Spontaneous Differentiation Research Articles

Improving the differentiation potential of pluripotent stem cells by optimizing culture conditions

Embryoid cells and induced pluripotent stem cells (iPSCs) are pluripotent stem cells (PSCs). They retain differentiation and self-renewal potential. However, the differentiation potential of PSCs can be changed by the culture medium. PSCs retain their differentiation potential when cultured with medium that supports the glycolytic pathway, showing high expression of chromodomain-helicase-DNA-binding protein 7 (CHD7), but lose their differentiation potential with medium that supports mitochondrial function, showing reduced levels of CHD7. Labeling cells by their copy number variant profile revealed that genetically different PSC populations can be cultured by medium selection. Another factor that defines the self-renewal potential of PSCs is culture condition. PSCs form colonies as they grow, and spontaneous differentiation inevitably occurs along the rim of these colonies in areas that lack cell-to-cell contact; because of this, undifferentiated cell populations would diminish if differentiated cells are not removed properly. Seeding cells on a less potent cell-binding material may minimize the inclusion of differentiated cells, exploiting the reduced adhesive properties of differentiated cells. Culturing cells with medium that supports the glycolytic pathway, using CHD7 as a biomarker for differentiation potential, and culturing cells on less sticky material can improve the differentiation potential of already established PSC clones.

Generation of two induced pluripotent stem cell lines (RCMGi005-A/B) from human skin fibroblasts of a cystic fibrosis patient with homozygous F508del mutation in CFTR gene

Induced pluripotent stem cells (iPSCs) was successfully generated from skin fibroblast obtained from patient with cystic fibrosis by using non-integrating, viral CytoTune™-iPS 2.0 Sendai Reprogramming Kit, which contain three vectors preparation: polycistronic Klf4–Oct3/4–Sox2, cMyc, and Klf4. Created iPSC lines showed a normal karyotype, expressed pluripotency markers and demonstrated the potential to differentiate into three germ layers in spontaneous differentiation assay.

A Novel Cell-Based Model for a Rare Disease: The Tks4-KO Human Embryonic Stem Cell Line as a Frank-Ter Haar Syndrome Model System.

Tyrosine kinase substrate with four SH3 domains (Tks4) scaffold protein plays roles in cell migration and podosome formation and regulates systemic mechanisms such as adult bone homeostasis and adipogenesis. Mutations in the Tks4 gene (SH3PXD2b) cause a rare developmental disorder called Frank-Ter Haar syndrome (FTHS), which leads to heart abnormalities, bone tissue defects, and reduced adiposity. We aimed to produce a human stem cell-based in vitro FTHS model system to study the effects of the loss of the Tks4 protein in different cell lineages and the accompanying effects on the cell signalome. To this end, we used CRISPR/Cas9 (clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR associated (Cas9)) to knock out the SH3PXD2b gene in the HUES9 human embryonic stem cell line (hESC), and we obtained stable homo- and heterozygous knock out clones for use in studying the potential regulatory roles of Tks4 protein in embryonic stem cell biology. Based on pluripotency marker measurements and spontaneous differentiation capacity assays, we concluded that the newly generated Tks4-KO HUES9 cells retained their embryonic stem cell characteristics. We propose that the Tks4-KO HUES9 cells could serve as a tool for further cell differentiation studies to investigate the involvement of Tks4 in the complex disorder FTHS. Moreover, we successfully differentiated all of the clones into mesenchymal stem cells (MSCs). The derived MSC cultures showed mesenchymal morphology and expressed MSC markers, although the expression levels of mesodermal and osteogenic marker genes were reduced, and several EMT (epithelial mesenchymal transition)-related features were altered in the Tks4-KO MSCs. Our results suggest that the loss of Tks4 leads to FTHS by altering cell lineage differentiation and cell maturation processes, rather than by regulating embryonic stem cell potential.

