Single Progenitor Cell Research Articles

Using Metalloporphyrin Nanosensors for In Situ Monitoring and Measurement of Nitric Oxide and Peroxynitrite in a Single Human Neural Progenitor Cell.

Nitric oxide (NO) is an inorganic signaling molecule that plays a crucial role in the regulation of numerous physiological functions. An oxidation product of the cytoprotective NO is cytotoxic peroxynitrite (ONOO-). In biological systems, the concentrations of NO and ONOO- are typically transient, ranging from nanomolar to micromolar, and these increases are normally followed by a swift return to their basal levels due to their short life spans. To understand the vital physiological role of NO and ONOO- in vitro and in vivo, sensitive and selective methods are necessary for direct and continuous NO and ONOO- measurements in real time. Because electrochemical methods can be adjusted for selectivity, sensitivity, and biocompatibility in demanding biological environments, they are suitable for real-time monitoring of NO and ONOO- release. Metalloporphyrin nanosensors, described here, have been designed to measure the concentration of NO and ONOO- produced by a single human neural progenitor cell (hNPC) in real time. These nanosensors (200-300 nm in diameter) can be positioned accurately in the proximity of 4-5 ± 1 μm from an hNPC membrane. The response time of the sensors is better than a millisecond, while detection limits for NO and ONOO- are 1 × 10-9 and 3 × 10-9 mol/L, respectively, with a linear concentration response of up to about 1 μM. The application of these metalloporphyrin nanosensors for the efficient measurement of the concentrations of NO and ONOO- in hNPCs is demonstrated, providing an opportunity to observe in real time the molecular changes of the two signaling molecules in situ.

A knock down strategy for rapid, generic, and versatile modelling of muscular dystrophies in 3D-tissue-engineered-skeletal muscle

BackgroundHuman iPSC-derived 3D-tissue-engineered-skeletal muscles (3D-TESMs) offer advanced technology for disease modelling. However, due to the inherent genetic heterogeneity among human individuals, it is often difficult to distinguish disease-related readouts from random variability. The generation of genetically matched isogenic controls using gene editing can reduce variability, but the generation of isogenic hiPSC-derived 3D-TESMs can take up to 6 months, thereby reducing throughput.MethodsHere, by combining 3D-TESM and shRNA technologies, we developed a disease modelling strategy to induce distinct genetic deficiencies in a single hiPSC-derived myogenic progenitor cell line within 1 week.ResultsAs proof of principle, we recapitulated disease-associated pathology of Duchenne muscular dystrophy and limb-girdle muscular dystrophy type 2A caused by loss of function of DMD and CAPN3, respectively. shRNA-mediated knock down of DMD or CAPN3 induced a loss of contractile function, disruption of tissue architecture, and disease-specific proteomes. Pathology in DMD-deficient 3D-TESMs was partially rescued by a candidate gene therapy treatment using micro-dystrophin, with similar efficacy compared to animal models.ConclusionsThese results show that isogenic shRNA-based humanized 3D-TESM models provide a fast, cheap, and efficient tool to model muscular dystrophies and are useful for the preclinical evaluation of novel therapies.

Применение моноклональных антител: проблемы и перспективы в ветеринарии

Monoclonal antibodies are the special type of antibodies that are produced by immune cells belonging to the same cell clone. Their specificity lies in the fact that they come from a single progenitor cell, unlike polyclonal antibodies. Monoclonal antibodies have unique properties – monospecificity and identity, therefore they are an ideal diagnostic drug. Monoclonal antibodies have been used in human medicine for decades, but until recently there were no similarly promising developments in veterinary medicine. This article analyzed various sources that describe the use of monoclonal antibodies in the veterinary field, including for the treatment of animal diseases. At the moment, not enough research has been conducted to assess the risk of using monoclonal antibodies for treatment, so studying this problem is truly urgent. It is worth mentioning various diagnostic drugs containing monoclonal antibodies. With their help, it is possible to conduct diagnostic studies that identify antibodies to an antigen or the antigens themselves. Such studies include enzyme immunoassay and immunochromatographic analysis. This article analyzes the problems and prospects of using monoclonal bodies in veterinary medicine.

