T cells and macrophages jointly modulate osteogenesis of mesenchymal stromal cells.

Approximately 5%-10% of fractures go on to delayed healing and nonunion, posing significant clinical, economic, and social challenges. Current treatment methods involving open bone harvesting and grafting are associated with considerable pain and potential morbidity at the donor site. Hence, there is growing interest in minimally invasive approaches such as bone marrow aspirate concentrate (BMAC), which contains mesenchymal stromal cells (MSCs), macrophages (Mφ), and T cells. However, the use of cultured or activated cells for treatment is not yet FDA-approved in the United States, necessitating further exploration of optimal cell types and proportions for effective bone formation. As our understanding of osteoimmunology advances, it has become apparent that factors from anti-inflammatory Mφ (M2) promote bone formation by MSCs. Additionally, M2 Mφ promote T helper 2 (Th2) cells and Treg cells, both of which enhance bone formation. In this study, we investigated the interactions among MSCs, Mφ, and T cells in bone formation and explored the potential of subsets of BMAC. Coculture experiments were conducted using primary MSCs, Mφ, and CD4+ T cells at specific ratios. Our results indicate that nonactivated T cells had no direct influence on osteogenesis by MSCs, while coculturing MSCs with Mφ and T cells at a ratio of 1:5:10 positively impacted bone formation. Furthermore, higher numbers of T cells led to increased M2 polarization and a higher proportion of Th2 cells in the early stages of coculture. These findings suggest the potential for enhancing bone formation by adjusting immune and mesenchymal cell ratios in BMAC. By understanding the interactions and effects of immune cells on bone formation, we can develop more effective strategies and protocols for treating bone defects and nonunions. Further studies are needed to investigate these interactions in vivo and explore additional factors influencing MSC-based therapies.

Rat nasal mucosa-derived ectodermal mesenchymal stem cells: A new therapeutic option for chronic rhinosinusitis.

OBJECTIVE: To investigate the effect of nasal mucosa-derived ectodermal mesenchymal stem cells (NM-EMSCs) on the inflammatory state of rats with chronic rhinosinusitis (CRS) and the underlying therapeutic mechanism. METHODS: NM-EMSCs were isolated and extracted to construct a rat model of CRS. Fifteen Sprague‒Dawley (SD) rats were randomly divided into three groups: CK + NS group rats were injected locally with saline in the nasal mucosa; CRS + NS group rats were injected locally with saline in the nasal mucosa; and CRS + EMSCs group rats were injected locally with NM-EMSCs in the nasal mucosa. One rat from the CRS + EMSCs group was randomly euthanized at 2, 4, and 6 days after injection, and the nasal mucosa tissues were collected for HE staining, Masson's trichrome staining, and periodic acid-Schiff staining. RESULTS: NM-EMSCs specifically expressing CD73, CD105, and CD90 were successfully isolated from the nasal mucosa of rats and were able to differentiate into adipocytes, osteoblasts, and chondrocytes. After saline and NM-EMSC injection, compared with those in the blank control CK + NS group, the nasal mucosa in the CRS + NS and CRS + EMSC groups exhibited obvious thickening, a large amount of inflammatory cell infiltration, and increased collagen and mucin distribution. Four days post-NM-EMSC injection, the thickening of the nasal mucosa in the CRS group was gradually alleviated, the inflammatory cell infiltration gradually decreased, and the distribution of collagen and mucin and the collagen-positive area gradually decreased. Moreover, only a small number of inflammatory cells were visible, and the distribution of mucins was limited to 6 days post-NM-EMSC injection. CONCLUSION: NM-EMSCs effectively attenuated inflammation in the nasal mucosa of CRS model rats.

Mesenchymal Stem Cells and Purinergic Signaling in Autism Spectrum Disorder: Bridging the Gap between Cell-Based Strategies and Neuro-Immune Modulation.

The prevalence of autism spectrum disorder (ASD) is still increasing, which means that this neurodevelopmental lifelong pathology requires special scientific attention and efforts focused on developing novel therapeutic approaches. It has become increasingly evident that neuroinflammation and dysregulation of neuro-immune cross-talk are specific hallmarks of ASD, offering the possibility to treat these disorders by factors modulating neuro-immunological interactions. Mesenchymal stem cell-based therapy has already been postulated as one of the therapeutic approaches for ASD; however, less is known about the molecular mechanisms of stem cell influence. One of the possibilities, although still underestimated, is the paracrine purinergic activity of MSCs, by which stem cells ameliorate inflammatory reactions. Modulation of adenosine signaling may help restore neurotransmitter balance, reduce neuroinflammation, and improve overall brain function in individuals with ASD. In our review article, we present a novel insight into purinergic signaling, including but not limited to the adenosinergic pathway and its role in neuroinflammation and neuro-immune cross-talk modulation. We anticipate that by achieving a greater understanding of the purinergic signaling contribution to ASD and related disorders, novel therapeutic strategies may be devised for patients with autism in the near future.

