Stem Cells

Development of motoneurons from stem cells is characterized by a change from glycolytic to oxidative metabolism. Since this transition remains poorly understood, we examined it at five distinct differentiation stages from hiPSC to motoneuron. While a direct comparison of hiPSCs and mature motoneurons confirmed the expected glycolytic-to-oxidative shift, the intermediate stages showed that the conversion was not monotonic. After an initial drop of glycolysis at the hiPSC-to-neuroepithelial transition, late neuroepithelial cells showed intermittent peaks of the glycolytic marker lactate dehydrogenase A and the metabolic regulator TIGAR. Furthermore, the lactate-produced-to-glucose-consumed ratio remained elevated. A fully oxidative phenotype was only assumed upon progress from neural progenitors to motoneurons, portrayed by a definitive drop of the lactate-produced-to-glucose-consumed ratio, an increase of mitochondrial membrane charging, and shifts from lactate dehydrogenase A to B, from pyruvate dehydrogenase to anaplerotic pyruvate carboxylase, and from Mitofusin 1 to 2. Together, our data show that metabolic maturation in human motoneurons does not occur as a simple switch. Instead, it unfolds through distinct stages in a directional yet nonlinear manner.

BackgroundGains of chromosome 20q11.21 are among the most common culture-acquired abnormalities in human pluripotent stem cells (hPSC), conferring a well-defined survival advantage while altering differentiation capacity. However, it remains unclear whether this advantage persists during differentiation, how the aneuploidy alters ectodermal and retinal pigment epithelium (RPE) lineage specification, and which genes within the minimal amplicon drive these effects. MethodsWe used three isogenic human embryonic stem cell line pairs (wild-type and 20q11.21 gain) and assessed their behaviour in two neuroectoderm differentiation systems: directed neuroectoderm induction (dual SMAD inhibition) and long-term spontaneous RPE differentiation. Competitive dynamics were measured in mixed cultures, and lineage outcomes were analysed using immunostaining, gene expression profiling and single-cell RNA sequencing. To identify driver genes, we generated BCL2L1 and ID1 overexpression lines and tested their effects under both directed and spontaneous differentiation conditions. ResultsAcross all lines and conditions, 20q cells expanded from a minor fraction to dominate mixed cultures, indicating that their competitive advantage persists beyond the undifferentiated state. Despite this dominance, pure 20q cells failed to specify to neuroectoderm or RPE. Single-cell transcriptomics revealed consistent diversion toward non-neural ectodermal and extraembryonic fates. Mechanistically, overexpression of BCL2L1 and ID1 alone or in combination impaired neuroectoderm specification, while synergistic effect of both genes promoted non-neural ectodermal outcomes under directed differentiation conditions. In spontaneous differentiation, both genes could disrupt differentiation. ConclusionsThe 20q11.21 gain couples a persistent survival advantage with a disruption of neural and RPE lineage competence, redirecting cells toward alternative ectodermal and extraembryonic fates. These effects arise from the combined action of two dosage-sensitive genes BCL2L1 and ID1 within the amplicon, illustrating how regional gene dosage can reshape developmental signalling responses in hPSC.

Mesenchymal stem cells (MSCs) have garnered significant attention over the past three decades due to their robust regenerative potential, primarily mediated by their paracrine activity by releasing soluble bioactive factors and extracellular vesicles (EVs). The MSC secretome plays a pivotal role in wound healing by influencing cellular migration, inflammation, angiogenesis, extracellular matrix (ECM) remodeling, and re-epithelialization. SPARC (Secreted Protein Acidic and Rich in Cysteine), a multifunctional ECM glycoprotein involved in tissue repair and remodeling, regulates key processes such as cell migration, proliferation, angiogenesis, and survival. Despite its known role in ECM dynamics, the impact of SPARC expression on the regenerative properties of MSCs remains underexplored. In this study, we hypothesized that SPARC overexpression in MSCs enhances their secretomes regenerative capacity. Using lentiviral systems, we generated SPARC-overexpressing (+SPARC) and SPARC-knockdown (KD-SPARC) MSCs to investigate SPARCs role in wound healing. Conditioned media (CM) derived from these MSCs were analyzed in vitro for their effects on human skin keratinocytes and fibroblasts. Our results revealed that SPARC expression significantly influences cell-specific migration and cell cycle. Furthermore, in an in vivo wound healing model, CM from +SPARC MSCs accelerated regeneration, while SPARC absence in MSCs CM delayed the healing process. These findings underscore the critical role of SPARC in modulating MSC secretome composition and enhancing its regenerative efficacy. This study highlights SPARC as a promising therapeutic target for the development of advanced regenerative therapies aimed at improving cutaneous wound healing outcomes.