Inverted Sinonasal Papilloma: Update and Literature Review

Inverted sinonasal papilloma is a benign neoplasm that usually affects the intra-nasal cavity and adjacent paranasal sinuses. Inverted papillomas have three unique features that distinguishes them from other sinonasal tumors including; a propensity for local destructive growth, a high rate of recurrence and risk of malignant transformation. The reported yearly incidence is about 0.6 to 1.5 cases per 100,000 people per year and they usually account for 0.5% to 4% of all nasal tumors. They occur at the following locations; both lateral nasal walls, ethmoidal cells, maxillary sinus, the frontal sinus, sphenoid sinuses and the nasal septum in decreasing order. It occurs most commonly in the fifth and sixth decades of life with a male-to-female ratio of 2-3:1. Inverted papilloma can arise from the entire Schneiderian membrane and molecular genetics have confirmed that it is an actual neoplasm that arises from a single progenitor cell. Most cases are diagnosed from clinical history and thorough physical examination. However, complete removal of all affected tissue with thorough histopathological evaluation is recommended to ensure a correct diagnosis. While there is yet no established pathognomonic histopathological feature to predict recurrence with all certainty, there is ongoing investigation for predictive markers of recurrence. Smoking seems to be a recognized risk factor for developing multiple recurrences and there is evidence that the presence of HPV serotypes 16 and 18 could be predictive of malignant transformation. The main aim of treatment is to achieve compete surgical resection and to prevent recurrence and malignant transformation. A period of clinical follow up is recommended after treatment. The aim of this review is to provide a comprehensive and updated overview of the clinical presentation, diagnosis, management and prognosis of this unique lesion.

Rescue of Alzheimer’s disease phenotype in a mouse model by transplantation of wild-type hematopoietic stem and progenitor cells

Alzheimer's disease (AD) is the most prevalent cause of dementia; microglia have been implicated in AD pathogenesis, but their role is still matter of debate. Our study showed that single systemic wild-type (WT) hematopoietic stem and progenitor cell (HSPC) transplantation rescued the AD phenotype in 5xFAD mice and that transplantation may prevent microglia activation. Indeed, complete prevention of memory loss and neurocognitive impairment and decrease of β-amyloid plaques in the hippocampus and cortex were observed in the WT HSPC-transplanted 5xFAD mice compared with untreated 5xFAD mice and with mice transplanted with 5xFAD HSPCs. Neuroinflammation was also significantly reduced. Transcriptomic analysis revealed a significant decrease in gene expression related to "disease-associated microglia" in the cortex and "neurodegeneration-associated endothelial cells" in the hippocampus of the WT HSPC-transplanted 5xFAD mice compared with diseased controls. This work shows that HSPC transplant has the potential to prevent AD-associated complications and represents a promising therapeutic avenue for this disease.

Deep learning exploration of single-cell and spatially resolved cancer transcriptomics to unravel tumour heterogeneity

Tumour heterogeneity is one of the critical confounding aspects in decoding tumour growth. Malignant cells display variations in their gene transcription profiles and mutation spectra even when originating from a single progenitor cell. Single-cell and spatial transcriptomics sequencing have recently emerged as key technologies for unravelling tumour heterogeneity. Single-cell sequencing promotes individual cell-type identification through transcriptome-wide gene expression measurements of each cell. Spatial transcriptomics facilitates identification of cell-cell interactions and the structural organization of heterogeneous cells within a tumour tissue through associating spatial RNA abundance of cells at distinct spots in the tissue section. However, extracting features and analyzing single-cell and spatial transcriptomics data poses challenges. Single-cell transcriptome data is extremely noisy and its sparse nature and dropouts can lead to misinterpretation of gene expression and the misclassification of cell types. Deep learning predictive power can overcome data challenges, provide high-resolution analysis and enhance precision oncology applications that involve early cancer prognosis, diagnosis, patient survival estimation and anti-cancer therapy planning. In this paper, we provide a background to and review of the recent progress of deep learning frameworks to investigate tumour heterogeneity using both single-cell and spatial transcriptomics data types.