Effects of extracellular vesicles for ischemic stroke: A meta­analysis of preclinical studies.

Ischemic stroke is a common occurrence worldwide, posing a severe threat to human health and leading to negative financial impacts. Currently available treatments still have numerous limitations. As research progresses, extracellular vesicles are being found to have therapeutic potential in ischemic stroke. In the present study, the literature on extracellular vesicle therapy in animal studies of ischemic stroke was screened by searching databases, including PubMed, Embase, Medline, Web of Science and the Cochrane Library. The main outcomes of the present study were the neurological function score, apoptotic rate and infarct volumes. The secondary outcomes were pro-inflammatory factors, including tumor necrosis factor-α (TNF-α), interleukin (IL)-1ß and IL-6. The study quality was assessed using the CAMARADES Checklist. Subgroup analyses were performed to evaluate factors influencing extracellular vesicle therapy. Review Man3ager5.3 was used for data analysis. A total of 20 relevant articles were included in the present meta-analysis. The comprehensive analysis revealed that extracellular vesicles exerted a significant beneficial effect on neurobehavioral function, reducing the infarct volume and decreasing the apoptotic rate. Moreover, extracellular vesicles were found to promote nerve recovery by inhibiting pro-inflammatory factors (TNF-α, IL-1ß and IL-6). On the whole, the present meta-analysis examined the combined effects of extracellular vesicles on nerve function, infarct volume, apoptosis and inflammation, which provides a foundation for the clinical study of extracellular vesicles.

Influential factors on stem cell therapy success in canine model of spinal cord Injury: A systematic review and meta-analysis.

Spinal cord injury (SCI) is a serious medical condition. The search for an effective cure remains a persistent challenge. Current treatments, unfortunately, are unable to sufficiently improve neurological function, often leading to lifelong disability. This systematic review and meta-analysis evaluated the effectiveness of stem cell therapy for SCI using canine models. It also explored the optimal protocol for implementing stem cell therapy. A comprehensive search of studies was conducted from 2000 to October 2022. This study focused on five outcomes: motor function score, histopathology, IHC, western blot, and SEP. The results demonstrated a significant improvement in locomotion post-SCI in dogs treated with stem cell therapy. The therapy also led to an average increase of 3.15 points in the Olby score of the treated dogs compared to the control group. These findings highlights stem cell therapy's potential as a promising SCI treatment. The meta-analysis suggests that using bone marrow stem cells, undergoing neural differentiation in vitro, applying a surgical implantation or intrathecal route of administration, associating matrigel in combination with stem cells, and a waiting period of two weeks before starting treatment can enhance SCI treatment effectiveness.

Emerging Strategies in Mesenchymal Stem Cell-Based Cardiovascular Therapeutics.

Cardiovascular diseases continue to challenge global health, demanding innovative therapeutic solutions. This review delves into the transformative role of mesenchymal stem cells (MSCs) in advancing cardiovascular therapeutics. Beginning with a historical perspective, we trace the development of stem cell research related to cardiovascular diseases, highlighting foundational therapeutic approaches and the evolution of cell-based treatments. Recognizing the inherent challenges of MSC-based cardiovascular therapeutics, which range from understanding the pro-reparative activity of MSCs to tailoring patient-specific treatments, we emphasize the need to refine the pro-regenerative capacity of these cells. Crucially, our focus then shifts to the strategies of the fourth generation of cell-based therapies: leveraging the secretomic prowess of MSCs, particularly the role of extracellular vesicles; integrating biocompatible scaffolds and artificial sheets to amplify MSCs' potential; adopting three-dimensional ex vivo propagation tailored to specific tissue niches; harnessing the promise of genetic modifications for targeted tissue repair; and institutionalizing good manufacturing practice protocols to ensure therapeutic safety and efficacy. We conclude with reflections on these advancements, envisaging a future landscape redefined by MSCs in cardiovascular regeneration. This review offers both a consolidation of our current understanding and a view toward imminent therapeutic horizons.

Optimization of methods for intrasplenic administration of human amniotic epithelial cells in order to perform safe and effective cell-based therapy for liver diseases.

