Early embryonic polarity establishment and implications for lineage differentiation.

Polarity establishment is one of the key factors affecting early embryonic development. Polarity establishment begins with myosin phosphorylation in the 8-cell embryo, and phosphorylation activates actin leading to its initiation of contractility. Subsequently, actin undergoes reorganization to form an apical domain rich in microvilli on the non-contacting surface of each blastomere, and form the actomyosin ring that marks the maturation of the apical domain in conjunction with polar protein complexes and others. From the process of polarity establishment, it can be seen that the formation of the apical domain is influenced by actin-related proteins and polar protein complexes. Some zygote genome activation (ZGA) and lineage-specific genes also regulate polarity establishment. Polarity establishment underlies the first cell lineage differentiation during early embryonic development. It regulates lineage segregation and morphogenesis by affecting asymmetric cell division, asymmetric localization of lineage differentiation factors, and activity of the Hippo signaling pathway. In this review, we systematically summarize the mechanisms of early embryonic polarity establishment and its impact on lineage differentiation in mammals, and discuss the shortcomings of the currently available studies in terms of regulatory mechanisms and species, thereby providing clues and systematic perspectives for elucidating early embryonic polarity establishment.

Discrepancy of flowering time between genetically close sublineages of Aegilops umbellulata Zhuk.

Aegilops umbellulata Zhuk., a wild diploid wheat-related species, has been used as a genetic resource for several important agronomic traits. However, its genetic variations have not been comprehensively studied. We sequenced RNA from 114 accessions of Ae. umbellulata to evaluate DNA polymorphisms and phenotypic variations. Bayesian clustering and phylogenetic analysis based on SNPs detected by RNA sequencing revealed two divergent lineages, UmbL1 and UmbL2. The main differences between them were in the sizes of spikes and spikelets, and culm diameter. UmbL1 is divided into two sublineages, UmbL1e and UmbL1w. These genetic differences corresponded to geographic distributions. UmbL1e, UmbL1w, and UmbL2 are found in Turkey, Iran/Iraq, and Greece, respectively. Although UmbL1e and UmbL1w were genetically similar, flowering time and other morphological traits were more distinct between these sublineages than those between the lineages. This discrepancy can be explained by the latitudinal and longitudinal differences in habitats. Specifically, latitudinal clines of flowering time were clearly observed in Ae. umbellulata, strongly correlated with solar radiation in the winter season. This observation implies that latitudinal differences are a factor in differences in the flowering times of Ae. umbellulata. Differences in flowering time could influence other morphological differences and promote genetic divergence between sublineages.

Oxidative-stress induced Bmp2-Smad1/5/8 signaling dependent differentiation of early cardiomyocytes from embryonic and adult epicardial cells.

Heart failure has become a major life-threatening cause affecting millions globally, characterized by the permanent loss of adult functional cardiomyocytes leading to fibrosis which ultimately deprives the heart of its functional efficacy. Here we investigated the reparative property of embryonic and adult epicardial cells towards cardiomyocyte differentiation under oxidative stress-induced conditions along with the identification of a possible molecular signaling pathway. Isolated epicardial cells from embryonic chick hearts subjected to oxidative stress and hypoxia induction. Initial assessment of successful injury induction reveals hypertrophy of isolated epicardial cells. Detailed marker gene expression analyses and inhibitor studies reveal Bone morphogenic protein (Bmp)2-Smad1/5/8 signaling dependent cardiomyocyte lineage specification via epithelial to mesenchymal transition (EMT) post-injury. EMT is further confirmed by increased proliferation, migration, and differentiation towards cardiomyocyte lineage. We have also established an in-vivo model in adult male rats using Isoproterenol. Successful oxidative stress-mediated injury induction in adult heart was marked by increased activated fibroblasts followed by apoptosis of adult cardiomyocytes. The detailed characterization of adult epicardial cells reveals similar findings to our avian in-vitro data. Both in-vitro and in-vivo results show a significant increase in the expression of cardiomyocyte specific markers indicative of lineage specificity and activation of epicardial cells post oxidative stress mediated injury. Our findings suggest an EMT-induced reactivation of epicardial cells and early cardiomyocyte lineage specification following oxidative stress in a Bmp2- Smad1/5/8 dependent manner. Overall, this regulatory mechanism of cardiomyocyte differentiation induced by oxidative stress may contribute to the field of cardiac repair and regenerative therapeutics.