CRISPR-Cas9 Genome Editing and Rapid Selection of Cell Pools.

The harnessing of the CRISPR-Cas9 system allows for quick and inexpensive genome editing in tissue culture models. Traditional CRISPR-Cas9 genome editing techniques rely on the ability of single progenitor cells to expand into new pools in a process known as clonal expansion. This is a significant technical challenge that is difficult to overcome for nontransformed cell culture models such as Drosophila ovarian somatic sheath cells (OSCs). OSCs are a unique ex vivo model for epigenetic regulation by PIWI-interacting RNAs (piRNAs) that establish restriction of mobile genetic elements in germ cells to protect genome integrity. Here, we provide a protocol to generate endogenously tagged proteins and gene knockouts without the need for clonal selection. We combine CRISPR-Cas genome editing and knockin of antibiotic selection markers to generate edited cell pools. At the example of Drosophila piwi in OSCs, we demonstrate a strategy that relies on the insertion of an artificial intron to accommodate a selection marker with minimal disturbance of the resulting mRNA. In brief, our donor cassette contains a peptide tag and an optimized intron that accommodates a selection marker driven by an independent promoter on the other genomic strand. The selection marker is transcribed as an independent mRNA, and the intron is efficiently removed from the mRNA encoding the endogenously tagged (endo-tagged) piwi gene. The endo-tagged Piwi protein is expressed at wild-type levels and appropriately localizes to the nucleus of OSCs. We also describe strategies for C-terminal tagging and generation of knockout alleles in OSCs and in human embryonic kidney cells, discuss different design strategies, and provide a plasmid toolkit (available at Addgene). Our protocol enables robust genome editing in OSCs for the first time and provides a simple and time-saving alternative for other cell culture systems. Published 2022. This article is a U.S. Government work and is in the public domain in the USA. Basic Protocol 1: Design and cloning of single-guide RNA plasmids Basic Protocol 2: Design and cloning of donor template plasmids for epitope tagging Alternate Protocol: Design and cloning of donor template plasmids for gene knockout Basic Protocol 3: Transfection and selection of edited cell pools.

Clonal Dynamics after Haematopoietic Stem Cell Transplantation Using Genome-Wide Somatic Mutations