The use of decellularized diseased livers in regenerative medicine is a promising approach for eliminating organ shortages. Bioengineering studies have shown that ECM can impact cell physiology, inducing cell activation, function, and ECM deposition, which suggests that the ECM has a "memory" that is involved in the outcome after recellularization. However, the effect of diseased ECM memory on new cells in vitro and in vivo has not been thoroughly investigated. Since it has been increasingly recognized that liver ECM changes due to different factors, it is comprehensively that diseased ECM obtained from discarded organs will ensure a distinct environment and impact cell survival and physiology. Thus, we aimed at investigating the impact of the memory of diseased ECM obtained from metabolic dysfunction-associated steatohepatitis (MASH)-derived organs on steatohepatitis establishment. To address this aim, we explored decellularized ECM obtained from rats and humans with MASH in different contexts. First, MASH ECM was characterized and then submitted to transplantation to investigate whether a MASH-derived ECM could be used as a scaffold for transplantation and to promote steatohepatitis features in control animals. Histological analysis revealed that the MASH-ECM was completely recellularized after transplantation in both control and MASH recipient rats. However, steatosis and fibrosis were observed in MASH ECM after transplantation in both groups. Molecular analysis showed that MASH ECM stimulates de novo lipogenesis and fibrosis 30 days after transplantation. Untargeted metabolomic analysis revealed that cells grown on MASH ECM had a similar metabolic profile, even when transplanted into healthy or MASH recipient rats. In addition, we observed that MASH ECM promoted impaired lipid oxidation and mitochondrial dysfunction when transplanted into healthy recipients. Altered lipid turnover and inflammatory signaling were observed in MASH ECM transplanted in MASH recipients. In vitro analysis revealed that MASH ECM induced lipid accumulation in HepG2 cells after 10 days of culture. Calcium signalling experiments obtained from HepG2 cells cultured in MASH ECM showed a lower response to ATP, a reduced calcium signalling amplitude, and a distinct response profile than that observed in healthy ECM. On the other hand, a diseased human-derived ECM could still provide an environment that allows cell development. Taken together, our data showed that MASH ECM impacts cell metabolism, promoting steatohepatitis maintenance. In conclusion, our data confirm that diseased ECM memory can impact cell physiology contributing to disease progression.

Mesenchymal stem cells (MSCs) cultured in hypoxic conditions have been suggested to have more therapeutic efficacy than those cultured under normoxic conditions, and there is growing interest in using hypoxic MSCs for clinical treatment, particularly human umbilical cord (hUC)-MSCs. We investigated how hUC-MSCs and human bone marrow (hBM)-MSCs change from normoxia to hypoxia (1% O2) for 2 weeks of culture. In the growth speed and population doubling time, hUC-MSCs cultured under hypoxia exhibited a significantly higher proliferation rate beyond cancerous cells, such as human glioblastoma and breast cancer cells, while hBM-MSCs did not show a significant difference between normoxia and hypoxia, and were statistically slower than these cancerous cells. Notably, hypoxic hUC-MSCs showed upregulation of genes related to metabolic reprogramming (cholesterol biosynthesis and fatty acid metabolism pathways) and cancer stem cell-like phenotype (factors related to Wnt and Hedgehog signaling pathways, cell proliferation drivers, and apoptosis-resistance), and lesser migration and homing to the traumatic brain injury than normoxic hUC-MSCs after intravenous injection. Thus, whether hUC-MSCs cultured under hypoxia offer clinical benefits and use are safe, given their extremely accelerated proliferation rate and partial cancer stem cell-like traits, requires comprehensive and careful investigation.