Dynamic and distinct histone modifications facilitate human trophoblast lineage differentiation.

The placenta serves as an essential organ for fetal growth throughout pregnancy. Histone modification is a crucial regulatory mechanism involved in numerous biological processes and development. Nevertheless, there remains a significant gap in our understanding regarding the epigenetic regulations that influence trophoblast lineage differentiation, a fundamental aspect of placental development. Here, through comprehensive mapping of H3K4me3, H3K27me3, H3K9me3, and H3K27ac loci during the differentiation of trophoblast stem cells (TSCs) into syncytiotrophoblasts (STs) and extravillous trophoblasts (EVTs), we reveal dynamic reconfiguration in H3K4me3 and H3K27ac patterns that establish an epigenetic landscape conducive to proper trophoblast lineage differentiation. We observe that broad H3K4me3 domains are associated with trophoblast lineage-specific gene expression. Unlike embryonic stem cells, TSCs lack robust bivalent domains. Notably, the repression of ST- and EVT-active genes in TSCs is primarily attributed to the weak H3K4me3 signal rather than bivalent domains. We also unveil the inactivation of TSC enhancers precedes the activation of ST enhancers during ST formation. Our results provide a comprehensive global map of diverse histone modifications, elucidating the dynamic histone modifications during trophoblast lineage differentiation.

Nr5a2 ensures inner cell mass formation in mouse blastocyst.

Recent studies have elucidated Nr5a2's role in activating zygotic genes during early mouse embryonic development. Subsequent research, however, reveals that Nr5a2 is not critical for zygotic genome activation but is vital for the gene program between the 4- and 8-cell stages. A significant gap exists in experimental evidence regarding its function during the first lineage differentiation's pivotal period. In this study, we observed that approximately 20% of embryos developed to the blastocyst stage following Nr5a2 ablation. However, these blastocysts lacked inner cell mass (ICM), highlighting Nr5a2's importance in first lineage differentiation. Mechanistically, using RNA sequencing and CUT&Tag, we found that Nr5a2 transcriptionally regulates ICM-specific genes, such as Oct4, to establish the pluripotent network. Interference with or overexpression of Nr5a2 in single blastomeres of 2-cell embryos can alter the fate of daughter cells. Our results indicate that Nr5a2 works as a doorkeeper to ensure ICM formation in mouse blastocyst.

An arms-race against resistance: leukemic stem cells and lineage plasticity.

Acute myeloid leukemia (AML) therapy is undergoing rapid development, but primary and acquired resistance to therapy complicates the prospect of a durable cure. Recent functional and single-cell multi-omics approaches have greatly expanded our knowledge of the diversity of lineage trajectories in AML settings. AML cells range from undifferentiated stem-like cells to more differentiated myeloid or megakaryocyte/erythroid cells. Current clinically relevant drugs predominantly target the myeloid progenitor lineage, while monocyte- or stem cell-like states can evade current AML treatment and may be targeted in the future with lineage-specific inhibitors. The extent of aberrant lineage plasticity upon therapeutic pressure in AML cells in conjunction with hijacking of normal differentiation pathways is still a poorly understood topic. Insights into the mechanisms of lineage plasticity of AML stem cells could identify both therapy-specific and cross-drug resistance pathways and reveal novel strategies to overcome them.

Chromatin state transitions in the Drosophila intestinal lineage identify principles of cell-type specification.