In animal experimental models the administration of stem cells into the spleen should ensure high effectiveness of their implantation in the liver due to a direct vascular connection between the two organs. The aim of this study was to update the methods of experimental intrasplenic cell transplantation using human amniotic epithelial cells (hAECs) which are promising cells in the treatment of liver diseases. BALB/c mice were administered intrasplenically with 0.5, 1, and 2 million hAECs by direct bolus injection (400 µl/min) and via a subcutaneous splenic port by fast (20 µl/min) and slow (10 µl/min) infusion. The port was prepared by translocating the spleen to the skin pocket. The spleen, liver, and lungs were collected at 3 h, 6 h, and 24 h after the administration of cells. The distribution of hAECs, histopathological changes in the organs, complete blood count, and biochemical markers of liver damage were assessed. It has been shown that the method of intrasplenic cell administration affects the degree of liver damage. The largest number of mice showing significant liver damage was observed after direct administration and the lowest after slow administration through a port. Liver damage increased with the number of administered cells, which, paradoxically, resulted in increased liver colonization efficiency. It was concluded that the administration of 1 × 106 hAECs by slow infusion via a subcutaneous splenic port reduces the incidence of complications at the expense of a slight decrease in the effectiveness of implantation of the transplanted cells in the liver.

Corrigendum: Assessment of mesenchymal stem/stromal cell-based therapy in K/BxN serum transfer-induced arthritis.

[This corrects the article DOI: 10.3389/fimmu.2022.943293.].

Efficacy of Engraftment and Safety of Human Umbilical Di-Chimeric Cell (HUDC) Therapy after Systemic Intraosseous Administration in an Experimental Model.

Cell-based therapies hold promise for novel therapeutic strategies in regenerative medicine. We previously characterized in vitro human umbilical di-chimeric cells (HUDCs) created via the ex vivo fusion of human umbilical cord blood (UCB) cells derived from two unrelated donors. In this in vivo study, we assessed HUDC safety and biodistribution in the NOD SCID mouse model at 90 days following the systemic intraosseous administration of HUDCs. Twelve NOD SCID mice (n = 6/group) received intraosseous injection of donor UCB cells (3.0 × 106) in Group 1, or HUDCs (3.0 × 106) in Group 2, without immunosuppression. Flow cytometry assessed hematopoietic cell surface markers in peripheral blood and the presence of HLA-ABC class I antigens in lymphoid and non-lymphoid organs. HUDC safety was assessed by weekly evaluations, magnetic resonance imaging (MRI), and at autopsy for tumorigenicity. At 90 days after intraosseous cell administration, the comparable expression of HLA-ABC class I antigens in selected organs was found in UCB control and HUDC therapy groups. MRI and autopsy confirmed safety by no signs of tumor growth. This study confirmed HUDC biodistribution to selected lymphoid organs following intraosseous administration, without immunosuppression. These data introduce HUDCs as a novel promising approach for immunomodulation in transplantation.

Biological scaffold as potential platforms for stem cells: Current development and applications in wound healing.

Wound repair is a complex challenge for both clinical practitioners and researchers. Conventional approaches for wound repair have several limitations. Stem cell-based therapy has emerged as a novel strategy to address this issue, exhibiting significant potential for enhancing wound healing rates, improving wound quality, and promoting skin regeneration. However, the use of stem cells in skin regeneration presents several challenges. Recently, stem cells and biomaterials have been identified as crucial components of the wound-healing process. Combination therapy involving the development of biocompatible scaffolds, accompanying cells, multiple biological factors, and structures resembling the natural extracellular matrix (ECM) has gained considerable attention. Biological scaffolds encompass a range of biomaterials that serve as platforms for seeding stem cells, providing them with an environment conducive to growth, similar to that of the ECM. These scaffolds facilitate the delivery and application of stem cells for tissue regeneration and wound healing. This article provides a comprehensive review of the current developments and applications of biological scaffolds for stem cells in wound healing, emphasizing their capacity to facilitate stem cell adhesion, proliferation, differentiation, and paracrine functions. Additionally, we identify the pivotal characteristics of the scaffolds that contribute to enhanced cellular activity.

Expanding the role of CAR T-cell therapy: From B-cell hematological malignancies to autoimmune rheumatic diseases.