Abstract P3101: Chromatin Remodeling BAF (SWI/SNF) Complex Controls Both Cardiogenesis And Neurogenesis From Human Pluripotent Stem Cells

Background: Early human heart and brain development simultaneously occur during embryogenesis. Notably, in human newborns, congenital heart defects strongly associate with neurodevelopmental abnormalities, suggesting a common gene or complex underlying both cardiogenesis and neurogenesis. However, due to lack of in vivo studies, the molecular mechanisms that govern both early human heart and brain development remain elusive. Results: Here, we report ARID1A, a DNA-binding subunit of the BAF(SWI/SNF) epigenetic complex, controls both neurogenesis and cardiogenesis from human embryonic stem cells (hESCs) through distinct mechanisms. Loss-of-ARID1A (ARID1A –/– ) leads to spontaneous differentiation of neural cells together with globally enhanced expression of neurogenic genes in undifferentiated hESCs. Additionally, when compared with WT hESCs, cardiac differentiation from ARID1A –/– hESCs is prominently suppressed, whereas neural differentiation is significantly promoted. Whole genome-wide scRNA-seq, ATAC-seq, and ChIP-seq analyses reveal that ARID1A is required to open chromatin accessibility on promoters of essential cardiogenic genes, and temporally associated with key cardiogenic transcriptional factors T and MEF2C during early cardiac development. However, during early neural development, transcription of most essential neurogenic genes is dependent on ARID1A, which can interact with a known neural restrictive silencer factor REST/NRSF. Conclusions: We uncover the key and opposite roles by the BAF(SWI/SNF) complex to govern early cardiac and neural development. Global chromatin accessibility on cardiogenic genes is dependent on ARID1A, whereas transcriptional activity of neurogenic genes is under control by ARID1A-REST/NRSF interaction.

Generation of induced pluripotent stem cell line (RCMGi008-A) from human skin fibroblasts of a cystic fibrosis patient with compound heterozygous F508del/CFTRdele2.3 mutations in CFTR gene

Skin fibroblasts obtained from a 20-year-old woman with clinically manifested and genetically proven (F508del/CFTRdele2.3) cystic fibrosis were successfully transformed into induced pluripotent stem cells (iPSCs) by using Sendai virus-based reprogramming vectors including the four Yamanaka factors, OCT3/4, SOX2, KLF4, and c-MYC. The iPSCs showed a normal karyotype, expressed pluripotency markers and exhibited the potential to differentiate into three germ layers in spontaneous differentiation assay. This iPSC line may be used for development of a personalized treatment including genome editing, disease modelling, cell differentiation and organoid formation, pharmacological investigations and drug screening.

Optimizing Tenogenic Differentiation of Equine Adipose-Derived Mesenchymal Stem Cells (eq-ASC) Using TGFB3 Along with BMP Antagonists.

Objective Tendon repair strategies usually are accompanied by pathological mineralization and scar tissue formation that increases the risk of re-injuries. This study aimed to establish an efficient tendon regeneration method simultaneously with a reduced risk of ectopic bone formation. Materials and MethodsIn this experimental study, tenogenic differentiation was induced through transforming growth factor- β3 (TGFB3) treatment in combination with the inhibiting concentrations of bone morphogenetic proteins (BMP) antagonists, gremlin-2 (GREM2), and a Wnt inhibitor, namely sclerostin (SOST). The procedure’s efficacy was evaluated using real-time polymerase chain reaction (qPCR) for expression analysis of tenogenic markers and osteo- chondrogenic marker genes. The expression level of two tenogenic markers, SCX and MKX, was also evaluated by immunocytochemistry. Sirius Red staining was performed to examine the amounts of collagen fibers. Moreover, to investigate the impact of the substrate on tenogenic differentiation, the nanofibrous scaffolds that highly resemble tendon extracellular matrix was employed.Results Aggregated features formed in spontaneous normal culture conditions followed by up-regulation of tenogenic and osteogenic marker genes, including SCX, MKX, COL1A1, RUNX2, and CTNNB1. TGFB3 treatment exaggerated morphological changes and markedly amplified tenogenic differentiation in a shorter period of time. Along with TGFB3 treatment, inhibition of BMPs by GREM2 and SOST delayed migratory events to some extent and dramatically reduced osteo-chondrogenic markers synergistically. Nanofibrous scaffolds increased tenogenic markers while declining the expression of osteo-chondrogenic genes. ConclusionThese findings revealed an appropriate in vitro potential of spontaneous tenogenic differentiation of eq- ASCs that can be improved by simultaneous activation of TGFB and inhibition of osteoinductive signaling pathways.