Background Allogeneic hematopoietic stem cell transplantation (HSCT) is a potentially curative treatment received by over 40,000 patients/year. Despite being an established treatment for 50 years, fundamental questions remain about the dynamics of long-term stem cell engraftment and immunological reconstitution in humans. How many of the >108 transplanted CD34+ cells contribute to long-term hematopoiesis? How does increasing age of donor and recipient reshape clonal dynamics of HSCT? Are the same HSCs responsible for both myeloid and lymphoid reconstitution? Does HSCT increase mutation rates or clonal evolution? Methods We used spontaneous somatic mutations occurring genome-wide in single hematopoietic stem and progenitor cells (HSPCs) to reconstruct high-resolution lineage trees of the hematopoietic system after HSCT. We analysed blood samples from both the donor (D) and recipient (R) for 10 HLA-matched sibling HSCTs - the duration of time elapsed since transplant ranged from 9 to 31 years, meaning that we could study genuinely long-term reconstitution in the recipient, compared to the unperturbed hematopoiesis in the donor. For each individual (D and R), peripheral blood CD34+ HSPC-derived colonies were grown on methylcellulose medium. Whole genome sequencing was performed on 100-300 colonies per individual, generating a final dataset of 3,413 genomes. Somatic mutations from each colony's founder cell were identified and used to build phylogenies for each D/R pair (Figure). For 7/10 pairs, we then used ultra-deep targeted sequencing to assess clonal contributions in sorted mature D/R cell subsets, including monocytes, B-cells and T-cells. Approximate Bayesian computation was used to estimate numbers of engrafting HSCs. RESULTSIn most cases, 10,000 - 50,000 HSCs engrafted and contributed to long-term reconstitution during HSCT. However, grafts from older donors, or those with lower CD34+ cell doses tended to have lower estimates, some as low as <1,000 cells.Recipients had decreased clonal diversity compared to their donors, equivalent to 8-10 years of additional ageing.We detected 93 individual clones that contributed >1% of the population of at least one mature cell type. Each of these expanded clones was detectable in both the donor and the recipient, confirming that they had originated prior to HSCT.Only ~10% of these expanded clones carried mutations in genes known to contribute to clonal hematopoiesis, suggesting that there are many other genes that can influence stem cell dynamics after HSCT.The sizes of these expanded clones often varied dramatically between donors and recipients, up to 25-fold differences in some instances.The population bottleneck caused by HSCT was not sufficient to explain the differences in clonal composition between recipient and donor, suggesting that there are additional HSCT-specific selective pressures during stem cell harvest, in vitro manipulation, infusion, homing and engraftment.While large proportions of mature myeloid and B-cell populations usually derived from the identified clones, this was not true for T-cell populations. This suggests that following HSCT, T-cells are largely derived from stem cells that do not contribute substantially to myeloid reconstitution.HSPC mutation burdens in recipients were similar to their donors, contradicting previous hypotheses that the proliferative stress of HSCT would lead to excess mutations. CONCLUSION Our findings reveal that tens of thousands of HSCs typically engraft during HSCT, but with significant loss of clonal diversity in recipients compared to their unperturbed donors. HSCT-specific selective pressures shape dynamics of hematopoietic reconstitution in the recipient, with substantial differences in clonal composition between myeloid and T-lymphoid populations. This study shows the power of using somatic mutations as clonal marks for lineage tracing in real-world HSCT in humans, an approach we believe will be a new gold standard for understanding the impact of different stem cell sources and transplant protocols. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Very large hidden genetic diversity in one single tumor: evidence for tumors-in-tumor.

Despite the concern of within-tumor genetic diversity, this diversity is in fact limited by the kinship among cells in the tumor. Indeed, genomic studies have amply supported the 'Nowell dogma' whereby cells of the same tumor descend from a single progenitor cell. In parallel, genomic data also suggest that the diversity could be >10-fold larger if tumor cells are of multiple origins. We develop an evolutionary hypothesis that a single tumor may often harbor multiple cell clones of independent origins, but only one would be large enough to be detected. To test the hypothesis, we search for independent tumors within a larger one (or tumors-in-tumor). Very high density sampling was done on two cases of colon tumors. Case 1 indeed has 13 independent clones of disparate sizes, many having heavy mutation burdens and potentially highly tumorigenic. In Case 2, despite a very intensive search, only two small independent clones could be found. The two cases show very similar movements and metastasis of the dominant clone. Cells initially move actively in the expanding tumor but become nearly immobile in late stages. In conclusion, tumors-in-tumor are plausible but could be very demanding to find. Despite their small sizes, they can enhance the within-tumor diversity by orders of magnitude. Such increases may contribute to the missing genetic diversity associated with the resistance to cancer therapy.

Proteome alterations during clonal isolation of established human pancreatic cancer cell lines

Clonal isolation is an integral step of numerous workflows in genome editing and cell engineering. It comprises the isolation of a single progenitor cell from a defined cell line population with subsequent expansion to obtain a monoclonal cell population. This process is associated with transient loss of cell–cell contacts and absence of a multicellular microenvironment. Previous studies have revealed transcriptomic changes upon clonal isolation with cell line specific extent. Since transcriptome alterations are only partially reflected on the proteome level, we sought to investigate the impact of clonal isolation on the cellular proteome to a depth of > 6000 proteins in three established pancreatic cancer cell lines. We show that clonal isolation does have an impact on the cellular proteome, however, with cell line specific extent, affecting different biological processes, and also depending on the isolation method. We demonstrate a different impact of clonal isolation on mesenchymal- and epithelial-derived cell lines mainly affecting cell proliferation, metabolism, cell adhesion and cellular stress. The results bear relevance to the field of genomic editing and cell engineering and highlight the need to consider the impact of clonal isolation when interpreting data stemming from experiments that include this step.

