. A larger randomized prospective study would be needed to evaluate further the safety and potential efficacy of this cell therapy for vision loss associated with RP.

The December 2024 US Food and Drug Administration (FDA) approval of Mesoblast's Ryoncil (remestemcel-L-rknd)-allogeneic bone marrow mesenchymal stromal cell (MSC(M)) therapy-in pediatric acute steroid-refractory graft-versus-host-disease finally ended a long-lasting drought on approved MSC clinical products in the United States. While other jurisdictions-including Europe, Japan, India, and South Korea-have marketed autologous or allogeneic MSC products, the United States has lagged in its approval. The sponsor's significant efforts and investments, working closely with the FDA addressing concerns regarding clinical efficacy and consistent MSC potency through an iterative process that spanned several years, was rewarded with this landmark approval. This approval will revive investment and enthusiasm in MSC products, further approvals in major markets, and will continue to foreshadow the long-predicted success of MSCs as a pharmaceutical.

Huntington’s disease (HD) is an autosomal dominant neurodegenerative disorder caused by a trinucleotide repeat expansion in exon 1 of the huntingtin (HTT) gene. Nuclease-deficient Cas9 protein (dCas9) epigenetic editing for targeted gene regulation is a promising therapeutic approach for HD through downregulation of the causative gene, HTT. A screen of several dCas9 variants with expanded PAM recognition was fused to KRAB and DNMT3A/L to assess the ability to downregulate total HTT. Surprisingly, only SpdCas9 could significantly downregulate HTT, while expanded PAM recognition variants dxCas9 and dCas9-VQR were less efficient or unable to reduce HTT expression. Using our lead construct with SpdCas9, DNA methylation changes were assessed through reduced representation bisulfite sequencing, showing high on-target increases in DNA methylation and few off-targets. In addition, HTT silencing was mitotically stable for up to 6 weeks in a rapidly dividing cell line. Finally, significant downregulation of HTT was achieved in patient-derived neuronal stem cells, showing the efficacy of this system in a disease-relevant cell type. This approach represents a novel therapeutic pathway for the treatment of HD.

Mesenchymal stromal cells (MSCs) have been tested in multiple clinical trials to treat peripheral artery disease, especially the more severe form called critical limb ischemia. However, MSCs have often not met the expected efficacy endpoints. We developed a more potent therapeutic by genetically modifying MSCs to overexpress Vascular Endothelial Growth Factor (VEGF-A165). Here, we report preclinical studies submitted to the Food and Drug Administration (FDA) as part of our Investigational New Drug submission package. In vitro studies included the characterization of cell banks, transcriptome and secretome analysis, and in vitro potency assays. In vivo studies using immune-deficient NSG mice include dose-finding efficacy studies using a Matrigel plug model, cell retention studies, measurements of circulating VEGF, and toxicology studies to rule out severe adverse events. Our results suggest both the safety and efficacy of MSC/VEGF and support a first-in-human clinical trial to test this new combined cell/gene therapy.

Human embryonic stem cells (hESCs) and induced pluripotent stem cells (hiPSCs) hold great potential in regenerative medicine. These cells can be expanded indefinitely in theory and are able to differentiate into different types of cells for cell therapies, drug screening, and basic biology studies. The reliable and effective propagation of hESCs and hiPSCs is important for their downstream applications. Basic fibroblast growth factor (bFGF) is critical to hESCs and hiPSCs for maintaining their pluripotency. Plant-produced growth factors are safe to use without potential contamination of infectious viruses and are less expensive to produce. In this study, we used rice cell-made basic fibroblast growth factor (RbFGF) to propagate hESCs and hiPSCs for at least eight passages. Both hESCs and hiPSCs cultured with RbFGF not only maintained the morphology but also the specific expression (OCT4, SSEA4, SOX2, and TRA-1-60) of PSCs, similar to those cultured with the commercial Escherichia coli-produced bFGF. Furthermore, both gene chip-based PluriTest and TaqMan hPSC Scorecard pluripotency analysis demonstrated the pluripotent expression profile of the hESCs cultured with RbFGF. In vitro trilineage assays further showed that these hESCs and hiPSCs cultured on RbFGF were capable of giving rise to cell derivatives of ectoderm, mesoderm, and endoderm, further demonstrating their pluripotency. Finally, chromosome stability was also maintained in hESCs cultured with RbFGF as demonstrated by normal karyotypes. This study suggests broad applications for plant-made growth factors in stem cell culture and regenerative medicine.