Restriction Checkpoint Controls Bradyzoite Development in Toxoplasma gondii.

ABSTRACTHuman toxoplasmosis is a life-threatening disease caused by the apicomplexan parasite Toxoplasma gondii. Rapid replication of the tachyzoite is associated with symptomatic disease, while suppressed division of the bradyzoite is responsible for chronic disease. Here, we identified the T. gondii cell cycle mechanism, the G1 restriction checkpoint (R-point), that operates the switch between parasite growth and differentiation. Apicomplexans lack conventional R-point regulators, suggesting adaptation of alternative factors. We showed that Cdk-related G1 kinase TgCrk2 forms alternative complexes with atypical cyclins (TgCycP1, TgCycP2, and TgCyc5) in the rapidly dividing developmentally incompetent RH and slower dividing developmentally competent ME49 tachyzoites and bradyzoites. Examination of cyclins verified the correlation of cyclin expression with growth dependence and development capacity of RH and ME49 strains. We demonstrated that rapidly dividing RH tachyzoites were dependent on TgCycP1 expression, which interfered with bradyzoite differentiation. Using the conditional knockdown model, we established that TgCycP2 regulated G1 duration in the developmentally competent ME49 tachyzoites but not in the developmentally incompetent RH tachyzoites. We tested the functions of TgCycP2 and TgCyc5 in alkaline induced and spontaneous bradyzoite differentiation (rat embryonic brain cells) models. Based on functional and global gene expression analyses, we determined that TgCycP2 also regulated bradyzoite replication, while signal-induced TgCyc5 was critical for efficient tissue cyst maturation. In conclusion, we identified the central machinery of the T. gondii restriction checkpoint comprised of TgCrk2 kinase and three atypical T. gondii cyclins and demonstrated the independent roles of TgCycP1, TgCycP2, and TgCyc5 in parasite growth and development.IMPORTANCE Toxoplasma gondii is a virulent and abundant human pathogen that puts millions of silently infected people at risk of reactivation of the chronic disease. Encysted bradyzoites formed during the chronic stage are resistant to current therapies. Therefore, insights into the mechanism of tissue cyst formation and reactivation are major areas of investigation. The fact that rapidly dividing parasites differentiate poorly strongly suggests that there is a threshold of replication rate that must be crossed to be considered for differentiation. We discovered a cell cycle mechanism that controls the T. gondii growth-rest switch involved in the conversion of dividing tachyzoites into largely quiescent bradyzoites. This switch operates the T. gondii restriction checkpoint using a set of atypical and parasite-specific regulators. Importantly, the novel T. gondii R-point network was not present in the parasite's human and animal hosts, offering a wealth of new and parasite-specific drug targets to explore in the future.

Age-dependent rise in IFN-γ competence undermines effective type 2 responses to nematode infection

The efficient induction of type 2 immune responses is central to the control of helminth infections. Previous studies demonstrated that strong Th1 responses driven by intracellular pathogens as well as a bias for type 1 activity in senescent mice impedes the generation of Th2 responses and the control of intestinal nematode infections. Here, we show that the spontaneous differentiation of Th1 cells and their expansion with age restrains type 2 immunity to infection with the small intestinal nematode H. polygyrus much earlier in life than previously anticipated. This includes the more extensive induction of IFN-γ competent, nematode-specific Th2/1 hybrid cells in BALB/c mice older than three months compared to younger animals. In C57BL/6 mice, Th1 cells accumulate more rapidly at steady state, translating to elevated Th2/1 differentiation and poor control of parasite fitness in primary infections experienced at a young age. Blocking of early IFN-γ and IL-12 signals during the first week of nematode infection leads to sharply decreased Th2/1 differentiation and promotes resistance in both mouse lines. Together, these data suggest that IFN-γ competent, type 1 like effector cells spontaneously accumulating in the vertebrate host progressively curtail the effectiveness of anti-nematode type 2 responses with rising host age.