P53 mutation in normal esophagus promotes multiple stages of carcinogenesis but is constrained by clonal competition

Aging normal human oesophagus accumulates TP53 mutant clones. These are the origin of most oesophageal squamous carcinomas, in which biallelic TP53 disruption is almost universal. However, how p53 mutant clones expand and contribute to cancer development is unclear. Here we show that inducing the p53R245W mutant in single oesophageal progenitor cells in transgenic mice confers a proliferative advantage and clonal expansion but does not disrupt normal epithelial structure. Loss of the remaining p53 allele in mutant cells results in genomically unstable p53R245W/null epithelium with giant polyaneuploid cells and copy number altered clones. In carcinogenesis, p53 mutation does not initiate tumour formation, but tumours developing from areas with p53 mutation and LOH are larger and show extensive chromosomal instability compared to lesions arising in wild type epithelium. We conclude that p53 has distinct functions at different stages of carcinogenesis and that LOH within p53 mutant clones in normal epithelium is a critical step in malignant transformation.

Cell Fate of Retinal Progenitor Cells: In Ovo UbC-StarTrack Analysis.

Clonal cell analysis outlines the ontogenic potential of single progenitor cells, allowing the elucidation of the neural heterogeneity among different cell types and their lineages. In this work, we analyze the potency of retinal stem/progenitor cells through development using the chick embryo as a model. We implemented in ovo the clonal genetic tracing strategy UbC-StarTrack for tracking retinal cell lineages derived from individual progenitors of the ciliary margin at E3.5 (HH21-22). The clonal assignment of the derived-cell progeny was performed in the neural retina at E11.5-12 (HH38) through the identification of sibling cells as cells expressing the same combination of fluorophores. Moreover, cell types were assessed based on their cellular morphology and laminar location. Ciliary margin derived-cell progenies are organized in columnar associations distributed along the peripheral retina with a limited tangential dispersion. The analysis revealed that, at the early stages of development, this region harbors multipotent and committed progenitor cells.

Diagnostic and prognostic significance of miRNA-511 and miRNA-16 expressions in adult patients with B-acute lymphoblastic leukemia

Background Acute lymphoblastic leukemia (ALL) is a neoplastic disease that results from multistep somatic mutations in a single lymphoid progenitor cell. MicroRNAs (miRNAs) are critical regulators of gene expression, tumor suppression, and oncogenesis. Aim To evaluate miRNA-511 and miRNA-16 expression in Egyptian adult patients with B-ALL. Patients and methods A total of 37 newly diagnosed adult patients with B-ALL admitted to Alexandria Main University Hospital in 2019 were included. Complete blood count, bone marrow aspiration, immunophenotyping, BCR-ABL testing, karyotyping, miRNA extraction using miRNeasy Mini followed by cDNA synthesis AQ6 (RQ-PCR combines cDNA synthesis from RNA templates using miScript II RT kit), and finally real-time PCR for miRNA-511 and miRNA-16 expression were among the investigations that were conducted. Results Mean age of patients with ALL was 30.65 ± 10.39 years, with male to female ratio of 1.4 : 1. Cytogenetic findings showed that only three patients had favorable risk, and the rest were either intermediate risk (19) or high risk (15). Among the high-risk group, there were 11 patients with Philadelphia chromosome (BCR-ABL 190) positive. Regarding the expression of miRNAs, most patients showed overexpression of both miRNA-16 and miRNA-511. MiRNA-511 was overexpressed in 81.1% (30) patients; among these patients, 43.3% (13) had adverse cytogenetic findings. MiRNA-16 was overexpressed in 70.3% (26) of patients, and half of them (13) had adverse cytogenetic findings. receiver operating characteristic curves showed diagnostic significance in B-ALL for miRNA-16, with sensitivity of 75.7% and specificity of 80%, and for miRNA-511, sensitivity was 89.2% and specificity was 90% (P&lt;0.05). Conclusion MiRNA-16 and miRNA-511 were significantly overexpressed in adult patients with B-ALL. They have a role in diagnosis but a weak role in patient prognosis.