Despite the growing prevalence of non-healing diabetic wounds, no current treatment options overcome multifactorial deficits in repair. To this end, a mesenchymal stromal cell-derived regenerative extracellular matrix (rECM) was evaluated for the ability to accelerate cutaneous wound repair in leptin receptor-deficient (db/db) diabetic mice with paired full-thickness dorsal skin defects. A single dose of rECM significantly accelerated wound closure compared with vehicle controls. Also, rECM dose-dependently improved overall histological healing scores and modulated granulation tissue dynamics, with the highest dose promoting rapid resolution of granulation tissue relative to wound area. Spatial transcriptomics and immunofluorescence revealed that rECM drove robust formation of de novo peripheral nerve clusters characterized by the Schwann cell marker, p75. The rECM also enhanced vascular maturation in healed wounds, increasing average blood vessel size, smooth muscle actin-positive vessels, and vessel density within myofibroblast-rich regions. In a complementary 3D angiogenic sprouting model, rECM accelerated endothelial invasion and filopodia extension, and at higher concentrations induced contraction of collagen matrices consistent with accelerated resolution of granulation tissue. These data demonstrate that rECM accelerates closure of diabetic skin defects by coordinating faster granulation tissue remodeling with enhanced peripheral nerve formation and vascular maturation.

Donor organ shortage remains the major barrier to transplantation resulting in deaths on the waiting list. For lungs, aspiration-related injury is a common cause of donor organ discard and increases the risk of primary graft dysfunction. Currently, no effective therapies exist to repair damaged donor lungs prior to transplantation. Here, we investigated whether mesenchymal stromal cells (MSCs) from bone marrow or full-term amniotic fluid could restore severely injured donor lungs in a porcine model integrating ex vivo lung perfusion, transplantation and post-transplant follow-up (n=48; 24 donors, 24 recipients). MSCs were administered either once during ex vivo lung perfusion or repeatedly across lung perfusion and the early post-transplant period and compared with placebo treated controls. A single dose conferred only partial benefit, whereas repeated dosing restored graft function, normalized gas exchange and haemodynamics, and prevented graft dysfunction. MSCs from both sources were similarly effective in repeated regimens. These findings identify dosing schedule, rather than cell source, as key determinant of durable organ rescue and support perfusion-guided cell therapy as potentially generalizable regenerative strategy across solid-organ transplantation.

Therapies based on mesenchymal stromal cells (MSCs) have high potential in the field of regenerative medicine due mainly to their immunomodulatory properties. However, their clinical translation is hampered by a lack of sufficiently standardised potency tests. Since macrophages comprise key mediators of the effects of MSCs, macrophage-based assays potentially provide a relevant in vitro tool for the evaluation of the activity of MSC products. This study involved the coculturing of canine adipose-derived mesenchymal stem cells (ASCs) with macrophages derived from human THP-1 and U937 monocyte cell lines, murine RAW264.7 macrophages and primary human macrophages. The M2 polarisation was assessed following stimulation with IL-4/IL-13. The mRNA expression of the pro- and anti-inflammatory markers was analysed applying qPCR. The ASC secretome acted to reduce the pro-inflammatory mRNA expression across all the macrophage models, albeit with a certain degree of model-dependent variability. Only the U937 macrophages responded consistently to the M2-polarising stimuli, while the RAW264.7 cells provided practical advantages in terms of routine screening. The results thus provided support for the application of macrophage-based potency assays as a suitable platform for the testing of MSC products; the U937 cells were found to be particularly suitable for the study of polarisation and the RAW264.7 cells for standardised screening.