Long-term hypoxia inhibits the passage-dependent stemness decrease and senescence increase of human dental pulp stem cells

Dental pulp stem cells (DPSCs) derived from discarded orthodontic teeth are easily obtained and have become a promising source for mesenchymal stem cell-based therapy. However, the pulp tissue is limited, and long-term culture induces cell senescence. Hypoxic culture was expected to be suitable for DPSC expansion, but the results have been contradictory. The aim of this study was to verify the effect of hypoxic culture on human DPSCs (hDPSCs). hDPSCs were isolated and cultured in normoxic (ambient O2 concentration) and hypoxic (5% O2) environments from passage 3 (P3) to P6. The biological characteristics of the cells at P4 (short-term culture) and P6 (long-term culture) were evaluated, including the expression of surface markers, cellular proliferation activity, cellular senescence, and spontaneous and induced differentiation. The results showed that the morphology, phenotype, and proliferation activity of hDPSCs were not affected by hypoxic culture. Long-term normoxic culture of hDPSCs induced cell stemness loss and cell senescence, while hypoxic culture could alleviate these effects. The expression of the stemness markers STRO-1 and OCT4 was increased and the number of senescent cells and the expression of the senescence-related genes P53 and TGF-β were reduced by long-term hypoxic culture. Spontaneous osteogenic and adipogenic differentiation did not occur during long-term normoxic culture. However, hypoxic culture suppressed the expression of the osteogenic markers ALP and RUNX-2 and the adipogenic markers PPAR-γ and FABP4. The induced osteogenic and adipogenic differentiation was apparently reduced by hypoxic culture as well. Our findings indicate that long-term hypoxia culture is beneficial to the maintenance of hDPSCs’ biological characteristics and provide some insights into their large-scale expansion.

Oscillatory calcium release and sustained store-operated oscillatory calcium signaling prevents differentiation of human oligodendrocyte progenitor cells

Endogenous remyelination in demyelinating diseases such as multiple sclerosis is contingent upon the successful differentiation of oligodendrocyte progenitor cells (OPCs). Signaling via the Gαq-coupled muscarinic receptor (M1/3R) inhibits human OPC differentiation and impairs endogenous remyelination in experimental models. We hypothesized that calcium release following Gαq-coupled receptor (GqR) activation directly regulates human OPC (hOPC) cell fate. In this study, we show that specific GqR agonists activating muscarinic and metabotropic glutamate receptors induce characteristic oscillatory calcium release in hOPCs and that these agonists similarly block hOPC maturation in vitro. Both agonists induce calcium release from endoplasmic reticulum (ER) stores and store operated calcium entry (SOCE) likely via STIM/ORAI-based channels. siRNA mediated knockdown (KD) of obligate calcium sensors STIM1 and STIM2 decreased the magnitude of muscarinic agonist induced oscillatory calcium release and attenuated SOCE in hOPCs. In addition, STIM2 expression was necessary to maintain the frequency of calcium oscillations and STIM2 KD reduced spontaneous OPC differentiation. Furthermore, STIM2 siRNA prevented the effects of muscarinic agonist treatment on OPC differentiation suggesting that SOCE is necessary for the anti-differentiative action of muscarinic receptor-dependent signaling. Finally, using a gain-of-function approach with an optogenetic STIM lentivirus, we demonstrate that independent activation of SOCE was sufficient to significantly block hOPC differentiation and this occurred in a frequency dependent manner while increasing hOPC proliferation. These findings suggest that intracellular calcium oscillations directly regulate hOPC fate and that modulation of calcium oscillation frequency may overcome inhibitory Gαq-coupled signaling that impairs myelin repair.

