Cell Differentiation Activity Research Articles

TGFβ1 Regulates Cellular Composition of In Vitro Cardiac Perivascular Niche Based on Cardiospheres.

The cardiac perivascular niche is a cellular microenvironment of a blood vessel. The principles of niche regulation are still poorly understood. We studied the effect of TGFβ1 on cells forming the cardiac perivascular niche using 3D cell culture (cardiospheres). Cardiospheres contained progenitor (c-Kit), endothelial (CD31), and mural (αSMA) cells, basement membrane proteins (laminin) and extracellular matrix proteins (collagen I, fibronectin). TGFβ1 treatment decreased the length of CD31+ microvasculature, VE cadherin protein level, and proportion of NG2+ cells, and increased proportion of αSMA+ cells and transgelin/SM22α protein level. We supposed that this effect is related to the stabilizing function of TGFβ1 on vascular cells: decreased endothelial cell proliferation, as shown for HUVEC, and activation of mural cell differentiation.

CD40L-Activated DC Promotes Th17 Differentiation and Inhibits Th2 Differentiation in Sepsis-Induced Lung Injury via cGAS-STING Signaling.

Immune hemostasis due to an infection plays a vital role in sepsis-induced multiple organ dysfunction. Dendritic cells (DC) and T helper (Th) cells are the key members of the immune system maintaining immune homeostasis. This study aimed to explore the effect and mechanism of CD40L on the activation of DC and activated DC-induced Th2/Th17 differentiation. A CD40L knockout and cecal ligation and puncture (CLP) mouse model was established via cecal ligation. HE staining was used to evaluate the pathological changes. The gene expressions were studied using quantitative real-time polymerase chain reaction (qRT-PCR), while a transwell system was used to perform the co-culture of DC and T-cells. Flow cytometry was performed to detect the subtype of T and DC cells. ELISA was used to assess the amount of inflammatory factors. CD40L was highly expressed in the plasma of CLP mice. Knock out of CD40L inhibited the activation of DC cell and Th17 differentiation while promoting the Th2 differentiation. The mechanistic investigations revealed that CD40L promoted the activation of cGAS-STING pathway. Rescue experiments indicated that CD40L mediated DC activation via cGAS-STING signaling. Moreover, co-culturing of CD and CD+4 T-cells demonstrated that silencing of CD40L in DC suppressed the DC activation and inhibited Th17 differentiation while promoting Th2 differentiation. These findings revealed a relationship between CD40L, DC activation, and Th2/Th17 differentiation balance in sepsis-induced acute lung injury for the first time. These findings are envisaged to provide novel molecular targets for sepsis-induced lung injury treatment.

Inactivation Pathway of Diterpenoid Regulator in the Moss Physcomitrium patens

AbstractThe endogenous levels of plant hormones, including gibberellins (GAs), are strictly regulated and maintained during growth and development in seed plants. The regulation of endogenous levels of bioactive GAs is mediated by the mechanisms of their biosynthesis and inactivation. The moss Physcomitrium patens harbors a partial GA biosynthetic pathway from geranylgeranyl diphosphate to ent-kaurenoic acid (KA). Recently, we have identified ent-3β-hydroxy kaurenoic acid (3OH-KA) as a biologically active metabolite of KA to control the protonemal cell differentiation. In addition, ent-2α-hydroxy kaurenoic acid catalyzed by KA 2-oxidase (KA2ox) was also identified as inactive product. Although the activation and inactivation pathways from KA have been identified, the inactivation pathway of 3OH-KA remains to be elucidated. Considering the GA inactivation mechanism of flowering plants, in which GA2ox hydroxylates the C-2 position of GAs as part of the biosynthetic pathway, it was presumed that 3OH-KA was converted to 2,3-dihydroxy KA by PpKA2ox; however, this work shows that PpKA2ox undergoes hydroxylation at the C-16 position to synthesize a new compound ent-3β,16β-dihydroxy kaurenoic acid (3,16diOH-KA) from 3OH-KA. The protonemal cell differentiation activity of 3,16diOH-KA was low, and 3,16diOH-KA was detected in wild-type strains. These results indicate that 3,16diOH-KA was the major inactivating metabolite of 3OH-KA.

Expression Quantitative Trait Locus of Wood Formation-Related Genes in Salix suchowensis.