Extracellular vesicles (EVs) are increasingly recognized as critical mediators of intercellular communication, not least during cellular stress or therapy. While EV signalling is well-studied in various tissues, its role in the prepubertal testicular environment is not well understood. Chemotherapy, commonly used in paediatric oncology, poses a significant risk to spermatogonial stem cells (SSCs) and may affect long-term fertility in cancer survivors. The role of EVs in chemotherapy-induced testicular damage in these patients is unknown and may be important for developing new fertility preservation methods. Immortalised murine Sertoli (TM4) and spermatogonial (GC1-spg) cell lines were used to investigate cisplatin-induced changes in EV biogenesis, release, and function in an in vitro model of the prepubertal testicular microenvironment. Our findings indicate that cisplatin significantly increases EV secretion and internalisation by recipient cells. Notably, EVs from cisplatin-exposed Sertoli cells exhibit a novel pro-apoptotic phenotype when co-cultured with chemotherapy-naive Sertoli cells. Proteomic profiling of these EVs shows enrichment of apoptosis-regulatory proteins including caspases, activating Caspase-3/7 in recipient Sertoli cells. Conversely, germ cells exposed to Sertoli cell-derived EVs displayed reduced levels of apoptosis as well as a chemoprotective role to germ cells undergoing treatment with cisplatin. These findings indicate a dual role for Sertoli cell-derived EVs in mediating (1) apoptosis in Sertoli cells and (2) protection of germ cells following cisplatin exposure. The presence of pro-apoptotic molecules, especially caspases, in chemotherapy-induced Sertoli cell EVs provides mechanism for the induction of somatic cell apoptosis. Furthermore, their protective effects on germ cells demonstrate the complexity of EV-mediated signalling between testicular cell types. Manipulating EV biogenesis and cargo loading could be a promising approach to reduce chemotherapy-related gonadotoxicity and preserve fertility in childhood cancer patients.

Somatic cell nuclear transfer (SCNT) holds great promise for regenerative medicine and agriculture, but its application is severely hampered by low efficiency, primarily attributable to aberrant epigenetic reprogramming. Although embryonic stem cells (ESCs) and trophoblast stem cells (TSCs) have been successfully derived from cloned embryos, an in vitro counterpart of the primitive endoderm (PrE) lineage has remained unavailable. To address this gap, this study reports the first successful establishment of extra-embryonic endoderm stem cell lines (XENs) from mouse SCNT-derived blastocysts (NT-XENs). Under conventional culture conditions, NT-XENs were generated from hybrid B6D2F1 blastocysts at a high efficiency of 55%, comparable to that of fertilization-derived XEN lines (FD-XENs, 50%), whereas derivation from inbred C57BL/6J SCNT-derived blastocysts was markedly lower (12.5%). Immunofluorescence and NanoString multiplex gene expression profiling confirmed that NT-XENs robustly expressed specific marker genes for PrE/XENs (e.g., Gata4, Gata6, Sox17), while exhibiting negligible or absent expression of pluripotency and trophoblast markers. Based on NanoString assay data, NT-XENs and FD-XENs shared highly similar global gene expression patterns, yet also exhibited some nonnegligible differences, exemplified by the differentially expressed genes (DEGs) Pecam1, Gtl2, Thbd and Xlr3b, which may suggest that the NT-XENs resided in a more differentiated state (potentially biased toward parietal endoderm (PE)) and retained SCNT-specific epigenetic imprinting errors, including aberrant X-chromosome inactivation and dysregulation of imprinted domains. In summary, this study successfully establishes NT-XEN cell lines, providing a valuable in vitro model for investigating the reprogramming scenarios of PrE lineage in SCNT and offering novel insights into the mechanisms underlying developmental failure of cloned embryos.

Interspecies chimeras comprising human tissues have potential for use in disease modeling and regenerative medicine. Here, we successfully transplanted human induced pluripotent stem cell (iPSC)-derived PDX1+ pancreatic progenitor cells into Pdx1-deficient mouse embryos via intraplacental injection. The engrafted human cells predominantly localized to the duodenum, produced insulin, and extended the lifespan of Pdx1-/- mice by up to 10 days after birth. Transcriptomic analyses confirmed human pancreatic gene expression in human cells engrafted into the mouse duodenum. Our findings demonstrated the feasibility of generating interspecies chimeras with functional human pancreatic cells through in utero transplantation of lineage-committed progenitors. This approach circumvents developmental barriers while minimizing ethical concerns associated with PSCs. However, the incomplete rescue of the Pdx1-/- phenotype highlights the need for further research to enhance human cell engraftment and tissue integration. Overall, this study provides a foundation for developing human-animal chimera models for studying human development and regenerative therapies.