Tissue homeostasis relies on rewiring of stem cell transcriptional programs into those of differentiated cells. Here, we investigate changes in chromatin occurring in a bipotent adult stem cells. Combining mapping of chromatin-associated factors with statistical modeling, we identify genome-wide transitions during differentiation in the adult Drosophila intestinal stem cell (ISC) lineage. Active, stem-cell-enriched genes transition to a repressive heterochromatin protein-1-enriched state more prominently in enteroendocrine cells (EEs) than in enterocytes (ECs), in which the histone H1-enriched Black state is preeminent. In contrast, terminal differentiation genes associated with metabolic functions follow a common path from a repressive, primed, histone H1-enriched Black state in ISCs to active chromatin states in EE and EC cells. Furthermore, we find that lineage priming has an important function in adult ISCs, and we identify histone H1 as a mediator of this process. These data define underlying principles of chromatin changes during adult multipotent stem cell differentiation.

Different Sources of Bone Marrow Mesenchymal Stem Cells: A Comparison of Subchondral, Mandibular, and Tibia Bone-derived Mesenchymal Stem Cells.

BACKGROUND: Stem cell properties vary considerably based on the source and tissue site of mesenchymal stem cells (MSCs). The mandibular condyle is a unique kind of craniofacial bone with a special structure and a relatively high remodeling rate. MSCs here may also be unique to address specific physical needs. OBJECTIVE: The aim of this study was to compare the proliferation and multidirectional differentiation potential among MSCs derived from the tibia (TMSCs), mandibular ramus marrow (MMSCs), and condylar subchondral bone (SMSCs) of rats in vitro. METHODS: Cell proliferation and migration were assessed by CCK-8, laser confocal, and cell scratch assays. Histochemical staining and real-time PCR were used to evaluate the multidirectional differentiation potential and DNA methylation and histone deacetylation levels. RESULTS: The proliferation rate and self-renewal capacity of SMSCs were significantly higher than those of MMSCs and TMSCs. Moreover, SMSCs possessed significantly higher mineralization and osteogenic differentiation potential. Dnmt2, Dnmt3b, Hdac6, Hdac7, Hdac9, and Hdac10 may be instrumental in the osteogenesis of SMSCs. In addition, SMSCs are distinct from MMSCs and TMSCs with lower adipogenic differentiation and chondrogenic differentiation potential. The multidirectional differentiation capacities of TMSCs were exactly the opposite of those of SMSCs, and the results of MMSCs were intermediate. CONCLUSION: This research offers a new paradigm in which SMSCs could be a useful source of stem cells for further application in stem cell-based medical therapies due to their strong cell renewal and osteogenic capacity.

Mesenchymal stem cells derived from the fibrotic tissue of atrophic nonunion or the bone marrow of iliac crest: A donor-matched comparison.

Purpose: Atrophic nonunion is one of the most difficult complications of fracture. The cellular factors that contribute to atrophic nonunion are poorly understood, and mesenchymal stem cells (MSCs) are recognized as the key contributor to bone formation. This study aimed to characterize the MSCs isolated from the fibrotic tissue of atrophic nonunion (AN-MSCs) from the perspective of proliferation, differentiation potential, senescence, and paracrine function. Methods: Human atrophic fibrotic tissue was obtained from four donors aged 29-37 for isolating AN-MSCs, and donor-matched bone marrow acquired from the iliac crest for isolating MSCs (IC-MSCs) as control. The AN-MSCs or IC-MSCs in passage 3 were applied for the following evaluations. The surface markers expressed on the two cells were evaluated using flow cytometry. The proliferation of the two cells for up to 11 days was comparatively investigated. After osteogenic, chondrogenic, or adipogenic induction, multi-lineage differentiation of AN-MSCs or IC-MSCs was comparatively evaluated using lineage-specific stains and lineage-specific gene expression. Enzyme-linked immunosorbent assay (ELISA) assessment was applied to evaluate the paracrine function of AN-MSCs or IC-MSCs. Cellular senescence of AN-MSCs or IC-MSCs was evaluated using senescence-associated ß-galactosidase (SA-ß-gal) staining. Results: AN-MSCs or IC-MSCs from the four donors showed morphologic and immunophenotypic characteristics of MSCs, with the expression of MSCs markers and negative expression of hematopoietic markers. In general, AN-MSCs showed similar proliferation and adipogenic capacity with IC-MSCs. In contrast, IC-MSCs showed significantly higher osteogenic and chondrogenic capacity compared to AN-MSCs. Moreover, the culture medium of IC-MSCs contains significantly higher levels of VEGF, TGF-ß1, PDGF-BB, and IGF-1 than the culture medium of AN-MSCs. Lastly, the AN-MSCs are more prone to cellular senescence than the IC-MSCs. Conclusions: In-vitro, AN-MSCs were similar to IC-MSCs in proliferation and adipogenic capacity, but inferior to IC-MSCs in osteogenic and chondrogenic capacity, paracrine function, and anti-senescence.