Shrub willows are widely planted for landscaping, soil remediation, and biomass production, due to their rapid growth rates. Identification of regulatory genes in wood formation would provide clues for genetic engineering of willows for improved growth traits on marginal lands. Here, we conducted an expression quantitative trait locus (eQTL) analysis, using a full sibling F1 population of Salix suchowensis, to explore the genetic mechanisms underlying wood formation. Based on variants identified from simplified genome sequencing and gene expression data from RNA sequencing, 16,487 eQTL blocks controlling 5505 genes were identified, including 2148 cis-eQTLs and 16,480 trans-eQTLs. eQTL hotspots were identified, based on eQTL frequency in genomic windows, revealing one hotspot controlling genes involved in wood formation regulation. Regulatory networks were further constructed, resulting in the identification of key regulatory genes, including three transcription factors (JAZ1, HAT22, MYB36) and CLV1, BAM1, CYCB2;4, CDKB2;1, associated with the proliferation and differentiation activity of cambium cells. The enrichment of genes in plant hormone pathways indicates their critical roles in the regulation of wood formation. Our analyses provide a significant groundwork for a comprehensive understanding of the regulatory network of wood formation in S. suchowensis.

RNA m6A modifications regulate crosstalk between tumor metabolism and immunity.

In recent years, m6A modifications in RNA transcripts have arisen as a hot topic in cancer research. Indeed, a number of independent studies have elaborated that the m6A modification impacts the behavior of tumor cells and tumor-infiltrating immune cells, altering tumor cell metabolism along with the differentiation and functional activity of immune cells. This review elaborates on the links between RNA m6A modifications, tumor cell metabolism, and immune cell behavior, discussing this topic from the viewpoint of reciprocal regulation through "RNA m6A-tumor cell metabolism-immune cell behavior" and "RNA m6A-immune cell behavior-tumor cell metabolism" axes. In addition, we discuss the various factors affecting RNA m6A modifications in the tumor microenvironment, particularly the effects of hypoxia associated with cancer cell metabolism along with immune cell-secreted cytokines. Our analysis proposes the conclusion that RNA m6A modifications support widespread interactions between tumor metabolism and tumor immunity. With the current viewpoint that long-term cancer control must tackle cancer cell malignant behavior while strengthening anti-tumor immunity, the recognition of RNA m6A modifications as a key factor provides a new direction for the targeted therapy of tumors. This article is categorized under: RNA Processing > RNA Editing and Modification RNA in Disease and Development > RNA in Disease RNA Interactions with Proteins and Other Molecules > Protein-RNA Interactions: Functional Implications.

Effects of Human Chorionic Gonadotropin on Differentiation and Functional Activity of Myeloid-Derived Suppressor Cells

Effects of Human Chorionic Gonadotropin on Differentiation and Functional Activity of Myeloid-Derived Suppressor Cells

Cellular Senescence in Cardiovascular Diseases: From Pathogenesis to Therapeutic Challenges.

Cellular senescence (CS), classically considered a stable cell cycle withdrawal, is hallmarked by a progressive decrease in cell growth, differentiation, and biological activities. Senescent cells (SNCs) display a complicated senescence-associated secretory phenotype (SASP), encompassing a variety of pro-inflammatory factors that exert influence on the biology of both the cell and surrounding tissue. Among global mortality causes, cardiovascular diseases (CVDs) stand out, significantly impacting the living quality and functional abilities of patients. Recent data suggest the accumulation of SNCs in aged or diseased cardiovascular systems, suggesting their potential role in impairing cardiovascular function. CS operates as a double-edged sword: while it can stimulate the restoration of organs under physiological conditions, it can also participate in organ and tissue dysfunction and pave the way for multiple chronic diseases under pathological states. This review explores the mechanisms that underlie CS and delves into the distinctive features that characterize SNCs. Furthermore, we describe the involvement of SNCs in the progression of CVDs. Finally, the study provides a summary of emerging interventions that either promote or suppress senescence and discusses their therapeutic potential in CVDs.

Rheum palmatum L. and Salvia miltiorrhiza Bge. Alleviates Acute Pancreatitis by Regulating Th17 Cell Differentiation: An Integrated Network Pharmacology Analysis, Molecular Dynamics Simulation and Experimental Validation.

