One-step cell biomanufacturing platform:porous gelatin microcarrier beads promote human embryonic stem cell-derived midbrain dopaminergic progenitor cell differentiation in vitro and survival after transplantation in vivo

Numerous studies have shown that cell replacement therapy can replenish lost cells and rebuild neural circuitry in animal models of Parkinson’s disease.Transplantation of midbrain dopaminergic progenitor cells is a promising treatment for Parkinson’s disease.However,transplanted cells can be injured by mechanical damage during handling and by changes in the transplantation niche.Here,we developed a one-step biomanufacturing platform that uses small-aperture gelatin microcarriers to produce beads carrying midbrain dopaminergic progenitor cells.These beads allow midbrain dopaminergic progenitor cell differentiation and cryopreservation without digestion,effectively maintaining axonal integrity in vitro.Importantly,midbrain dopaminergic progenitor cell bead grafts showed increased survival and only mild immunoreactivity in vivo compared with suspended midbrain dopaminergic progenitor cell grafts.Overall,our findings show that these midbrain dopaminergic progenitor cell beads enhance the effectiveness of neuronal cell transplantation.

Combining neural progenitor cell transplant and rehabilitation for enhanced recovery after cervical spinal cord injury

Efforts to promote recovery of function after human spinal cord injury(SCI) will likely require interventions to rgeting the corticospinal tract(CST) motor system:the most important pathway for voluntary motor control in humans.This system has historically been the most refractory to regenerative efforts after SCI.The "nonregeneration" of the CST changed when robust regeneration of the CST into spared tissue was demonstrated by the inactivation of phosphatase and tensin homolog and delivery of inosine.

Aberrant adult neurogenesis in intractable epilepsy:can GABAergic progenitor transplantation normalize this process?

Temporal lobe epilepsy(TLE) is a common type of focal epilepsy characterized by seizure foci within the temporal lobes.While surgical resection of the foci is an established and effective approach for controlling seizures,both temporal lobes cannot be removed,due to their prominent roles in learning and memory.Additionally,seizures induce changes to the temporal lobes that contribute to hyperexcitability,including mossy fiber sprouting,astrogliosis.

Perilipin-2 mediates ferroptosis in oligodendrocyte progenitor cells and myelin injury after ischemic stroke

Differentiation of oligodendrocyte progenitor cells into mature myelin-forming oligodendrocytes contributes to remyelination.Failure of remyelination due to oligodendrocyte progenitor cell death can result in severe nerve damage.Ferroptosis is an iron-dependent form of regulated cell death caused by membrane rupture induced by lipid peroxidation,and plays an important role in the pathological process of ischemic stroke.However,there are few studies on oligodendrocyte progenitor cell ferroptosis.We analyzed transcriptome sequencing data from GEO databases and identified a role of ferroptosis in oligodendrocyte progenitor cell death and myelin injury after cerebral ischemia.Bioinformatics analysis suggested that perilipin-2(PLIN2)was involved in oligodendrocyte progenitor cell ferroptosis.PLIN2 is a lipid storage protein and a marker of hypoxia-sensitive lipid droplet accumulation.For further investigation,we established a mouse model of cerebral ischemia/reperfusion.We found significant myelin damage after cerebral ischemia,as well as oligodendrocyte progenitor cell death and increased lipid peroxidation levels around the infarct area.The ferroptosis inhibitor,ferrostatin-1,rescued oligodendrocyte progenitor cell death and subsequent myelin injury.We also found increased PLIN2 levels in the peri-infarct area that co-localized with oligodendrocyte progenitor cells.Plin2 knockdown rescued demyelination and improved neurological deficits.Our findings suggest that targeting PLIN2 to regulate oligodendrocyte progenitor cell ferroptosis may be a potential therapeutic strategy for rescuing myelin damage after cerebral ischemia.