Uranium-bearing dust induces differentiation and expansion of enteroendocrine cells in human colonoids.

Chronic exposure to environmental toxins and heavy metals has been associated with intestinal inflammation, increased susceptibility to pathogen-induced diseases, and higher incidences of colorectal cancer, all of which have been steadily increasing in prevalence for the past 40 years. The negative effects of heavy metals on barrier permeability and inhibition of intestinal epithelial healing have been described; however, transcriptomic changes within the intestinal epithelial cells and impacts on lineage differentiation are largely unknown. Uranium exposure remains an important environmental legacy and physiological health concern, with hundreds of abandoned uranium mines located in the Southwestern United States largely impacting underserved indigenous communities. Herein, using human colonoids, we defined the molecular and cellular changes that occur in response to uranium bearing dust (UBD) exposure. We used single cell RNA sequencing to define the molecular changes that occur to specific identities of colonic epithelial cells. We demonstrate that this environmental toxicant disrupts proliferation and induces hyperplastic differentiation of secretory lineage cells, particularly enteroendocrine cells (EEC). EECs respond to UBD exposure with increased differentiation into de novo EEC sub-types not found in control colonoids. This UBD-induced EEC differentiation does not occur via canonical transcription factors NEUROG3 or NEUROD1. These findings highlight the significance of crypts-based proliferative cells and secretory cell differentiation as major colonic responses to heavy metal-induced injury.

Elucidating the cell metabolic heterogeneity during hematopoietic lineage differentiation based on Met-Flow.

Cell metabolism is critically involved in the differentiation of the hematopoietic lineage and, therefore, has attracted the attention of researchers, however, in-depth studies on cellular metabolic activity of hematopoietic cells (HCs) require attention. This investigation compared the metabolic activity of HCs at critical lineage differentiation stages, including hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), and differentiated blood cells, via multiple methods and basic reference values. Primary metabolic processes of HCs, including anabolism, catabolism, phosphate, and glucose metabolism, were analyzed, and their maps were drawn. The data revealed that GLUT1 expression in HSCs was substantially higher than in all progenitor cells and mature myeloid blood cells, indicating their strong glucose uptake capacity. In myeloid differentiation, the ACAC expression of HPC2 was markedly higher than in neutrophils and monocytes. The ACAC, ASS1, ATP5A, and PRDX2 of HPC2 expression in lymphoid differentiation was substantially greater than in B and Natural-killer cells. CLP, CMP, GMP, MEP, and HPC1 inherit increased glucose uptake stem cell properties. In lymphocyte subsets, the expression of ACAC, ASS1, ATP5A, CPT1A, and PRDX2 in CD4+ T subgroups (naive and memory CD4+ T and nTreg) were elevated than in B subgroups (pro-, pre-, immature and mature Bs) and CD8+ T subgroups. Furthermore, leukemia stem cells (LSCs) had increased levels of ACAC, CPT1A, G6PD, IDH2, and PRDX2 than leukemia cells, indicating a stronger metabolic capacity of LSCs than differentiated leukemia cells.