To identify the core targets of Rheum palmatum L. and Salvia miltiorrhiza Bge., (Dahuang-Danshen, DH-DS) and the mechanism underlying its therapeutic efficacy in acute pancreatitis (AP) using a network pharmacology approach and validate the findings in animal experiments. Network pharmacology analysis was used to elucidate the mechanisms underlying the therapeutic effects of DH-DS in AP. The reliability of the results was verified by molecular docking simulation and molecular dynamics simulation. Finally, the results of network pharmacology enrichment analysis were verified by immunohistochemistry, Western blot analysis and real-time quantitative PCR, respectively. Sixty-seven common targets of DH-DS in AP were identified and mitogen-activated protein kinase 3 (MAPK3), Janus kinase 2 (JAK2), signal transducer and activator of transcription 3 (STAT3), protein c-Fos (FOS) were identified as core targets in the protein interaction (PPI) network analysis. Gene ontology analysis showed that cellular response to organic substance was the main functions of DH-DS in AP, and Kyoto Encyclopedia of Genes and Genomes analysis showed that the main pathway included Th17 cell differentiation. Molecular docking simulation confirmed that DH-DS binds with strong affinity to MAPK3, STAT3 and FOS. Molecular dynamics simulation revealed that FOS-isotanshinone II and STAT3-dan-shexinkum d had good binding capacity. Animal experiments indicated that compared with the AP model group, DH-DS treatment effectively alleviated AP by inhibiting the expression of interleukin-1β, interleukin-6 and tumor necrosis factor-α, and blocking the activation of Th17 cell differentiation (P<0.01). DH-DS could inhibit the expression of inflammatory factors and protect pancreatic tissues, which would be functioned by regulating Th17 cell differentiation-related mRNA and protein expressions.

Epithelial TNF controls cell differentiation and CFTR activity to maintain intestinal mucin homeostasis.

The gastrointestinal tract relies on the production, maturation, and transit of mucin to protect against pathogens and to lubricate the epithelial lining. Although the molecular and cellular mechanisms that regulate mucin production and movement are beginning to be understood, the upstream epithelial signals that contribute to mucin regulation remain unclear. Here, we report that the inflammatory cytokine tumor necrosis factor (TNF), generated by the epithelium, contributes to mucin homeostasis by regulating both cell differentiation and cystic fibrosis transmembrane conductance regulator (CFTR) activity. We used genetic mouse models and noninflamed samples from patients with inflammatory bowel disease (IBD) undergoing anti-TNF therapy to assess the effect of in vivo perturbation of TNF. We found that inhibition of epithelial TNF promotes the differentiation of secretory progenitor cells into mucus-producing goblet cells. Furthermore, TNF treatment and CFTR inhibition in intestinal organoids demonstrated that TNF promotes ion transport and luminal flow via CFTR. The absence of TNF led to slower gut transit times, which we propose results from increased mucus accumulation coupled with decreased luminal fluid pumping. These findings point to a TNF/CFTR signaling axis in the adult intestine and identify epithelial cell-derived TNF as an upstream regulator of mucin homeostasis.

Regulation of early seedling establishment and root development in Arabidopsis thaliana by light and carbohydrates

Main conclusionRoot development is regulated by sucrose and light during early seedling establishment through changes in the auxin response and chromatin topology.Light is a key environmental signal that regulates plant growth and development. The impact of light on development is primarily analyzed in the above-ground tissues, but little is known about the mechanisms by which light shapes the architecture of underground roots. Our study shows that carbohydrate starvation during skotomorphogenesis is accompanied by compaction of nuclei in the root apical meristem, which prevents cell cycle progression and leads to irreversible root differentiation in the absence of external carbohydrates, as evidenced by the lack of DNA replication and increased numbers of nuclei with specific chromatin characteristics. In these conditions, induction of photomorphogenesis was unable to restore seedling growth, as overall root growth was compromised. The addition of carbohydrates, either locally or systemically by transferring seedlings to sugar-containing medium, led to the induction of adventitious root formation with rapid recovery of seedling growth. Conversely, transferring in vitro carbohydrate-grown seedlings from light to dark transiently promoted cell elongation and significantly reduced root meristem size, but did not primarily affect cell cycle kinetics. We show that, in the presence of sucrose, dark incubation does not affect zonation in the root apical meristem but leads to shortening of the proliferative and transition zones. Sugar starvation led to a rapid increase in lysine demethylation of histone H3 at position K9, which preceded a rapid decline in cell cycle activity and activation of cell differentiation. In conclusion, carbohydrates are required for cell cycle activity, epigenetics reprogramming and for postmitotic cell elongation and auxin-regulated response in the root apical meristem.

Effects of Human Chorionic Gonadotropin on Differentiation and Functional Activity of Myeloid-Derived Suppressor Cells

Effects of Human Chorionic Gonadotropin on Differentiation and Functional Activity of Myeloid-Derived Suppressor Cells

Alpha-Fetoprotein as a Factor of Differentiation and Functional Activity of Myeloid-Derived Suppressor Cells.