Chimeric antigen receptor (CAR) T-cell therapy is a form of immunotherapy where the lymphocytes, mostly T-cells, are redirected to specifically recognize and eliminate a target antigen by coupling them with CARs. The binding of CAR and target cell surface antigens leads to vigorous T cell activation and robust anti-tumor immune responses. Areas of implication of CAR T-cell therapies include mainly hematological malignancies (i.e., advanced B-cell cancers); however, recent studies have proven the unprecedented success of the new immunotherapy also in autoimmune rheumatic diseases. We aim to review the recent advances in CAR T-cell therapies in rheumatology but also to address the limitations of their use in the real clinical practice based on the data on their efficacy and safety.

Human Nasal Inferior Turbinate-Derived Neural Stem Cells Improve the Niche of Substantia Nigra Par Compacta in a Parkinson's Disease Model by Modulating Hippo Signaling.

BACKGROUND: Parkinson's disease (PD) is one of the most prevalent neurodegenerative diseases, following Alzheimer's disease. The onset of PD is characterized by the loss of dopaminergic neurons in the substantia nigra. Stem cell therapy has great potential for the treatment of neurodegenerative diseases, and human nasal turbinate-derived stem cells (hNTSCs) have been found to share some characteristics with mesenchymal stem cells. Although the Hippo signaling pathway was originally thought to regulate cell size in organs, recent studies have shown that it can also control inflammation in neural cells. METHODS: Dopaminergic neuron-like cells were differentiated from SH-SY5Y cells (DA-Like cells) and treated with 1-Methyl-4-phenylpyridinium iodide to stimulate Reactive oxidative species (ROS) production. A transwell assay was conducted to validate the effect of hNTSCs on the Hippo pathway. We generated an MPTP-induced PD mouse model and transplanted hNTSCs into the substantia nigra of PD mice via stereotaxic surgery. After five weeks of behavioral testing, the brain samples were validated by immunoblotting and immunostaining to confirm the niche control of hNTSCs. RESULTS: In-vitro experiments showed that hNTSCs significantly increased cell survival and exerted anti-inflammatory effects by controlling ROS-mediated ER stress and hippocampal signaling pathway factors. Similarly, the in-vivo experiments demonstrated an increase in anti-inflammatory effects and cell survival rate. After transplantation of hNTSCs, the PD mouse model showed improved mobility and relief from PD symptoms. CONCLUSION: hNTSCs improved the survival rate of dopaminergic neurons by manipulating the hippocampal pathway through Yes-associated protein (YAP)/transcriptional coactivator with a PDZ-binding motif (TAZ) by reducing inflammatory cytokines. In this study, we found that controlling the niche of hNTSCs had a therapeutic effect on PD lesions.

Identification and removal of unexpected proliferative off-target cells emerging after iPSC-derived pancreatic islet cell implantation.

Differentiation of pancreatic endocrine cells from human pluripotent stem cells (PSCs) has been thoroughly investigated for application in cell therapy against diabetes. In the context of induced pancreatic endocrine cell implantation, previous studies have reported graft enlargement resulting from off-target pancreatic lineage cells. However, there is currently no documented evidence of proliferative off-target cells beyond the pancreatic lineage in existing studies. Here, we show that the implantation of seven-stage induced PSC-derived pancreatic islet cells (s7-iPICs) leads to the emergence of unexpected off-target cells with proliferative capacity via in vivo maturation. These cells display characteristics of both mesenchymal stem cells (MSCs) and smooth muscle cells (SMCs), termed proliferative MSC- and SMC-like cells (PMSCs). The frequency of PMSC emergence was found to be high when 108 s7-iPICs were used. Given that clinical applications involve the use of a greater number of induced cells than 108, it is challenging to ensure the safety of clinical applications unless PMSCs are adequately addressed. Accordingly, we developed a detection system and removal methods for PMSCs. To detect PMSCs without implantation, we implemented a 4-wk-extended culture system and demonstrated that putative PMSCs could be reduced by compound treatment, particularly with the taxane docetaxel. When docetaxel-treated s7-iPICs were implanted, the PMSCs were no longer observed. This study provides useful insights into the identification and resolution of safety issues, which are particularly important in the field of cell-based medicine using PSCs.

In vitro anti-leukemic effect of Wharton's jelly derived mesenchymal stem cells.