Small extracellular vesicles derived from human induced pluripotent stem cell-differentiated neural progenitor cells mitigate retinal ganglion cell degeneration in a mouse model of optic nerve injury

Several studies have found that transplantation of neural progenitor cells(NPCs)promotes the survival of injured neurons.However,a poor integration rate and high risk of tumorigenicity after cell transplantation limits their clinical application.Small extracellular vesicles(sEVs)contain bioactive molecules for neuronal protection and regeneration.Previous studies have shown that stem/progenitor cell-derived sEVs can promote neuronal survival and recovery of neurological function in neurodegenerative eye diseases and other eye diseases.In this study,we intravitreally transplanted sEVs derived from human induced pluripotent stem cells(hiPSCs)and hiPSCs-differentiated NPCs(hiPSC-NPC)in a mouse model of optic nerve crush.Our results show that these intravitreally injected sEVs were ingested by retinal cells,especially those localized in the ganglion cell layer.Treatment with hiPSC-NPC-derived sEVs mitigated optic nerve crush-induced retinal ganglion cell degeneration,and regulated the retinal microenvironment by inhibiting excessive activation of microglia.Component analysis further revealed that hiPSC-NPC derived sEVs transported neuroprotective and anti-inflammatory miRNA cargos to target cells,which had protective effects on RGCs after optic nerve injury.These findings suggest that sEVs derived from hiPSC-NPC are a promising cell-free therapeutic strategy for optic neuropathy.

Revolutionizing tumor immunotherapy:unleashing the power of progenitor exhausted T cells

In exploring persistent infections and malignancies, a distinctive subgroup of CD8^(+) T cells, progenitor exhausted CD8^(+) T(Tpex) cells, has been identified. These Tpex cells are notable for their remarkable self-renewal and rapid proliferation abilities. Recent strides in immunotherapy have demonstrated that Tpex cells expand and differentiate into responsive exhausted CD8^(+) T cells, thus underscoring their critical role in the immunotherapeutic retort. Clinical examinations have further clarified a robust positive correlation between the proportional abundance of Tpex cells and enhanced clinical prognosis. Tpex cells have found noteworthy applications in the formulation of inventive immunotherapeutic approaches against tumors. This review describes the functions of Tpex cells in the tumor milieu, particularly their potential utility in tumor immunotherapy. Precisely directing Tpex cells may be essential to achieving successful outcomes in immunotherapy against tumors.

Single-cell sequencing technology in diabetic wound healing:New insights into the progenitors-based repair strategies

Diabetes mellitus(DM),an increasingly prevalent chronic metabolic disease,is characterised by prolonged hyperglycaemia,which leads to long-term health consequences.Although much effort has been put into understanding the pathogenesis of diabetic wounds,the underlying mechanisms remain unclear.The advent of single-cell RNA sequencing(scRNAseq)has revolutionised biological research by enabling the identification of novel cell types,the discovery of cellular markers,the analysis of gene expression patterns and the prediction of develop-mental trajectories.This powerful tool allows for an in-depth exploration of pathogenesis at the cellular and molecular levels.In this editorial,we focus on progenitor-based repair strategies for diabetic wound healing as revealed by scRNAseq and highlight the biological behaviour of various healing-related cells and the alteration of signalling pathways in the process of diabetic wound healing.ScRNAseq could not only deepen our understanding of the complex biology of diabetic wounds but also identify and validate new targets for inter-vention,offering hope for improved patient outcomes in the management of this challenging complication of DM.

Hypoxic pre-conditioned adipose-derived stem/progenitor cells embedded in fibrin conduits promote peripheral nerve regeneration in a sciatic nerve graft model

Recent results emphasize the supportive effects of adipose-derived multipotent stem/progenitor cells(ADSPCs)in peripheral nerve recovery.Cultivation under hypoxia is considered to enhance the release of the regenerative potential of ADSPCs.This study aimed to examine whether peripheral nerve regeneration in a rat model of autologous sciatic nerve graft benefits from an additional custom-made fibrin conduit seeded with hypoxic pre-conditioned(2%oxygen for 72 hours)autologous ADSPCs(n=9).This treatment mode was compared with three others:fibrin conduit seeded with ADSPCs cultivated under normoxic conditions(n=9);non-cell-carrying conduit(n=9);and nerve autograft only(n=9).A 16-week follow-up included functional testing(sciatic functional index and static sciatic index)as well as postmortem muscle mass analyses and morphometric nerve evaluations(histology,g-ratio,axon density,and diameter).At 8 weeks,the hypoxic pre-conditioned group achieved significantly higher sciatic functional index/static sciatic index scores than the other three groups,indicating faster functional regeneration.Furthermore,histologic evaluation showed significantly increased axon outgrowth/branching,axon density,remyelination,and a reduced relative connective tissue area.Hypoxic pre-conditioned ADSPCs seeded in fibrin conduits are a promising adjunct to current nerve autografts.Further studies are needed to understand the underlying cellular mechanism and to investigate a potential application in clinical practice.