We studied the role of alpha-fetoprotein (AFP) in regulation of differentiation and functional activity of human myeloid-derived suppressor cells (MDSC) in vitro. To obtain MDSC, CD11b+ cells were isolated from the peripheral blood of healthy donors followed by cytokine induction (IL-1β+GM-CSF) into the MDSC phenotype. The cell functions were assessed by the expression of indoleamine 2,3-dioxygenase (IDO) and arginase-1 (Arg1) and cytokine profile of the cell cultures. Native AFP did not affect the total number of MDSC and the percentage of polymorphonuclear MDSC (PMN-MDSC), but increased the number of monocytic MDSC (M-MDSC). AFP did not change the expression of Arg1, but in low concentrations (10 and 50 U/ml) increased the number of IDO-containing cells. AFP modulated the cytokine profile of CD11b+ cells: it reliably decreased the level of IL-19 (50 and100 U/ml) and showed a tendency to decrease the levels of IL-34, MMP-2, sCD163, CHI3L1, OPN and to increase the levels of IL-29, IL-32, APRIL, PTX3, and sTNF-R1. Thus, we have demonstrated a regulatory effect of native AFP at the level of MDSC generated from CD11b+ cells under conditions of cytokine induction in vitro.

Decellularized umbilical cord stroma in tissue engineering and regenerative medicine: a systematic review

Despite great progress in the field of biomaterials for tissue engineering and regenerative medicine, the high requirements placed on artificial matrices (matrices, carriers, scaffolds) are the reason for the ongoing search for natural or synthetic extracellular matrix mimetics. Among such materials, decellularized umbilical cord (UC) stroma appears to be very attractive – it has a high content of hyaluronic acid, cytokines, and growth factors, and there are no ethical restrictions for its production. Decellularized UC stroma has been found to promote cartilage, liver tissue and nerve tissue repair, as well as wound healing. The review critically analyzes and summarizes published data on the ability of decellularized UC stroma to maintain the necessary conditions for adhesion, migration, differentiation and functional activity of adherent cells, thus stimulating the internal (physiological) regenerative potential of tissues. Literature was searched for in the following electronic databases: Medline/PubMed (www/ncbi. nlm.nih.gov/pubmed), Cochrane library (https://www.cochrane.org), and eLIBRARY/Russian Science Citation Index (https://www.elibrary.ru). Inclusion criteria were the presence of biomaterials obtained from decellularized human UC stroma. Exclusion criteria for papers included research objects as decellularized umbilical cord vessels (veins and arteries) and umbilical cord cell cultures. Twenty-five original articles in English and Russian were selected for analysis of the products obtained, their applications, decellularization methods and research results. The review also discusses the prospects for decellularized umbilical cord in medicine.

Vitamin D in Keloids: An alternative to steroids?

Madam, Keloids are wound healing scars with well-demarcated overgrowth of fibrotic tissue, mainly involving excessive collagen proliferation produced by fibroblasts. They extend beyond the original injury site, and are associated with pain and pruritus (1,2). Few treatments available few for Keloids include occlusive dressings, compression therapy and intralesional corticosteroid injections (1). Triamcinolone acetate, a corticosteroid injection, is considered the first line treatment as it possesses anti-inflammatory and anti-mitotic activities, which reduce scar volume and prevent a recurrence. However, approximately 50percent of keloids are considered to be steroid resistant (3, 4). Moreover, as per literature, the usage of intralesional corticosteroid injections can cause several local and systemic adverse effects such as telangiectasias, skin necrosis, skin and subcutaneous fat atrophy, and Cushing’s syndrome (5) In light of the above, researchers are keen on finding other treatment modalities for treating these lesions, and among them Vitamin D, is being investigated. Vitamin D is a steroid hormone that normally plays a role in regulating serum calcium, inhibiting cellular proliferation and promoting cell differentiation and apoptotic activities. Transforming growth factor-beta (TGF-B), an inflammatory marker, has been implicated in the pathogenesis of keloids. TGF-B is believed to be inhibited with localised Vitamin D. Furthermore, Vitamin D deficiency plays a significant role in keloidogenesis, a process mediated by dysregulation of koebnerisin, an antimicrobial polypeptide (2). A study was conducted recently, where 40 adults, aged 18 to 60 years, were administered intralesional Vitamin D, on keloids sized atleast 5 cm, with sessions carried out every week for a maximum of 3-4 sessions. This study showed a highly significant reduction in the Vancouver Scar Scale, a scarring scoring system (1). However, further trials, specially comparative studies, are needed to effectively weigh in the efficacy of Vitamin D over steroids. Advantages of Vitamin D are evident and promising so far – it’s cost-friendly, and shows mild side effects such as burning, pain, swelling and tenderness that shortly disappears on its own.