Single-cell imaging of the cell cycle reveals CDC25B-induced heterogeneity of G1 phase length in neural progenitor cells.

Although lengthening of the cell cycle and G1 phase is a generic feature of tissue maturation during development, the underlying mechanism remains poorly understood. Here, we develop a time-lapse imaging strategy to measure the four cell cycle phases in single chick neural progenitor cells in their endogenous environment. We show that neural progenitors are widely heterogeneous with respect to cell cycle length. This variability in duration is distributed over all phases of the cell cycle, with the G1 phase contributing the most. Within one cell cycle, each phase duration appears stochastic and independent except for a correlation between S and M phase duration. Lineage analysis indicates that the majority of daughter cells may have a longer G1 phase than mother cells, suggesting that, at each cell cycle, a mechanism lengthens the G1 phase. We identify that the CDC25B phosphatase known to regulate the G2/M transition indirectly increases the duration of the G1 phase, partly through delaying passage through the restriction point. We propose that CDC25B increases the heterogeneity of G1 phase length, revealing a previously undescribed mechanism of G1 lengthening that is associated with tissue development.

Shh and Olig2 sequentially regulate oligodendrocyte differentiation from hiPSCs for the treatment of ischemic stroke.

Rationale: Demyelination is a major component of white matter injury, characterized by oligodendrocyte (OL) death and myelin sheath loss, which result in memory loss and cognitive impairment in the context of ischemic stroke. Accumulating evidence has shown that OLs can be generated by the direct activation of defined transcription factors (TFs) in human induced pluripotent stem cells (hiPSCs); however, the rapid acquisition of single TF-induced OL progenitor cells (OPCs) as cell therapy for ischemic stroke remains to be thoroughly explored.Methods: A stable, chemically defined protocol was used to generate a substantial number of transplantable and functional OLs through the partial inhibition of sonic hedgehog (Shh) activity by GANT61 during neural induction from hiPSCs and sequential induction of TF Olig2 overexpression. Transcriptome and metabolome analyses further revealed a novel molecular event in which Olig2 regulates OL differentiation from hiPSC-derived neural progenitor cells (NPCs). Olig2-induced NG2+ OPCs (Olig2-OPCs) were then evaluated for their therapeutic potential in cell-based therapy for ischemic stroke.Results: GANT61 treatment resulted in a motor neuron (MN)-OL fate switch during neural induction, and induced overexpression of Olig2 accelerated oligodendroglial lineage cell specification. Olig2-OPCs expressed typical oligodendroglial lineage marker genes, including NKX2.2, CSPG4, and ST8SIA1, and displayed superior ability to differentiate into mature OLs in vitro. Mechanistically, Olig2-OPCs showed increased gene expression of the peroxisome proliferator-activated receptor γ (PPARγ) signaling pathway, and activated CEPT1-mediated phospholipogenesis. Functionally, inhibiting PPARγ and knocking down CEPT1 further compromised the terminal differentiation of Olig2-OPCs. Most importantly, when transplanted into a rat model of transient middle cerebral artery occlusion (tMCAO), Olig2-OPCs efficiently promoted neurological functional recovery by reducing neuronal death, promoting remyelination, and rescuing spatial memory decline.Conclusions: We developed a stable, chemically defined protocol to generate OPCs/OLs with partial inhibition of Shh activity by GANT61 from hiPSCs and sequentially induced the expression of the single TF Olig2. Olig2-OPC transplantation may be an ideal alternative approach for ischemic stroke rehabilitation therapy.