Toll-like receptors 2 and 4 differentially regulate the self-renewal and differentiation of spinal cord neural precursor cells

BackgroundToll-like receptors (TLRs) represent critical effectors in the host defense response against various pathogens; however, their known function during development has also highlighted a potential role in cell fate determination and neural differentiation. While glial cells and neural precursor cells (NPCs) of the spinal cord express both TLR2 and TLR4, their influence on self-renewal and cell differentiation remains incompletely described.MethodsTLR2, TLR4 knock-out and the wild type mice were employed for spinal cord tissue analysis and NPCs isolation at early post-natal stage. Sox2, FoxJ1 and Ki67 expression among others served to identify the undifferentiated and proliferative NPCs; GFAP, Olig2 and β-III-tubulin markers served to identify astrocytes, oligodendrocytes and neurons respectively after NPC spontaneous differentiation. Multiple comparisons were analyzed using one-way ANOVA, with appropriate corrections such as Tukey's post hoc tests used for comparisons.ResultsWe discovered that the deletion of TLR2 or TLR4 significantly reduced the number of Sox2-expressing NPCs in the neonatal mouse spinal cord. While TLR2-knockout NPCs displayed enhanced self-renewal, increased proliferation and apoptosis, and delayed neural differentiation, the absence of TLR4 promoted the neural differentiation of NPCs without affecting proliferation, producing long projecting neurons. TLR4 knock-out NPCs showed significantly higher expression of Neurogenin1, that would be involved in the activation of this neurogenic program by a ligand and microenvironment-independent mechanism. Interestingly, the absence of both TLR2 and TLR4, which induces also a significant reduction in the expression of TLR1, in NPCs impeded oligodendrocyte precursor cell maturation to a similar degree.ConclusionsOur data suggest that Toll-like receptors are needed to maintain Sox2 positive neural progenitors in the spinal cord, however possess distinct regulatory roles in mouse neonatal spinal cord NPCs—while TLR2 and TLR4 play a similar role in oligodendrocytic differentiation, they differentially influence neural differentiation.

Generation of induced pluripotent stem cell line derived from FNAIT patient with CD36 deficiency mutations

Fetal and neonatal alloimmune thrombocytopenia (FNAIT) caused by anti-CD36 isoantibodies is a common disease and the frequency of type I CD36 deficiency is relatively high in eastern Asian populations.Currently, patient-specific induced pluripotent stem cells (hiPSC) are believed to be useful tools for studying anti-CD36 mediated FNAIT and finding new therapeutic approaches to the disease.We generated an iPSC line from peripheral blood mononuclear cells of a patient carrying a 329-330delAC of the CD36 gene.The iPSC expressed pluripotency markers, gave rise to derivatives of three germ layers during spontaneous differentiation, had a normal karyotype, and retained the patient-specific mutation.

Bioorthogonally Cross-Linked Hyaluronan-Laminin Hydrogels for 3D Neuronal Cell Culture and Biofabrication.

Laminins (LNs) are key components in the extracellular matrix of neuronal tissues in the developing brain and neural stem cell niches. LN-presenting hydrogels can provide a biologically relevant matrix for the 3D culture of neurons toward development of advanced tissue models and cell-based therapies for the treatment of neurological disorders. Biologically derived hydrogels are rich in fragmented LN and are poorly defined concerning composition, which hampers clinical translation. Engineered hydrogels require elaborate and often cytotoxic chemistries for cross-linking and LN conjugation and provide limited possibilities to tailor the properties of the materials. Here a modular hydrogel system for neural 3D cell cultures, based on hyaluronan and poly(ethylene glycol), that is cross-linked and functionalized with human recombinant LN-521 using bioorthogonal copper-free click chemistry, is shown. Encapsulated human neuroblastoma cells demonstrate high viability and grow into spheroids. Long-term neuroepithelial stem cells (lt-NES) cultured in the hydrogels can undergo spontaneous differentiation to neural fate and demonstrate significantly higher viability than cells cultured without LN. The hydrogels further support the structural integrity of 3D bioprinted structures and maintain high viability of bioprinted and syringe extruded lt-NES, which can facilitate biofabrication and development of cell-based therapies.

Stemness Maintenance and Massproduction of Neural Stem Cells on Poly L-Lactic Acid Nanofibrous Membrane Based on Piezoelectriceffect.