Absence of Toll-like receptor 5 confers survival in mice bearing ovarian tumors treated with anti-PD-L1

Abstract Ovarian cancer accounts for more deaths than any other cancer of the female reproductive system. Patients bearing ovarian tumors infiltrated with high frequencies of T cells associate with a greater survival probability. However, therapies targeting T cells in ovarian cancers are ineffective. We observed in models of late-stage murine ovarian cancer that in absence of Toll-Like Receptor 5 (TLR5) signaling, anti-PD-L1 promotes significant survival and protection against tumor rechallenge. Ovarian tumors from rechallenged TLR5KO mice exhibited significantly higher frequency/number of CD8 T cells expressing functional and activation markers. Thus, in the absence of TLR5 signaling, T cells mount/maintain a stronger response to ovarian tumors after inhibitory receptor blockade. Furthermore, protection against tumor progression in TLR5KO mice corresponds with a significant increase in tumor infiltrating cross presenting and IL-12 producing Dendritic cells (DCs). Additionally, in CD11c Cre x TLR5 fl/fl mice, whose DCs lack TLR5 expression, PD-L1-blockade mediated extended survival in ovarian tumor-bearing mice. Using a mixed bone marrow chimera approach, we demonstrate that direct TLR5 signaling functionally alters DCs to become more suppressive in the tumor microenvironment, impacting T cell differentiation and anti-tumor activity, thereby reducing survival during aPD-L1 therapy. Clinically, roughly 7.5% of the general population harbor a TLR5 SNP that diminishes TLR5 signaling and is associated with increased long-term survival for ovarian cancer patients. Therefore, patients who express the TLR5 SNP may immediately benefit from anti-PDL1 therapy and those without the SNP, TLR5 antagonism. NIH/NCI 1R01CA253285 UVA Farrow Fellowship

Eosinophil-derived TGFβ1 controls the new bone formation in chronic rhinosinusitis with nasal polyps

Chronic rhinosinusitis with nasal polyps (CRSwNP) is characterized by chronic eosinophilic inflammation and new bone formation (NBF). These processes may be associated with each other in the pathogenesis and influence the severity and prognosis of the disease. However, it is still unclear how eosinophilic inflammation is involved in the NBF. Sinus bone cells were isolated from ethmoid bone tissues of patients with CRSwNP and controls. Transforming growth factor beta 1 (TGFβ1) and alkaline phosphatase (ALP) expression in sinus bone cells was determined using quantitative RT-PCR, immunoblotting, and immunohistochemistry. The co-localization of TGFβ1 with eosinophils was assessed by immunofluorescence staining. Sinus bone cells were co-cultured with eosinophils (Eol-1 cell line), which were differentiated with butyrate, to measure the osteoblast differentiation activity of sinus bone cells. TGFβ1 expression was increased in sinus bone tissues and correlated with CT scores in CRSwNP. TGFβ1 was also increased in the submucosa of CRSwNP and co-localized predominantly with eosinophils compared with neutrophils Differentiated Eol-1 cells-derived TGFβ1 increased ALP expression in sinus bone cells. Treatment with a TGFβ inhibitor attenuated TGFβ1-induced ALP expression and staining in sinus bone cells of CRSwNP, leading to loss of bone formation. Eosinophil-derived TGFβ1 was enriched in the submucosa of CRSwNP, which induced ALP expression in sinus bone cells and NBF. Therefore, eosinophil-derived TGFβ1 may mediate aberrant bone remodeling in CRSwNP.