Induced pluripotent stem cell (iPSC) models of neurodevelopmental disorders (NDDs) have promoted an understanding of commonalities and differences within or across patient populations by revealing the underlying molecular and cellular mechanisms contributing to disease pathology. Here, we focus on developing a human model for PPP2R5D-related NDD, called Jordan syndrome, which has been linked to Early-Onset Parkinson's Disease (EOPD). Here we sought to understand the underlying molecular and cellular phenotypes across multiple cell states and neuronal subtypes in order to gain insight into Jordan syndrome pathology. Our work revealed that iPSC-derived midbrain neurons from Jordan syndrome patients display significant differences in dopamine-associated pathways and neuronal architecture. We then evaluated a CRISPR-based approach for editing heterozygous dominant G-to-A mutations at the transcript level in patient-derived neural stem cells. Our findings show that site-directed RNA editing is influenced by sgRNA length and cell type. These studies support the potential for a CRISPR RNA editor system to selectively edit mutant transcripts harboring G-to-A mutations in neural stem cells while providing an alternative editing technology for those suffering from NDDs.

Ischemic injury causes dynamic damage to the native extracellular matrix (ECM), which plays a key role in tissue homeostasis and regeneration by providing structural support, facilitating force transmission, and transducing key signals to cells. The main approach aimed at repairing injury to ischemic tissues is restoration of vascular function. Due to their potential to form capillary niches, endothelial cells (ECs) are of greatest interest for vascular regeneration. Integrin binding to ECM is crucial for cell anchorage to the surrounding matrix, spreading, migration, and further activation of intracellular signaling pathways. In this study, we proposed to establish an in-situ engineering strategy to remodel the ECM at the ischemic site to guide EC endogenous binding and establish effective EC/ECM interactions to promote revascularization. We designed and constructed a dual-function molecule (LXW7)2-SILY, which is comprised of two functional domains: the first one (LXW7) binds to integrin αvβ3 expressed on ECs, and the second one (SILY) binds to collagen. In vitro, we confirmed (LXW7)2-SILY improved EC adhesion and survival. After in situ injection, (LXW7)2-SILY showed stable retention at the injured area and promoted revascularization, blood perfusion, and tissue regeneration in a mouse hindlimb ischemia model. • A dual-function peptide developed for in-situ engineering native extracellular matrix. • The dual-function peptide specifically anchors endogenous endothelial cells to extracellular matrix. • The dual-function peptide promotes vascularized tissue regeneration.

Introduction: CD19 chimeric antigen receptor (CAR) T-cells have transformed the landscape of treatment for relapsed/refractory (R/R) diffuse large b-cell lymphoma (DLBCL); however, only ~40% of patients have durable responses (Neelapu 2023). Essential to a favorable environment for CAR T-cells to expand, exert anti-tumor activity, and persist is lymphodepleting chemotherapy. Cyclophosphamide and fludarabine is a frequently used combination of lymphodepletion; however, the dosing of these agents varies and there is limited data prospectively comparing these doses with efficacy and safety. Rituximab has also demonstrated synergism with CAR T-cells in pre-clinical models and is also believed to mediate peripheral B-cell depletion, facilitating better tumor targeting by CARs, but it's role in lymphodepletion is not well described. In addition, timely access to this potentially curable therapy remains a challenge. As such, point of care manufacturing with rapid vein-to-vein time is critical to expanding access to CAR T-cell therapy. Methods: This is a phase I, single-center 3+3 dose escalation study of locally manufactured CD19 CAR T-cells at our university's GMP facility. The primary objectives include feasibility of manufacturing CD19 CAR T-cells, safety of escalating doses of lymphodepleting chemotherapy with or without rituximab, and safety of CD19 CAR T-cells in subjects with R/R DLBCL. Overall response rate (ORR), complete response (CR), partial response (PR), progression free survival (PFS), and overall survival (OS) are secondary objectives. This is an autologous CD19-directed CAR created with a lentiviral vector (Miltenyi Biotec), a 4-1BB costimulatory domain, and a CD3 zeta intracellular domain. Patients with R/R DLBCL after > 2 prior lines of therapy are eligible. Subjects receive escalating doses of lymphodepleting chemotherapy with cyclophosphamide and fludarabine with or without rituximab in 6 dose levels (DL). The fludarabine dose remains constant at 25 mg/m2 Days -5 to -3, and the cyclophosphamide dose escalates from 500 mg/m2 x 3 doses in DL 1 and 2, to 60 mg/kg x 1 dose in DL 3 and 4, and to 60 mg/kg x 2 doses in DL 5 and 6. Rituximab is given in DL 2, 4, and 6. A CAR T-cell dose of 1x106 cells/kg is given in each DL. The CAR T-cells were initially harvested at Day 12 then thawed from a cryopreserved product; they are currently given as a fresh infusion after an 8-day manufacturing period. Results: At time of data cutoff, 20 subjects enrolled and 15 are evaluable: 3 on DL 1, 4 on DL 2, 5 on DL 3, and 3 on DL 4. 6 patients have received fresh cell infusion since the manufacturing time was shortened. 11 (73%) subjects are female; median age is 63 (range 28-77). 9 subjects (60%) had primary refractory disease; 5 (33.3%) relapsed > 12 months from initial therapy and 1 (6.7%) relapsed < 12 months from initial therapy. 2 subjects had double hit lymphoma, 4 were double expressors, and 3 transformed from follicular lymphoma. 5 subjects have GCB immunophenotype, 8 have non-GCB, and 2 are unknown. Stage of disease includes 3 subjects with stage 2, 2 with stage 3, and 10 with stage IV. The median number of prior lines of therapy is 3 (range 2-8). 5 subjects had prior autologous stem cell transplantation (SCT) and 1 had prior allogeneic SCT. Day +30 PET shows ORR 86% (13/15), CR 46.7% (7/15), and PR 40% (6/15). On Day +90 PET, 3 additional subjects converted to CR (10/15; 67.7%); 2 subjects received no additional therapy and 1 started lenalidomide between Day +30 and +90. The median follow-up time is 9 months. At time of data cutoff (30 months; July 21, 2024), PFS is 67.7% (10/15) and OS 73.3% (11/15). 4 patients have expired: 3 from disease progression and 1 from infection. 6 subjects (40%) experienced CRS, with 4 (26.7%) Grade 1 events in DL 2, 3, and 4, and 2 (13.3%) Grade 2 events in DL 2 and 3. 1 subject (6.7%) had Grade 2 ICANS in DL 3. There have been no Grade 3 or 4 CRS or ICANS events. There are no dose limiting toxicities to date. Conclusions: These results demonstrate the safety and efficacy of CD19 CAR T-cells with escalating doses of lymphodepleting cyclophosphamide and fludarabine with and without rituximab. There have been no dose limiting toxicities and no Grade 3-4 CRS or ICANS. The feasibility of point of care manufacturing with vein-to-vein time of 8 days is demonstrated in this phase I study. The efficacy of the CARs is maintained and comparable to commercial CD19 products without any severe CRS or ICANS. Enrollment continues on DL 5.