The bone marrow microenvironment forms a highly specialized niche that houses hematopoietic stem and progenitor cells (HSPCs). Within bone, two anatomically distinct regions, the medullary cavity and the trabecular compartment, differ in their cellular and physical composition, with the potential to differentially regulate influence on resident HSPCs. We hypothesized that HSPCs enriched from the medullary cavity (BM) and trabeculae (TB) represent functionally distinct populations. Contrary to this, functional assessment of HSPCs revealed comparable cellular outputs between BM- and TB-derived HSPCs. To investigate whether microenvironmental signaling contributes to functional regulation, we examined the effects of extracellular vesicles (EVs) isolated from medullary BM and TB. Notably, TB-derived EVs inhibited cell cycle progression, directing HSPCs toward a quiescent state. Together, these findings demonstrate that while isolated BM- and TB-derived HSPCs exhibit similar cell-intrinsic properties, EVs enriched from the TB specifically promote HSPC quiescence, supporting a protective regulatory role for the trabecular microenvironment.

Blood and immune cell regeneration is sustained by hematopoietic stem and progenitor cells (HSPCs), which form the therapeutic basis of bone marrow transplantation. While the functional hierarchy of mouse HSPC subsets is well characterized, the distinct roles of human HSPC populations remain less well defined, particularly at clonal resolution and in the context of transplantation conditioning. While clonal tracking in humans and non-human primates has significantly advanced our understanding of hematopoietic dynamics, prior studies predominantly focused on CD34+ cells, a heterogeneous population of HSPCs. Moreover, secondary transplantation is considered the gold standard for distinguishing hematopoietic stem cells (HSCs) from multipotent progenitors (MPPs) in mice, but it has not been effectively utilized to study human HSPC populations. To address this knowledge gap, we performed quantitative clonal tracking of purified human HSCs (hHSCs) and human MPPs (hMPPs) in NSGW41 mice across primary and secondary transplantation under no conditioning, busulfan, and irradiation. Consistent with prior studies, both hHSCs and hMPPs sustained long-term multilineage reconstitution and differed in engraftment rates. Our quantitative clonal analysis further revealed that hHSC clones generated more blood cells, initiated lymphoid production earlier, and exhibited more robust multilineage differentiation than hMPP clones. hHSC clones were also less sensitive to conditioning, maintaining stable lineage biases. Notably, busulfan and irradiation differentially affected the magnitude, lineage bias, and timing of hematopoietic reconstitution without altering engraftment. During secondary transplantation, hHSCs and hMPPs contributed comparably to hematopoietic reconstitution, but their overall output, particularly monocytes and T cells, was substantially reduced. In contrast to primary recipients, human chimerism of secondary recipients in the peripheral blood was diminished relative to the bone marrow and spleen, and more hHSPC clones contributed to hematopoiesis. Extramedullary hematopoiesis was observed in all secondary recipients, with comparable contributions from hHSC and hMPP clones. Overall, this study provides insights into the distinct functions of hHSCs and hMPPs, the influence of conditioning, and the inefficiency of human hematopoiesis through serial transplantation. These findings advance our understanding of human hematopoiesis and provide a framework for utilizing and optimizing experimental models, improving transplantation conditioning strategies, and informing the preclinical evaluation of HSC-based cell and gene therapies.