E3 ubiquitin ligase on the biological properties of hematopoietic stem cell.

Hematopoietic stem cells are a group of heterogeneity cells with the potential to differentiate into various types of mature blood cells. Their basic biological properties include quiescence, self-renewal, multilineage differentiation, and homing ability, with the homing of exogenous hematopoietic stem cells after transplantation becoming a new focus, while the first three properties share some similarity in mechanism due to connectivity. In various complex mechanisms, the role of E3 ubiquitin ligases in hematopoietic homeostasis and malignant transformation is receiving increasing attention. As a unique part, E3 ubiquitin ligases play an important role in physiological regulation mechanism of posttranslational modification. In this review, we focus on the recent progress of the crucial role of E3 ubiquitin ligases that target specific proteins for ubiquitination to regulate biological properties of hematopoietic stem cells. Additionally, this paper deals with E3 ubiquitin ligases that affect the biological properties through aging and summarizes the relevant applications of targeting E3 ligases in hematopoietic malignancies. We present some ideas on the clinical application of E3 ubiquitin ligase to regulate hematopoietic stem cells and also believe that it is meaningful to study the upstream signal of these E3 ubiquitin ligases because hematopoietic stem cell dysfunction is caused by deficiency of some E3 ligases.

[CUP syndrome-diagnostics from the perspective of pathology]. / CUP-Syndrom ­ Diagnostik aus Sicht der Pathologie.

PROBLEM: Histologic and immunohistologic workup of tumor material from metastases of a previously unknown primary tumor is important for identifying their origin, but is often insufficient for this purpose without clinical oncologic and radiologic evaluation. PROCEDURE: In the initial cancer of unknown primary (CUP) situation, histologic and immunohistochemical workup together with clinicoradiologic correlations contribute significantly to the identification of the primary tumor. There are now accepted guidelines to follow when there is an initial CUP situation. Molecular diagnostic tools can be used to investigate changes at the nucleic acid level, which can provide clues about the primary tumor, including potential targets for therapy. If, despite broad and interdisciplinary diagnostics, it is not possible to identify the primary tumor, the diagnosis is CUP syndrome. If a true CUP situation is present, it is important to assign the tumor to a tumor class or a specific therapy-sensitive subgroup as best as possible so that the best possible treatment can be given. However, for a final assignment to a primary tumor or a final classification as CUP, a comparison with medical oncological and imaging findings is indispensable. CONCLUSION: When CUP is suspected, close interdisciplinary collaboration between pathology, medical oncology, and imaging is essential to achieve a viable classification as CUP or identification of a presumptive primary tumor, in the interest of providing the most specific and effective therapy for affected individuals.

The Effect of Boric Acid and Sodium Pentaborate Pentahydrate-Treated Foreskin Derived Mesenchymal Stem Cells on Liver Fibrosis.

Liver fibrosis is a worldwide public health problem due to its life-threatening complications, including portal hypertension, liver failure, cirrhosis, and hepatocellular carcinoma (HCC). Liver fibrosis is the net result of a complex excessive accumulation of extracellular matrix (ECM). Activation of hepatic stellate cells (HSCs) are the cause of deposition of ECM and are commonly recognized as a key step in liver fibrosis. The aim of this study was to investigate the effect of foreskin-derived mesenchymal stem cells treated with boron compounds on liver fibrosis. Rats were injected intraperitoneally with thioacetamide (TAA) at a dose of 150 mg/kg except sham and control groups' rats. Thioacetamide (TAA), foreskin-derived mesenchymal stem cells (TAA + FSDMSC), FSDMSC treated with boric acid (TAA + FSDMSC + BA), FSDMSC treated with sodium pentaborate pentahydrate (TAA + FSDMSC + NaB), control and sham groups were studied. Boron compound treated foreskin-derived mesenchymal stem cells were injected into the tail vein, and evaluations were conducted after 4 weeks and liver tissues were obtained for structural, immunohistochemical, and western blot studies and blood samples were taken for biochemical analysis. FSDMSC (BA) alleviates TAA-induced rats liver fibrosis, and BA showed a positive effect on foreskin-derived mesenchymal stem cells viability. After using BA-treated mesenchymal stem cells, we observed that there was regression in the fibrotic areas at TAA-induced liver fibrosis. The result demonstrates that the contribution of TAA + FSDMSC and TAA + FSDMSC (NaB) at the level of structure is not effective in regression of fibrosis in TAA-generated liver fibrosis. We concluded that FSDMSC treated with BA may be a factor in the regression of fibrosis.