Clonal hematopoiesis and its role in the development of hematological diseases

Introduction. The formation of blood cells in a healthy individual is ensured by polyclonal hematopoiesis. Recent studies have shown that with age, large clones with a common genetic marker are found in the peripheral blood, i. e. cells originating from a single progenitor cell. This phenomenon is called clonal hematopoiesis. In some cases, people with clonal hematopoiesis develop hematological diseases.Aim — to describe and summarize current data on the relationship between clonal hematopoiesis and hematological diseases.Main findings. This review describes the history of detection of clonal hematopoiesis, its main properties, the most frequent mutations in hematopoietic clones associated with the risk of transformation into myelodysplastic syndrome, and acute myeloid leukemia. The meaning and possible pathogenesis of tumor transformation are discussed.

Comparison of the Effects of Intratubal Injection of Adipose-Derived Mesenchymal Stem Cells in a Rat Sciatic Nerve Transection: An Experimental Study.

This study was designed to evaluate the efficacy of epineural tubulization (ENT) with or without intratubal application of adipose-derived mesenchymal stem cells (ASCs) in the rat model of sciatic nerve transection. After formation of 1-cm defect in the left sciatic nerve and ENT, 32 adults female Wistar albino rats were separated into 4 groups (n = 8 for each) including ENT per se (group 1; ENT group) and ENT plus intratubal ASC injection groups killed on day 21 (group 2; ENT-ASC-21-day group), 60 days (group 3; ENT-ASC-60-day group), and 120 days (group 4; ENT-ASC-120-day group). Functional (sciatic function index, hip circumference, withdrawal reflex latency, muscle weight ratio), electrophysiological, histomorphometric, and immunohistochemical analyses were performed in each group. Sciatic function index was significantly higher (-51.98 ± 5.94, P < 0.01) and withdrawal reflex latency was shorter (-6.21 ± 2.14, P < 0.01), in the group 4 as compared with all other groups on day 21. Amplitude of contraction was significantly lower in the group 4 as compared with all other groups (0.22 ± 0.05 vs 0.34 ± 0.07, 0.50 ± 0.11, and 0.61 ± 0.16, P < 0.01 for each). Immunohistochemical analysis revealed presence of green fluorescent protein, vimentin-stained cells, and single neural progenitor cells indicating that induction of neuronal differentiation by ASCs and direct involvement of ASCs within the axonal structure alongside extension of ASCs to the muscular layer of the group 4. In conclusion, our findings revealed that use of ENT plus intratubal ASC injection in a rat sciatic nerve transection model was associated with satisfactory functional outcome and improved peripheral axonal regeneration along with stem cell neural differentiation.

Retrospective assessment of clonality of a legacy cell line by analytical subcloning of the master cell bank.

In recent years, assurance of clonality of the production cell line has been emphasized by health authorities during review of regulatory submissions. When insufficient assurance of clonality is provided, augmented control strategies may be required for a commercial production process. In this study, we conducted a retrospective assessment of clonality of a legacy cell line through analysis of subclones from the master cell bank (MCB). Twenty-four subclones were randomly selected based on a predetermined acceptance sampling plan. All these subclones share a conserved integration junction, thus providing a high level of assurance that the cell population in the MCB was derived from a single progenitor cell. However, Southern blot analysis indicates that at least four subpopulations possibly exist in the MCB. Additional characterization of these four subpopulations demonstrated that the resulting changes in product quality attributes of some subclones are not related to the genetic heterogeneity observed in Southern blot hybridization. Furthermore, process consistency, process comparability, and analytical comparability have been demonstrated in batches produced across varying manufacturing processes, scales, facilities, cell banks, and cell ages. Finally, process and product consistency together with a high level of assurance of clonal origin of the MCB helped clear the hurdle for regulatory approval without requirement of additional control strategies.