Neural progenitor cells derived from fibroblasts induced by small molecule compounds under hypoxia for treatment of Parkinson’s disease in rats

Neural progenitor cells(NPCs) capable of self-renewal and differentiation into neural cell lineages offer broad prospects for cell therapy for neurodegenerative diseases. However, cell therapy based on NPC transplantation is limited by the inability to acquire sufficient quantities of NPCs. Previous studies have found that a chemical cocktail of valproic acid, CHIR99021, and Repsox(VCR) promotes mouse fibroblasts to differentiate into NPCs under hypoxic conditions. Therefore, we used VCR(0.5 mM valproic acid, 3 μM CHIR99021, and 1 μM Repsox) to induce the reprogramming of rat embryonic fibroblasts into NPCs under a hypoxic condition(5%). These NPCs exhibited typical neurosphere-like structures that can express NPC markers, such as Nestin, SRY-box transcription factor 2, and paired box 6(Pax6), and could also differentiate into multiple types of functional neurons and astrocytes in vitro. They had similar gene expression profiles to those of rat brain-derived neural stem cells. Subsequently, the chemically-induced NPCs(ciNPCs) were stereotactically transplanted into the substantia nigra of 6-hydroxydopamine-lesioned parkinsonian rats. We found that the ciNPCs exhibited long-term survival, migrated long distances, and differentiated into multiple types of functional neurons and glial cells in vivo. Moreover, the parkinsonian behavioral defects of the parkinsonian model rats grafted with ciNPCs showed remarkable functional recovery. These findings suggest that rat fibroblasts can be directly transformed into NPCs using a chemical cocktail of VCR without introducing exogenous factors, which may be an attractive donor material for transplantation therapy for Parkinson’s disease.

Soxllb regulates the migration and fate determination of Müller glia-derived progenitors during retina regeneration in zebrafish

The transcription factor Sox11 plays important roles in retinal neurogenesis during vertebrate eye development.However,its function in retina regeneration remains elusive.Here we report that Sox11 b,a zebrafish Sox11 homolog,regulates the migration and fate determination of Müller glia-derived progenitors(MGPCs)in an adult zebrafish model of mechanical retinal injury.Following a stab injury,the expression of Sox11 b was induced in proliferating MGPCs in the retina.Sox11 b knockdown did not affect MGPC formation at 4 days post-injury,although the nuclear morphology and subsequent radial migration of MGPCs were alte red.At 7 days post-injury,Sox11 b knockdown res ulted in an increased proportion of MGPCs in the inner retina and a decreased propo rtion of MGPCs in the outer nuclear layer,compared with controls.Furthermore,Sox11 b knockdown led to reduced photoreceptor regeneration,while it increased the numbe rs of newborn amacrines and retinal ganglion cells.Finally,quantitative polymerase chain reaction analysis revealed that Sox11 b regulated the expression of Notch signaling components in the retina,and Notch inhibition partially recapitulated the Sox11 b knockdown phenotype,indicating that Notch signaling functions downstream of Sox11 b.Our findings imply that Sox11 b plays key roles in MGPC migration and fate determination during retina regeneration in zebrafish,which may have critical im plications for future explorations of retinal repair in mammals.