BACKGROUND: Mesenchymal stem cells (MSCs) have the ability to self-renew and are multi-potent. They are a primary candidate for cell-based therapy due to their potential anti-cancer effects. The aim of this study was to evaluate the in vitro anti-leukemic effect of Wharton's Jelly-derived MSC (WJ-MSC) on the leukemic cell lines K562 and HL-60. METHODS: In this present study, WJ-MSCs were isolated from human umbilical cord. The cells were incubated according to the standard culture conditions and characterized by flow cytometry. For experiments, WJ-MSC and leukemic cells were incubated in the direct co-culture at a ratio of 1:5 (leukemia cells: WJ-MSC). HUVEC cells were used as a non-cancerous cell line model. The apoptotic effect of WJ-MSCs on the cell lines was analyzed using Annexin V/PI apoptosis assay. RESULTS: After the direct co-culture of WJ-MSCs on leukemic cell lines, we observed anti-leukemic effects by inducing apoptosis. We had two groups of determination apoptosis with and without WJ-MSCs for all cell lines. Increased apoptosis rates were observed in K562 and HL-60 cell lines, whereas the apoptosis rates in HUVEC cells were low. CONCLUSIONS: MSCs are known to inhibit the growth of tumors of both hematopoietic and non-hematopoietic origin in vitro. In our study, WJ-MSC treatment strongly inhibited the viability of HL-60 and K562 and induced apoptosis. Our results also provided new insights into the inhibition of tumor growth by WJ-MSCs in vitro. In the future, WJ-MSCs could be used to inhibit cancer cells in clinical applications.

Organoid-based personalized medicine: from tumor outcome prediction to autologous transplantation.

Inter-individual variation largely influences disease susceptibility, as well as response to therapy. In a clinical context, the optimal treatment of a disease should consider inter-individual variation and formulate tailored decisions at an individual level. In recent years, emerging organoid technologies promise to capture part of an individual's phenotypic variability and prove helpful in providing clinically relevant molecular insights. Organoids are stem cell-derived 3-dimensional models that contain multiple cell types that can self-organize and give rise to complex structures mimicking the organization and functionality of the tissue of origin. Organoids therefore represent a more faithful recapitulation of the dynamics of the tissues of interest, compared to conventional monolayer cultures, thus supporting their use in evaluating disease prognosis, or as a tool to predict treatment outcomes. Additionally, the individualized nature of patient-derived organoids enables the use of autologous organoids as a source of transplantable material not limited by histocompatibility. An increasing amount of preclinical evidence has paved the way for clinical trials exploring the applications of organoid-based technologies, some of which are in phase I/II. This review focuses on the recent progress concerning the use of patient-derived organoids in personalized medicine, including (1) diagnostics and disease prognosis, (2) treatment outcome prediction to guide therapeutic advice, and (3) organoid transplantation or cell-based therapies. We discuss examples of these potential applications and the challenges associated with their future implementation.

PD-1 Receptor (+) T cells are associated with the efficacy of the combined treatment with regulatory t cells and rituximab in type 1 diabetes children via regulatory t cells suppressive activity amelioration.

An imbalance between exaggerated autoaggressive T cell responses, primarily CD8 + T cells, and impaired tolerogenic mechanisms underlie the development of type 1 diabetes mellitus. Disease-modifying strategies, particularly immunotherapy focusing on FoxP3 + T regulatory cells (Treg), and B cells facilitating antigen presentation for T cells, show promise. Selective depletion of B cells may be achieved with an anti-CD20 monoclonal antibody (mAb). In a 2-year-long flow cytometry follow-up, involving 32 peripheral blood T and B cell markers across three trial arms (Treg + rituximab N = 12, Treg + placebo N = 13, control N = 11), we observed significant changes. PD-1 receptor (+) CD4 + Treg, CD4 + effector T cells (Teffs), and CD8 + T cell percentages increased in the combined regimen group by the end of follow-up. Conversely, the control group exhibited a notable reduction in PD-1 receptor (+) CD4 + Teff percentages. Considering clinical endpoints, higher PD-1 receptor (+) expression on T cells correlated with positive responses, including a higher mixed meal tolerance test AUC, and reduced daily insulin dosage. PD-1 receptor (+) T cells emerged as a potential therapy outcome biomarker. In vitro validation confirmed that successful Teff suppression was associated with elevated PD-1 receptor (+) Treg levels. These findings support PD-1 receptor (+) T cells as a reliable indicator of treatment with combined immunotherapy consisting of Tregs and anti-CD20 mAb efficacy in type 1 diabetes mellitus.

Human dental pulp stem/stromal cells in clinical practice.