Tri-Lineage Differentiation Potential of Osteosarcoma Cell Lines and Human Bone Marrow Stromal Cells from Different Anatomical Locations.

The bone cancer osteosarcoma, found mainly in adolescents, routinely forms around the growth plate/metaphysis of long bones. Bone marrow composition changes with age, shifting from a more hematopoietic to an adipocyte-rich tissue. This conversion occurs in the metaphysis during adolescence, implicating a link between bone marrow conversion and osteosarcoma initiation. To assess this, the tri-lineage differentiation potential of human bone marrow stromal cells (HBMSCs) isolated from the femoral diaphysis/metaphysis (FD) and epiphysis (FE) was characterized and compared to two osteosarcoma cell lines, Saos-2 and MG63. Compared to FE-cells, FD-cells showed an increase in tri-lineage differentiation. Additionally, differences were found between the Saos-2 cells exhibiting higher levels of osteogenic differentiation, lower adipogenic differentiation, and a more developed chondrogenic phenotype than MG63, with the Saos-2 being more comparable to FD-derived HBMSCs. The differences found between the FD and FE derived cells are consistent with the FD region containing more hematopoietic tissue compared to the FE. This may be related to the similarities between FD-derived cells and Saos-2 cells during osteogenic and chondrogenic differentiation. These studies reveal distinct differences in the tri-lineage differentiations of 'hematopoietic' and 'adipocyte rich' bone marrow, which correlate with specific characteristics of the two osteosarcoma cell lines.

hCINAP regulates the differentiation of embryonic stem cells by regulating NEDD4 liquid-liquid phase-separation-mediated YAP1 activation.

YAP1 functions in lineage differentiation of pluripotent embryonic stem cells (ESCs); however, the detailed mechanisms underlying the regulation of YAP1 activity during ESC differentiation remain elusive. Here, we report that hCINAP serves as a negative regulator of YAP1 during ESC fate decisions. The expression of mCINAP, the murine homolog of hCINAP, is downregulated during the differentiation process of murine ESC (mESC) ectoderm lineage, leading to liquid-liquid phase separation (LLPS) of NEDD4 and activation of YAP1. Mechanistically, hCINAP interacts with and prevents NEDD4 from forming cytoplasmic condensates that compartmentalize YAP1 and its kinase NLK, facilitating YAP1 phosphorylation at Ser128 and promoting YAP1 activation. mCINAP depletion leads to the formation of NEDD4 condensates and YAP1 activation, which impedes endoderm differentiation of mESCs. Our study shows that hCINAP is a vital regulator of YAP1 activity and is essential for stem cell fate decisions, which provides mechanistic insight into early embryogenesis.

Impaired Human Cardiac Cell Development due to NOTCH1 Deficiency.