The characteristics of mRNA m^(6)A methylomes in allopolyploid Brassica napus and its diploid progenitors

Genome duplication events,comprising whole-genome duplication and single-gene duplication,produce a complex genomic context leading to multiple levels of genetic changes.However,the characteristics of m^(6)A modification,the most widespread internal eukaryotic mRNA modification,in polyploid species are still poorly understood.This study revealed the characteristics of m^(6)A methylomes within the early formation and following the evolution of allopolyploid Brassica napus.We found a complex relationship between m^(6)A modification abundance and gene expression level depending on the degree of enrichment or presence/absence of m^(6)A modification.Overall,the m^(6)A genes had lower gene expression levels than the non-m^(6)A genes.Allopolyploidization may change the expression divergence of duplicated gene pairs with identical m^(6)A patterns and diverged m^(6)A patterns.Compared with duplicated genes,singletons with a higher evolutionary rate exhibited higher m^(6)A modification.Five kinds of duplicated genes exhibited distinct distributions of m^(6)A modifications in transcripts and gene expression level.In particular,tandem duplication-derived genes showed unique m^(6)A modification enrichment around the transcript start site.Active histone modifications(H3K27ac and H3K4me3)but not DNA methylation were enriched around genes of m^(6)A peaks.These findings provide a new understanding of the features of m 6A modification and gene expression regulation in allopolyploid plants with sophisticated genomic architecture.

Gene-modified neural progenitor cells for the treatment of neuropathic lysosomal storage diseases

Lysosomal storage diseases:Lysosomal storage diseases(LSDs) are a family of about 70 disorders,with an overall incidence of 1:7000 live births.They are caused by dysfunctional lysosomal hydrolases,eventually leading to the accumulation of undegraded substrate into the lysosome.This results in a wide array of symptoms,which may include:the presence of dysmorphic features,cardio-respiratory disease,bone and joint disease,organomegaly,developmental delay and neurocognitive decline.The majority of these diseases have a neurological component and in the absence of treatment.

CD34^(+ )progenitor cells as diagnostic and therapeutic targets in Alzheimer's disease

Alzheimer’s disease(AD)is the main neurodegenerative disease leading to dementia and cognitive impairment in the elderly.Considering AD to be an epidemic,an increase from the current 50 million to more than 150 million patients is expected by the year 2050.

Nutrient-regulated dynamics of chondroprogenitors in the postnatal murine growth plate

Longitudinal bone growth relies on endochondral ossification in the cartilaginous growth plate,where chondrocytes accumulate and synthesize the matrix scaffold that is replaced by bone.The chondroprogenitors in the resting zone maintain the continuous turnover of chondrocytes in the growth plate.Malnutrition is a leading cause of growth retardation in children;however,after recovery from nutrient deprivation,bone growth is accelerated beyond the normal rate,a phenomenon termed catch-up growth.Although nutritional status is a known regulator of long bone growth,it is largely unknown whether and how chondroprogenitor cells respond to deviations in nutrient availability.Here,using fate-mapping analysis in Axin2CreERT2 mice,we showed that dietary restriction increased the number of Axin2+chondroprogenitors in the resting zone and simultaneously inhibited their differentiation.Once nutrient deficiency was resolved,the accumulated chondroprogenitor cells immediately restarted differentiation and formed chondrocyte columns,contributing to accelerated growth.Furthermore,we showed that nutrient deprivation reduced the level of phosphorylated Akt in the resting zone and that exogenous IGF-1 restored the phosphorylated Akt level and stimulated differentiation of the pooled chondroprogenitors,decreasing their numbers.Our study of Axin2CreERT2 revealed that nutrient availability regulates the balance between accumulation and differentiation of chondroprogenitors in the growth plate and further demonstrated that IGF-1 partially mediates this regulation by promoting the committed differentiation of chondroprogenitor cells.

Premature aging of skeletal stem/progenitor cells rather than osteoblasts causes bone loss with decreased mechanosensation

A distinct population of skeletal stem/progenitor cells(SSPCs)has been identified that is indispensable for the maintenance and remodeling of the adult skeleton.However,the cell types that are responsible for age-related bone loss and the characteristic changes in these cells during aging remain to be determined.Here,we established models of premature aging by conditional depletion of Zmpste24(Z24)in mice and found that Prx1-dependent Z24 deletion,but not Osx-dependent Z24 deletion,caused significant bone loss.However,Acan-associated Z24 depletion caused only trabecular bone loss.Single-cell RNA sequencing(sc RNA-seq)revealed that two populations of SSPCs,one that differentiates into trabecular bone cells and another that differentiates into cortical bone cells,were significantly decreased in Prx1-Cre;Z24^(f/f)mice.Both premature SSPC populations exhibited apoptotic signaling pathway activation and decreased mechanosensation.Physical exercise reversed the effects of Z24depletion on cellular apoptosis,extracellular matrix expression and bone mass.This study identified two populations of SSPCs that are responsible for premature aging-related bone loss.The impairment of mechanosensation in Z24-deficient SSPCs provides new insight into how physical exercise can be used to prevent bone aging.