Prolonged cytopenias are a frequent complication of chimeric antigen receptor (CAR) T-cell therapies and are associated with increased infection risk and non-relapse mortality. Although inflammatory cytokines released during CAR-T cell activation have been implicated in immune effector cell-associated hematotoxicity (ICAHT), their direct effects on human hematopoietic stem and progenitor cells' (HSPCs) function remains incompletely understood. Here, we established a reductionist model of CAR-T-associated hematotoxicity using conditioned media (CM) derived from activated CD19 CAR-T cells. Sustained exposure of human HSPCs to CAR-T-derived inflammatory secretome impaired HSPC expansion and reduced long-term repopulating capacity in xenotransplantation assays. In contrast, short-term exposure did not abrogate HSPC function, indicating that brief inflammatory signals can initiate durable reprogramming events, with functional consequences emerging during subsequent proliferative expansion. Mechanistically, CAR-T CM induced IFN gamma- (IFNg) and TNF alpha- (TNFa) responsive transcriptional programs in HSPCs and promoted inflammatory myeloid skewing without evidence of apoptosis-dependent stem cell loss. Combined inhibition of IFNg and TNFa restored HSPC expansion, normalized lineage output, reversed inflammatory transcriptional signatures, and rescued in vivo repopulating capacity without impairing CAR-T cytotoxic activity. These findings demonstrate that CAR-T-derived inflammatory signaling can directly impair human HSC function and identify dual IFNg/TNFa blockade as a potential strategy to mitigate CAR-T-associated hematotoxicity while preserving antitumor efficacy.

BackgroundCompetitive transplantation is essential for defining intrinsic repopulating capacity of murine hematopoietic stem and progenitor cells (HSPCs), yet comparable assays for human cells have been limited by the lack of a robust in vivo platform. MethodsHere, we describe a novel competitive transplantation method in humanized NOD.Cg-KitW-41J Tyr + Prkdcscid Il2rgtm1Wjl/ThomJ (NBSGW) mice that enables simultaneous engraftment and longitudinal tracking of distinct human grafts within a shared microenvironment. ResultsUsing human leukocyte antigen-mismatched donor CD34+ cells, this method facilitates standard flow cytometry panels to track multiple donor cell chimerism, lineage output, and HSPC composition. The experimental framework may be adapted to different mouse models, conditioning strategies, donor sources, and treatments. ConclusionsOverall, this humanized competitive repopulation assay fills a critical translational gap and offers a flexible foundation for advancing mechanistic discovery in human hematopoietic biology and improving clinical strategies for stem cell transplantation.

Primordial germ cells (PGCs) are the population of cells that, in the human embryo, specify day 12 post-fertilization, and form the precursor cells for the future egg or sperm cells. Although in vitro differentiation of PGCs from human stem cells has been achieved, these primordial germ cell-like cells (hPGCLCs) fail to further mature. The reason for this is unclear. Previous studies in mice revealed that several specific metabolic changes occur during the maturation of these cells, which are essential for their developmental progress. However, very little is known about the metabolic profile of human primordial germ cells. In the severe scarcity of human PGCs, hPGCLCs serve as a research model to study PGC formation. To investigate this, we differentiated hPGCLCs using induced-pluripotent stem cells and performed a mass spectrometry analysis to establish their metabolome and proteome. These cells revealed distinct metabolic profile, with changes particularly at the proteome level. This included a shift between canonical and non-canonical citric acid cycle in hPGCLC, downregulation of late-stage glycolysis and reduction of nucleotide de novo synthesis. By providing an integrative map of these metabolic networks, we aim to provide insight on the influence of metabolism on human PGC development that could help improve methods for in vitro differentiation and maturation hPGCLCs.

Insufficient vascularization remains a major obstacle to the success of cell-based therapies for diabetes. Building on prior findings that loss of the phosphatase PTP1B enhances VEGFA production and improves graft vascularization, we investigated sodium tungstate (NaW), a pharmacological phosphatase inhibitor, as a strategy to improve transplantation outcomes. Using human fibroblast-derived insulin-producing cells and human stem cell-derived islets transplanted into the anterior chamber of the eye in immunodeficient mice, we show that NaW treatment significantly increases both vascularized area and insulin-positive tissue area, while reducing apoptosis within transplanted cells. Mechanistically, NaW upregulates VEGFA expression in transplanted cells and amplifies VEGFA-induced endothelial cell proliferation, migration, and tubulogenesis via MAPK/ERK signalling. These dual effects, which encompass stimulating both endocrine and endothelial compartments, lead to enhanced integration and function of transplanted cells. Importantly, the pro-angiogenic effects of NaW occur independently of exogenous endothelial cell supplementation, relying solely on the host endogenous endothelial cells. These findings position NaW, and potentially other phosphatase inhibitors, as promising adjuncts to improve vascularization, survival, and therapeutic efficacy in clinical cell-based transplantation protocols for diabetes. One-sentence summaryProangiogenic role of sodium tungstate in transplantation