BACKGROUND: NOTCH1 pathogenic variants are implicated in multiple types of congenital heart defects including hypoplastic left heart syndrome, where the left ventricle is underdeveloped. It is unknown how NOTCH1 regulates human cardiac cell lineage determination and cardiomyocyte proliferation. In addition, mechanisms by which NOTCH1 pathogenic variants lead to ventricular hypoplasia in hypoplastic left heart syndrome remain elusive. METHODS: CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 genome editing was utilized to delete NOTCH1 in human induced pluripotent stem cells. Cardiac differentiation was carried out by sequential modulation of WNT signaling, and NOTCH1 knockout and wild-type differentiating cells were collected at day 0, 2, 5, 10, 14, and 30 for single-cell RNA-seq. RESULTS: Human NOTCH1 knockout induced pluripotent stem cells are able to generate functional cardiomyocytes and endothelial cells, suggesting that NOTCH1 is not required for mesoderm differentiation and cardiovascular development in vitro. However, disruption of NOTCH1 blocks human ventricular-like cardiomyocyte differentiation but promotes atrial-like cardiomyocyte generation through shortening the action potential duration. NOTCH1 deficiency leads to defective proliferation of early human cardiomyocytes, and transcriptomic analysis indicates that pathways involved in cell cycle progression and mitosis are downregulated in NOTCH1 knockout cardiomyocytes. Single-cell transcriptomic analysis reveals abnormal cell lineage determination of cardiac mesoderm, which is manifested by the biased differentiation toward epicardial and second heart field progenitors at the expense of first heart field progenitors in NOTCH1 knockout cell populations. CONCLUSIONS: NOTCH1 is essential for human ventricular-like cardiomyocyte differentiation and proliferation through balancing cell fate determination of cardiac mesoderm and modulating cell cycle progression. Because first heart field progenitors primarily contribute to the left ventricle, we speculate that pathogenic NOTCH1 variants lead to biased differentiation of first heart field progenitors, blocked ventricular-like cardiomyocyte differentiation, and defective cardiomyocyte proliferation, which collaboratively contribute to left ventricular hypoplasia in hypoplastic left heart syndrome.

In Vitro Model Systems to Study Human T Cell Development.

Not only is human T cell development characterized by unique changes in surface marker expression, but it also requires specific growth factors and conditions to mimic and study T cell development in vitro. In this chapter, we provide an overview of the specific aspects that need attention when performing T cell differentiation cultures with human hematopoietic and T cell progenitors.

Multi-lineage Differentiation from Hematopoietic Stem Cells.

The hematopoietic stem cells (HSCs) have the ability to differentiate and give rise to all mature blood cells. Commitment to differentiation progressively limits the self-renewal potential of the original HSCs by regulating the level of lineage-specific gene expression. In this review, we will summarize the current understanding of the molecular mechanisms underlying HSC differentiation toward erythroid, myeloid, and lymphocyte lineages. Moreover, we will decipher how the single-cell technologies advance the lineage-biased HSC subpopulations and their differentiation potential.

Effervescent Atomizer as Novel Cell Spray Technology to Decrease the Gas-to-Liquid Ratio.

Cell spraying has become a feasible application method for cell therapy and tissue engineering approaches. Different devices have been used with varying success. Often, twin-fluid atomizers are used, which require a high gas velocity for optimal aerosolization characteristics. To decrease the amount and velocity of required air, a custom-made atomizer was designed based on the effervescent principle. Different designs were evaluated regarding spray characteristics and their influence on human adipose-derived mesenchymal stromal cells. The arithmetic mean diameters of the droplets were 15.4−33.5 µm with decreasing diameters for increasing gas-to-liquid ratios. The survival rate was >90% of the control for the lowest gas-to-liquid ratio. For higher ratios, cell survival decreased to approximately 50%. Further experiments were performed with the design, which had shown the highest survival rates. After seven days, no significant differences in metabolic activity were observed. The apoptosis rates were not influenced by aerosolization, while high gas-to-liquid ratios caused increased necrosis levels. Tri-lineage differentiation potential into adipocytes, chondrocytes, and osteoblasts was not negatively influenced by aerosolization. Thus, the effervescent aerosolization principle was proven suitable for cell applications requiring reduced amounts of supplied air. This is the first time an effervescent atomizer was used for cell processing.