Dental pulp stem/stromal cells (DPSCs) are fibroblast-like, neural crest-derived, and multipotent cells that can differentiate into several lineages. They are relatively easy to isolate from healthy and inflamed pulps, with little ethical concerns and can be successfully cryopreserved and thawed. The therapeutic effects of DPSCs derived from animal or human sources have been extensively studied through in-vitro and in-vivo animal experiments and the findings indicated that DPSCs are effective not only for dental diseases but also for systemic diseases. Understanding that translational research is a critical step through which the fundamental scientific discoveries could be translated into applicable diagnostics and therapeutics that directly benefit humans, several clinical studies were carried out to generate evidence for the efficacy and safety of autogenous or allogeneic human DPSCs (hDPSCs) as a treatment modality for use in cell-based therapy, regenerative medicine/dentistry and tissue engineering. In clinical medicine, hDPSCs were effective for treating acute ischemic stroke and human exfoliated deciduous teeth-conditioned medium (SHED-CM) repaired vascular damage of the corpus cavernous, which is the main cause of erectile dysfunction. Whereas in clinical dentistry, autologous SHED was able to regenerate necrotic dental pulp after implantation into injured teeth, and micrografts enriched with autologous hDPSCs and collagen sponge were considered a treatment option for human intrabony defects. In contrast, hDPSCs did not add a significant regenerative effect when they were used for the treatment of post-extraction sockets. Large-scale clinical studies across diverse populations are still lacking to provide robust evidence on the safety and efficacy of hDPSCs as a new treatment option for various human diseases including dental-related problems.

Human Omental Mature Adipocytes used as Paclitaxel Reservoir for Cell-Based Therapy in Ovarian Cancer.

Primary human omental adipocytes and ovarian cancer(OC) cells establish a bidirectional communication in which tumor driven lipolysis is induced in adipocytes and the resulting fatty acids are delivered to cancer cells within the tumor microenvironment. Despite meaningful improvement in the treatment of OC, its efficacy is still limited by hydrophobicity and untargeted effects related to chemotherapeutics. Herein, omental adipocytes are firstly used as a reservoir for paclitaxel, named Living Paclitaxel Bullets (LPB) and secondly benefit from the established dialogue between adipocytes and cancer cells to engineer a drug delivery process that target specifically cancer cells. These results show that mature omental adipocytes can successfully uptake paclitaxel and deliver it to OC cells in a transwell coculture based in vitro model. In addition, the efficacy of this proof-of-concept has been demonstrated in vivo and induces a significant inhibition of tumor growth on a xenograft tumor model. The use of mature adipocytes can be suitable for clinical prospection in a cell-based therapy system, due to their mature and differentiated state, to avoid risks related to uncontrolled cell de novo proliferation capacity after the delivery of the antineoplastic drug as observed with other cell types when employed as drug carriers.

Rilparencel (Renal Autologous Cell Therapy-REACT®) for Chronic Kidney Disease and Type 1 and Type 2 Diabetes: Phase 2 Trial Design Evaluating Bilateral Kidney Dosing and Redosing Triggers.

INTRODUCTION: Autologous cell-based therapies (CBT) to treat chronic kidney disease (CKD) with diabetes are novel and can potentially preserve renal function and decelerate disease progression. CBT dosing schedules are in early development and may benefit from individual bilateral organ dosing and kidney-dependent function to improve efficacy and durability. The objective of this open-label, phase 2 randomized controlled trial (RCT) is to evaluate participants' responses to rilparencel (Renal Autologous Cell Therapy-REACT®) following bilateral percutaneous kidney injections into the kidney cortex with a prescribed dosing schedule versus redosing based on biomarker triggers. METHODS: Eligible participants with type 1 or 2 diabetes and CKD, eGFR 20-50 mL/min/1.73 m2, urine albumin-to-creatinine ratio (UACR) 30-5,000 mg/g, hemoglobin >10 g/dL, and glycosylated hemoglobin <10% were enrolled. After a percutaneous kidney biopsy and bioprocessing ex vivo expansion of selected renal cells, participants were randomized 1:1 into two cohorts determined by the dosing scheme. Cohort 1 receives 2 cell injections, one in each kidney 3 months apart, and cohort 2 receives one injection and the second dose only if there is a sustained eGFR decline of ≥20 mL/min/1.73 m2 and/or UACR increase of ≥30% and ≥30 mg/g, confirmed by re-testing. CONCLUSION: The trial is fully enrolled with fifty-three participants. Cell injections and follow-up clinical visits are ongoing. This multicenter phase 2 RCT is designed to investigate the efficacy and safety of rilparencel with bilateral kidney dosing and compare two injection schedules with the potential of preserving or improving kidney function and delaying kidney disease progression among patients with stages 3a-4 CKD with diabetes.