Background Despite promising results in preclinical studies, mesenchymal stromal cells (MSCs) face significant challenges in clinical translation. A scoping review by our group highlighted two key issues contributing to this gap: i) the lack of clear and consensus definition for MSCs and ii) under reporting of critical parameters in MSC clinical studies. To address these issues, we conducted a modified Delphi study to establish and implement a consensus definition for MSCs and develop reporting guidelines for MSC clinical studies. Methods A steering committee of 22 international experts including stakeholders from different MSC research fields participated in the 3 Delphi rounds. For the first round, to obtain a broad perspective, additional investigators recommended by the steering committee were invited to participate. The first 2 rounds consisted of online surveys, while the 3rd round took the form of a virtual meeting. Participants were asked to rate a series of potential defining characteristics of MSCs and items for reporting guidelines. Consensus was defined as at least 80% of the participants rating the item in the same category of importance. Results Eighty-seven international participants participated in the first-round survey (Spring 2023), 17 participants in the second online survey (Fall 2023) and 15 participants in the final virtual consensus meeting (January 2024). For the MSC definition, 20 items were considered and 9 reached consensus. Items included terminology (1 item), cell marker expression (5 items), tissue origin (1 item), stemness (1 item) and description of critical quality attributes (1 item). For the reporting guidelines, on the 28 initial items and with additional items suggested during round 1, a total of 33 items to report were included. This included items on MSC intervention group and control (i.e., MSC product, dose, and administration, etc.), MSC characteristics (i.e., MSCs provenance, “fitness and viability, immune compatibility, etc.), and MSC culture conditions (i.e., oxygen environment, culture medium, use of serum, etc.). Conclusion By applying a Delphi method to establish a consensus definition for MSCs and reporting guidelines for MSC-based clinical, this work represents a significant advance in improving transparency and reproducibility in the conduct and reporting of MSC research.

Mesenchymal stem cells (MSCs) are novel therapeutics for the treatment of Crohn’s disease. However, their mechanism of action is unclear, especially in disease-relevant chronic models of inflammation. Thus, we used SAMP-1/YitFc (SAMP), a chronic and spontaneous murine model of small intestinal inflammation, to study the therapeutic effects and mechanism of action of human bone marrow-derived MSCs (hMSC). hMSC dose-dependently inhibited naïve T lymphocyte proliferation via prostaglandin E2 (PGE2) secretion and reprogrammed macrophages to an anti-inflammatory phenotype. We found that the hMSCs promoted mucosal healing and immunologic response early after administration in SAMP when live hMSCs are present (until day 9) and resulted in a complete response characterized by mucosal, histological, immunologic, and radiological healing by day 28 when no live hMSCs are present. hMSCs mediate their effect via modulation of T cells and macrophages in the mesentery and mesenteric lymph nodes (mLN). Sc-RNAseq confirmed the anti-inflammatory phenotype of macrophages and identified macrophage efferocytosis of apoptotic hMSCs as a mechanism that explains their long-term efficacy. Taken together, our findings show that hMSCs result in healing and tissue regeneration in a chronic model of small intestinal inflammation and despite being short-lived, exert long-term effects via sustained anti-inflammatory programming of macrophages via efferocytosis.