Neural stem cells (NSCs) therapy is promising for treating neurodegenerative disorders and neural injuries. However, the limited in vitro expansion, spontaneous differentiation, and decrease in stemness obstruct the acquisition of high quantities of NSCs, restricting the clinical application of cell-based therapies and tissue engineering. This article reports a facile method of promoting NSCs expansion and maintaining stemness using wireless electrical stimulation triggered by piezoelectric nanomaterials. A nanofibrous membrane of poly L-lactic acid (PLLA) is prepared by electrostatic spinning, and the favorable piezoelectric property of PLLA facilitates the freeing of electrons after transformation. These self-powered electric signals generated by PLLA significantly enhance NSCs proliferation. Further, an undifferentiated cellular state is maintained in the NSCs cultured on the surfaces of PLLA nanofibers exposed to ultrasonic vibration. In addition, the neural differentiation potencies and functions of NSCs expanded by piezoelectric-driven localized electricity are not attenuated. Moreover, cell stemness can be maintained by wireless electric stimulation. Taken together, the electronic signals mediated by PLLA nanofibers facilitate NSCs proliferation. This efficient and simple strategy can maintain the stemness of NSCs during proliferation, which is essential for their clinical application, and opens up opportunities for the mass production of NSCs for use in cell therapy.

The MuvB complex safeguards embryonic stem cell identity through regulation of the cell cycle machinery

Increasing evidences indicate that unlimited capacity for self-renewal and pluripotency, two unique properties of embryonic stem cells (ESCs), are intrinsically linked to cell cycle control. However, the precise mechanisms coordinating cell fate decisions and cell cycle regulation remain to be fully explored. Here, using CRISPR/Cas9-mediated genome editing, we show that in ESCs, deficiency of components of the cell cycle regulatory MuvB complex Lin54 or Lin52, but not Lin9 or Lin37, triggers G2/M arrest, loss of pluripotency, and spontaneous differentiation. Further dissection of these phenotypes demonstrated that this cell cycle arrest is accompanied by the gradual activation of mesoendodermal lineage-specifying genes. Strikingly, the abnormalities observed in Lin54-null ESCs were partially but significantly rescued by ectopic coexpression of genes encoding G2/M proteins Cyclin B1 and Cdk1. Thus, our study provides new insights into the mechanisms by which the MuvB complex determines cell fate through regulation of the cell cycle machinery.

Establishment of SIAISi018-A, an induced pluripotent stem cell (iPSC) line from a healthy 45-year-old Chinese Han

A 45-year-old Han Male from China contributed peripheral blood mononuclear cells induced by reprogramming human OKSM transcription factors (OCT3/4, KLF4 SOX2 and C-MYC) with a non-integrated additional vector system. Immunological markers confirmed the pluripotent nature of IPSC. Spontaneous tridermal differentiation confirmed the differentiation ability of IPSC with normal karyotype.

Establishment of an induced pluripotent stem cell line DHMi005-A from a healthy male proband

We generated an induced pluripotent stem cell (iPSC) line from a healthy male 29-year-old proband. Adipose fibroblasts (AFs) were reprogrammed using Sendai virus. Generated iPSCs showed typical stem cell morphology. From passage 9 on, iPSCs were free of virus. Pluripotency in the iPSCs was verified and spontaneous differentiation showed expression of all three germ layers. Karyotyping indicated no anomalies for the generated iPSCs. Many patient-specific iPSCs are generated from subcutaneous fat fibroblasts obtained during surgical procedure. The described control iPSC line was generated equally and therefore serves as an ideal control for adipose-fibroblast-based patient-specific iPSC lines in disease modeling.