Tppp3~+synovial/tendon sheath progenitor cells contribute to heterotopic bone after trauma

Heterotopic ossification(HO)is a pathological process resulting in aberrant bone formation and often involves synovial lined tissues.During this process,mesenchymal progenitor cells undergo endochondral ossification.Nonetheless,the specific cell phenotypes and mechanisms driving this process are not well understood,in part due to the high degree of heterogeneity of the progenitor cells involved.Here,using a combination of lineage tracing and single-cell RNA sequencing(sc RNA-seq),we investigated the extent to which synovial/tendon sheath progenitor cells contribute to heterotopic bone formation.For this purpose,Tppp3(tubulin polymerization-promoting protein family member 3)-inducible reporter mice were used in combination with either Scx(Scleraxis)or Pdgfra(platelet derived growth factor receptor alpha)reporter mice.Both tendon injury-and arthroplasty-induced mouse experimental HO models were utilized.Sc RNA-seq of tendon-associated traumatic HO suggested that Tppp3 is an early progenitor cell marker for either tendon or osteochondral cells.Upon HO induction,Tppp3 reporter^(+)cells expanded in number and partially contributed to cartilage and bone formation in either tendon-or joint-associated HO.In double reporter animals,both Pdgfra^(+)Tppp3^(+)and Pdgfra^(+)Tppp3^(-) progenitor cells gave rise to HO-associated cartilage.Finally,analysis of human samples showed a substantial population of TPPP3^(-) expressing cells overlapping with osteogenic markers in areas of heterotopic bone.Overall,these data demonstrate that synovial/tendon sheath progenitor cells undergo aberrant osteochondral differentiation and contribute to HO after trauma.

Prim-O-glucosylcimifugin ameliorates aging-impaired endogenous tendon regeneration by rejuvenating senescent tendon stem/progenitor cells

Adult tendon stem/progenitor cells(TSPCs)are essential for tendon maintenance,regeneration,and repair,yet they become susceptible to senescence with age,impairing the self-healing capacity of tendons.In this study,we employ a recently developed deep-learning-based efficacy prediction system to screen potential stemness-promoting and senescence-inhibiting drugs from natural products using the transcriptional signatures of stemness.The top-ranked candidate,prim-O-glucosylcimifugin(POG),a saposhnikovia root extract,could ameliorate TPSC senescent phenotypes caused by long-term passage and natural aging in rats and humans,as well as restore the self-renewal and proliferative capacities and tenogenic potential of aged TSPCs.In vivo,the systematic administration of POG or the local delivery of POG nanoparticles functionally rescued endogenous tendon regeneration and repair in aged rats to levels similar to those of normal animals.Mechanistically,POG protects TSPCs against functional impairment during both passage-induced and natural aging by simultaneously suppressing nuclear factor-κB and decreasing mTOR signaling with the induction of autophagy.Thus,the strategy of pharmacological intervention with the deep learning-predicted compound POG could rejuvenate aged TSPCs and improve the regenerative capacity of aged tendons.

Organoid-derived human retinal progenitor cells promote early dedifferentiation of Müller glia in Royal College of Surgeons rats