Single-cell analysis at the protein level delineates intracellular signaling dynamic during hematopoiesis

BackgroundHematopoietic stem and progenitor cell (HSPC) subsets in mice have previously been studied using cell surface markers, and more recently single-cell technologies. The recent revolution of single-cell analysis is substantially transforming our understanding of hematopoiesis, confirming the substantial heterogeneity of cells composing the hematopoietic system. While dynamic molecular changes at the DNA/RNA level underlying hematopoiesis have been extensively explored, a broad understanding of single-cell heterogeneity in hematopoietic signaling programs and landscapes, studied at protein level and reflecting post-transcriptional processing, is still lacking. Here, we accurately quantified the intracellular levels of 9 phosphorylated and 2 functional proteins at the single-cell level to systemically capture the activation dynamics of 8 signaling pathways, including EGFR, Jak/Stat, NF-κB, MAPK/ERK1/2, MAPK/p38, PI3K/Akt, Wnt, and mTOR pathways, during mouse hematopoiesis using mass cytometry.ResultsWith fine-grained analyses of 3.2 million of single hematopoietic stem and progenitor cells (HSPCs), and lineage cells in conjunction with multiparameter cellular phenotyping, we mapped trajectories of signaling programs during HSC differentiation and identified specific signaling biosignatures of cycling HSPC and multiple differentiation routes from stem cells to progenitor and lineage cells. We also investigated the recovery pattern of hematopoietic cell populations, as well as signaling regulation in these populations, during hematopoietic reconstruction. Overall, we found substantial heterogeneity of pathway activation within HSPC subsets, characterized by diverse patterns of signaling.ConclusionsThese comprehensive single-cell data provide a powerful insight into the intracellular signaling-regulated hematopoiesis and lay a solid foundation to dissect the nature of HSC fate decision. Future integration of transcriptomics and proteomics data, as well as functional validation, will be required to verify the heterogeneity in HSPC subsets during HSC differentiation and to identify robust markers to phenotype those HSPC subsets.

ALL-443: Diagnostic and Prognostic Significance of miRNA-511 and miRNA-16 Expression in Adult B-Acute Lymphoblastic Leukemia Patients

Acute lymphoblastic leukemia (ALL) is a neoplastic disease that results from multistep somatic mutations in a single lymphoid progenitor cell. MicroRNAs (miRNAs) are critical regulators of gene expression, tumor suppression, and oncogenesis. <h3>Aim:</h3> To evaluate miRNA-511 and miRNA-16 expression in Egyptian adult B-ALL. <h3>Subjects and Methods:</h3> 37 newly diagnosed adult B-ALL patients admitted to Alexandria Main University Hospital in 2019. Investigations included CBC, bone marrow aspiration, immunophenotyping, BCR-ABL testing, karyotyping, miRNA extraction using miRNeasy Mini Kit, qRT-PCR combined with cDNA synthesis from RNA templates using miScript II RT Kit, followed by real-time PCR for miR-511 and miR-16 expression analysis. <h3>Results:</h3> Mean age of ALL patients was 30.65 ± 10.39 years, with a male to female ratio of 1.4:1. Cytogenetic findings showed only 3 patients had favorable risk, and the rest were either intermediate risk (19) or high risk (15). Among the high-risk group, 11 patients were Philadelphia (BCR-ABL 190)-positive. Regarding the expression of the miRNAs, most patients showed the overexpression of both miR-16 and miR-511. MiR-511 was overexpressed in 81.1% (30) of patients; among these patients, 43.3% (13) had adverse cytogenetic findings. MiR-16 was overexpressed in 70.3% (26) of patients; half of them (13) had adverse cytogenetic findings. ROC curves showed the diagnostic significance in B-ALL of miR-16, with a sensitivity of 75.7% and a specificity of 80%; for miR-511, the sensitivity was 89.2%, and specificity was 90%, (p<0.05). Among the studied group, 9 (24%) patients developed COVID-19 viral infection, and of those, 3 (33%) patients died, comprising 21% of all deaths. The three patients were either refractory or relapsed and treated mostly as palliative cases. <h3>Conclusion:</h3> MiR-16 and miR-511 were significantly overexpressed in adult B-ALL. They have a role in diagnosis but a weak role in patients' prognosis.