Comparison of expression profiles between undifferentiated and differentiated porcine IPEC-J2 cells

BackgroundThe intestinal porcine enterocyte cell line (IPEC-J2) is a well-established model to study porcine intestinal physiology. IPEC-J2 cells undergo spontaneous differentiation during culture while changes in expression patterns of differentiated IPEC-J2 remain unclear. Therefore, this study was aimed to investigate the expression profiles of IPEC-J2 cells at the transcriptional level. Differentially expressed genes (DEGs), enriched pathways and potential key genes were identified. Alkaline phosphatase (AKP) and percentages of apoptotic cells were also measured.ResultsOverall, a total of 988 DEGs were identified, including 704 up-regulated and 284 down-regulated genes. GO analysis revealed that epithelial cell differentiation, apoptotic signaling pathway, regulation of cytokine production and immune system process, regulation of cell death and proliferation, cell junction complexes, and kinase binding were enriched significantly. Consistently, KEGG, REACTOME, and CORUM analysis indicated that cytokine responses modulation may be involved in IPEC-J2 differentiation. Moreover, AKP activity, a recognized marker of enterocyte differentiation, was significantly increased in IPEC-J2 after 14 days of culture. Meanwhile, annexin V-FITC/PI assay demonstrated a remarkable increase in apoptotic cells after 14 days of culture. Additionally, 10 hub genes were extracted, and STAT1, AKT3, and VEGFA were speculated to play roles in IPEC-J2 differentiation.ConclusionsThese findings may contribute to the molecular characterization of IPEC-J2, and may progress the understanding of cellular differentiation of swine intestinal epithelium.

Small Molecule Screening of Primary Human Acute Myeloid Leukemia Using Co-culture and Multiplexed FACS Analysis.

Ex vivo culture of primary acute myeloid leukemia (AML) cells is notoriously difficult due to spontaneous differentiation and cell death, which hinders mechanistic and translational studies. To overcome this bottleneck, we have implemented a co-culture system, where the OP9-M2 stromal cells support the growth, but most notably limit the differentiation of primary AML cells, thus allowing for mechanistic studies in vitro. Additionally, the co-culture on OP9-M2 stromal is superior in preserving surface marker expression of primary (adult and pediatric) AML cells in comparison to stroma-free culture. Thus, by combining the co-culture with multicolor, high-throughput FACS, we can evaluate the effect of hundreds of small molecules on multi-parametric processes including: cell survival, stemness (leukemic stem cells), and myeloid differentiation on the primary AML cells at a single-cell level. This method streamlines the identification of potential therapeutic agents, but also facilitates combinatorial screening aiming, for instance, at dissecting the regulatory pathways in a patient-specific manner. Graphic abstract: Schematic representation of the ex vivo small molecule screening of primary human acute myeloid leukemia. Irradiated, sub-confluent OP9-M2 stromal cells are plated in half-area 96 wells plates 4-16 h prior to adding primary AML cells. Compounds are added 36-48 h later and effects on cell number, leukemic stem cell population, and myeloid differentiation are quantifed by FACS after 4 days of treatment.

Proteomic and electron microscopy study of myogenic differentiation of alveolar mucosa multipotent mesenchymal stromal cells in three-dimensional culture.

Myocyte differentiation is featured by adaptation processes, including mitochondria repopulation and cytoskeleton re-organization. The difference between monolayer and spheroid cultured cells at the proteomic level is uncertain. We cultivated alveolar mucosa multipotent mesenchymal stromal cells in spheroids in a myogenic way for the proper conditioning of ECM architecture and cell morphology, which induced spontaneous myogenic differentiation of cells within spheroids. Electron microscopy analysis was used for the morphometry of mitochondria biogenesis, and proteomic was used complementary to unveil events underlying differences between two-dimensional/three-dimensional myoblasts differentiation. The prevalence of elongated mitochondria with an average area of 0.097 μm2 was attributed to monolayer cells 7 days after the passage. The population of small mitochondria with a round shape and area of 0.049 μm2 (p < 0.05) was observed in spheroid cells cultured under three-dimensional conditions. Cells in spheroids were quantitatively enriched in proteins of mitochondria biogenesis (DNM1L, IDH2, SSBP1), respiratory chain (ACO2, ATP5I, COX5A), extracellular proteins (COL12A1, COL6A1, COL6A2), and cytoskeleton (MYL6, MYL12B, MYH10). Most of the Rab-related transducers were inhibited in spheroid culture. The proteomic assay demonstrated delicate mechanisms of mitochondria autophagy and repopulation, cytoskeleton assembling, and biogenesis. Differences in the ultrastructure of mitochondria indicate active biogenesis under three-dimensional conditions.