AIM:To explore whether the subretinal transplantation of retinal progenitor cells from human embryonic stem cell-derived retinal organoid(h ERO-RPCs)could promote Müller glia dedifferentiation and transdifferentiation,thus improving visual function and delaying retinal degenerative progression.METHODS:h ERO-RPCs were subretinally transplanted into Royal College of Surgeons(RCS)rats.Electroretinography(ERG)recording was performed at 4 and 8wk postoperation to assess retinal function.Using immunofluorescence,the changes in outer nuclear layer(ONL)thickness and retinal Müller glia were explored at 2,4,and 8wk postoperation.To verify the effect of h ERO-RPCs on Müller glia in vitro,we cocultured h ERO-RPCs with Müller glia with a Transwell system.After coculture,Ki67 staining and quantitative polymerase chain reaction(q PCR)were performed to measure the proliferation and m RNA levels of Müller glia respectively.Cell migration experiment was used to detect the effect of h ERO-RPCs on Müller glial migration.Comparisons between two groups were performed by the unpaired Student’s t-test,and comparisons among multiple groups were made with one-way ANOVA followed by Tukey’s multiple comparison test.RESULTS:The visual function and ONL thickness of RCS rats were significantly improved by transplantation of h ERO-RPCs at 4 and 8wk postoperation.In addition to inhibiting gliosis at 4 and 8wk postoperation,h ERO-RPCs significantly increased the expression of dedifferentiation-associated transcriptional factor in Müller glia and promoted the migration at 2,4 and 8wk postoperation,but not the transdifferentiation of these cells in RCS rats.In vitro,using the Transwell system,we found that h ERO-RPCs promoted the proliferation and migration of primary rat Müller glia and induced their dedifferentiation at the m RNA level.CONCLUSION:These results show that h ERO-RPCs might promote early dedifferentiation of Müller glia,which may provide novel insights into the mechanisms of stem cell therapy and Müller glial reprogramming,contributing to the development of novel therapies for retinal degeneration disorders.

Influence of hypoxia on retinal progenitor and ganglion cells in human induced pluripotent stem cell-derived retinal organoids

AIM:To observe the effect of low oxygen concentration on the neural retina in human induced pluripotent stem cell(hiPSC)-derived retinal organoids(ROs).METHODS:The hiPSC and a three-dimensional culture method were used for the experiments.Generated embryoid bodies(EBs)were randomly and equally divided into hypoxic and normoxic groups.Photographs of the EBs were taken on days 38,45,and 52,and the corresponding volume of EBs was calculated.Simultaneously,samples were collected at these three timepoints,followed by fixation,sectioning,and immunofluorescence.RESULTS:The proportion of Ki67-positive proliferating cells increased steadily on day 38;this proliferationpromoting effect tended to increase tissue density rather than tissue volume.On days 45 and 52,the two groups had relatively similar ratios of Ki67-positive cells.Further immunofluorescence analysis showed that the ratio of SOX2-positive cells significantly increased within the neural retina on day 52(P<0.05).In contrast,the percentage of PAX6-and CHX10-positive cells significantly decreased following hypoxia treatment at all three timepoints(P<0.01),except for CHX10 at day 45(P>0.05).Moreover,the proportion of PAX6-/TUJ1+cells within the neural retinas increased considerably(P<0.01,<0.05,<0.05 respectively).CONCLUSION:Low oxygen promotes stemness and proliferation of neural retinas,suggesting that hypoxic conditions can enlarge the retinal progenitor cell pool in hiPSC-derived ROs.

Role of brahma-related gene 1/brahma-associated factor subunits in neural stem/progenitor cells and related neural developmental disorders

Different fates of neural stem/progenitor cells(NSPCs)and their progeny are determined by the gene regulatory network,where a chromatin-remodeling complex affects synergy with other regulators.Here,we review recent research progress indicating that the BRG1/BRM-associated factor(BAF)complex plays an important role in NSPCs during neural development and neural developmental disorders.Several studies based on animal models have shown that mutations in the BAF complex may cause abnormal neural differentiation,which can also lead to various diseases in humans.We discussed BAF complex subunits and their main characteristics in NSPCs.With advances in studies of human pluripotent stem cells and the feasibility of driving their differentiation into NSPCs,we can now investigate the role of the BAF complex in regulating the balance between self-renewal and differentiation of NSPCs.Considering recent progress in these research areas,we suggest that three approaches should be used in investigations in the near future.Sequencing of whole human exome and genome-wide association studies suggest that mutations in the subunits of the BAF complex are related to neurodevelopmental disorders.More insight into the mechanism of BAF complex regulation in NSPCs during neural cell fate decisions and neurodevelopment may help in exploiting new methods for clinical applications.