During embryonic development, neural crest cells (NCCs) represent a multipotent population characterized by an inherently transient nature, rapidly differentiating into various lineages. This instability has presented a fundamental challenge, as it is exceedingly difficult to maintain these cells in a stable, multipotent state in vitro. In this study, we report a robust culture system dependent on Wnt and FGF signaling that enables the long-term (>6 months) expansion of human iPSC-derived neural crest stem cells (NCSCs). These NCSCs retain their self-renewal and differentiation capacity, validated at the single-cell clonal level. ATAC-seq analysis indicated that posterior NCSCs maintain a more permissive chromatin structure at neuronal gene loci. Furthermore, ChIP-seq analysis revealed that the key transcription factor SOX10 binds to the regulatory regions of genes involved in both maintenance and differentiation. This system provides a stable source of human NCSCs, offering a valuable platform for developmental biology, disease modeling, and regenerative medicine.

BackgroundVascular mural cells (MC) are essential components of vasculature, playing critical roles in tissue regeneration and cell therapy. The use of animal derived ancillary materials, like fetal bovine serum (FBS), in the induction of MC from human pluripotent stem cells (hPSCs), represents one of the biggest limitations to guarantee preclinical safety standards required to use this products in clinical settings. This study aimed to validate human platelet lysate (hPL) as a serum-free alternative for MC differentiation from hPSCs. MethodsComparison of MC differentiation efficiency from hiPSC using FBS vs hPL supplemented cultures was performed, along with functionality and gene expression assessment through bulk RNA sequencing. ResultsOptimization of hPL concentration identified hPL1% as the most effective condition, yielding PDGFR-{beta}+/CNN1+ MC, with a comparable efficiency to FBS10% and similar interaction with endothelial cells in vascular formation assays. However, distinct transcriptional profiles revealed that FBS10% and hPL1% drive differentiation toward different MC subphenotypes; hPL1% promoted contractile gene expression, while FBS10% enriched extracellular matrix pathways. Higher hPL concentrations further shifted differentiation toward cardiomyocytes. ConclusionIn monolayer in vitro differentiation of MC from hiPSC, the differentiation efficiency using hPL 1% supplementation is equivalent to FBS 10%, while supporting a more contractile phenotype. These findings establish hPL as a xeno-minimized, clinically compliant substitute for FBS for hPSC-derived MC differentiation, an important breakthrough for regenerative medicine.

Autologous cell suspension (ACS)-based therapies are an established strategy to enhance wound repair, yet limitations in preparation workflows and donor skin requirements remain barriers to wider clinical implementation. We have previously developed VeritaCell, a rapid enzymatic disaggregation-based approach that generates highly viable skin cell populations, including epidermal stem cell-enriched fractions, and demonstrated their pro-regenerative biological properties in vitro. Here, we have evaluated the in vivo efficacy of VeritaCell-derived ACS using a rat full-thickness excisional wound model. ACS preparations were applied at donor-to-wound area ratios of 1:1, 1:10, and 1:20, and wound progression was monitored through longitudinal image-based quantification alongside histological assessment of tissue architecture. ACS-treated wounds exhibited enhanced early wound closure dynamics, with significant within-group improvements evident by Day 6. Histological analysis demonstrated improved neo-epithelial organisation and reduced epidermal thickening in the 1:10 and 1:20 groups, with the 1:10 condition showing tissue architecture most closely resembling unwounded skin. Notably, beneficial effects were observed even at low estimated cell numbers, suggesting that cell viability and biological activity may be key determinants of therapeutic efficacy. Collectively, these findings provide in vivo validation of VeritaCell-derived ACS and support the use of biologically informed donor-to-wound coverage ratios. This approach may enable effective wound repair while minimising donor skin requirements, with potential relevance for the treatment of extensive injuries such as burns.