Chromosome-Level Genome Assembly of the Rough-Toothed Dolphin (Steno bredanensis)

The rough-toothed dolphin (Steno bredanensis), the single extant species of the genus Steno, inhabits tropical and subtropical oceans. It is an attractive species for studying aquatic adaptation and evolution. The latest advances in high-throughput sequencing are transforming the study of marine mammals and contributing to understanding various phenomena at the species and population level by determining high-quality genomes. Here, to comprehensively understand the genetic features and explore the molecular basis of aquatic adaption, the chromosome-level genome assembly and comparative genomics analyses of S. bredanensis were performed. The 2.30 Gb final genome assembly of S. bredanensis (scaffold N50 length of 105.53 Mb) was obtained using single-tube long fragment read (stLFR) and Hi-C technologies. The genome assembly clearly revealed the preservation of large chromosomal fragments between S. bredanensis and the melon-headed whale (Peponocephala electra). The S. bredanensis genome contained 19,451 predicted protein-coding genes, of which about 92.33% have functional annotations. The genome assembly and gene sets showed high completeness, with a BUSCO score of 90.6% and 97.3%, respectively. We also identified several positively selected genes specific to S. bredanensis, which may be related to fat cell differentiation, tooth morphogenesis, and immunoregulatory activity. Finally, the demographic dynamics of S. bredanensis were estimated by the pairwise sequentially Markovian coalescent (PSMC) model and found that the population was affected by the climate at the time. We demonstrated that improved continuity and accuracy of the assembled sequence warranted the adoption of this chromosome-level genome as the reference genome and advanced the understanding of genetic features of the rough-toothed dolphin, which will be essential for future evolutionary studies and the protection of this species.

General Characteristics of Brain Immunohistochemical Markers

Neurons and neuroglia are an important object of medical and biological studies of the brain in normal and pathological conditions. The complexity of the cyto- and myeloarchitectonic of the central nervous system requires adequate methods for studying it. Immunohistochemical methods are among the most highly informative in establishing the morphological and functional characteristics of the nervous system, including during ontogenesis. The purpose of this review is to summarize and systematize literature data on immunohistochemical markers of neuro- and gliogenesis, proliferation, differentiation, and functional activity of nerve cells.

The immunomodulatory role of matrix metalloproteinases in colitis-associated cancer.

Matrix metalloproteinases (MMPs) are an important class of enzymes in the body that function through the extracellular matrix (ECM). They are involved in diverse pathophysiological processes, such as tumor invasion and metastasis, cardiovascular diseases, arthritis, periodontal disease, osteogenesis imperfecta, and diseases of the central nervous system. MMPs participate in the occurrence and development of numerous cancers and are closely related to immunity. In the present study, we review the immunomodulatory role of MMPs in colitis-associated cancer (CAC) and discuss relevant clinical applications. We analyze more than 300 pharmacological studies retrieved from PubMed and the Web of Science, related to MMPs, cancer, colitis, CAC, and immunomodulation. Key MMPs that interfere with pathological processes in CAC such as MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-12, and MMP-13, as well as their corresponding mechanisms are elaborated. MMPs are involved in cell proliferation, cell differentiation, angiogenesis, ECM remodeling, and the inflammatory response in CAC. They also affect the immune system by modulating differentiation and immune activity of immune cells, recruitment of macrophages, and recruitment of neutrophils. Herein we describe the immunomodulatory role of MMPs in CAC to facilitate treatment of this special type of colon cancer, which is preceded by detectable inflammatory bowel disease in clinical populations.

344Laser bioprinting of human iPSC-derived neural stem cells and neurons: Effect on cell survival, multipotency, differentiation, and neuronal activity.

Generation of human neuronal networks by three-dimensional (3D) bioprinting is promising for drug testing and hopefully will allow for the understanding of cellular mechanisms in brain tissue. The application of neural cells derived from human induced-pluripotent stem cells (hiPSCs) is an obvious choice, since hiPSCs provide access to cells unlimited in number and cell types that could be generated by differentiation. The questions in this regard include which neuronal differentiation stage is optimal for printing of such networks, and to what extent the addition of other cell types, especially astrocytes, supports network formation. These aspects are the focus of the present study, in which we applied a laser-based bioprinting technique and compared hiPSC-derived neural stem cells (NSCs) with neuronal differentiated NSCs, with and without the inclusion of co-printed astrocytes. In this study, we investigated in detail the effects of cell types, printed droplet size, and duration of differentiation before and after printing on viability, as well as proliferation, stemness, differentiation potential, formation of dendritic extensions and synapses, and functionality of the generated neuronal networks. We found a significant dependence of cell viability after dissociation on differentiation stage, but no impact of the printing process. Moreover, we observed a dependence of the abundance of neuronal dendrites on droplet size, a marked difference between printed cells and normal cell culture in terms of further differentiation of the cells, especially differentiation into astrocytes, as well as neuronal network formation and activity. Notably, there was a clear effect of admixed astrocytes on NSCs but not on neurons.