scholarly journals Single-cell time-series mapping of cell fate trajectories reveals an expanded developmental potential for human PSC-derived distal lung progenitors

2019 ◽  
Author(s):  
Killian Hurley ◽  
Jun Ding ◽  
Carlos Villacorta-Martin ◽  
Michael J. Herriges ◽  
Anjali Jacob ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Developmental Plasticity ◽  
Target Cells ◽  
Developmental Potential ◽  
Continuous State ◽  
Lung Progenitors ◽  
Over Time

AbstractAlveolar epithelial type 2 cells (AEC2s) are the facultative progenitors responsible for maintaining lung alveoli throughout life, yet are difficult to access from patients for biomedical research or lung regeneration applications. Here we engineer AEC2s from human induced pluripotent stem cells (iPSCs) in vitro and use single cell RNA sequencing (scRNA-seq) to profile the detailed kinetics of their differentiation over time. We focus on both the desired target cells as well as those that appear to diverge to alternative endodermal fates. By combining scRNA-seq with lentiviral barcoding to trace differentiating clones, we reveal the bifurcating cell fate trajectories followed as primordial lung progenitors differentiate into mature AEC2s. We define the global transcriptomic signatures of primary developing human AEC2s from fetal through adult stages in order to identify the subset of in vitro differentiating cells that appear to recapitulate the path of in vivo development. In addition, we develop computational methods based on Continuous State Hidden Markov Models (CSHMM) to identify the precise timing and type of signals, such as over-exuberant Wnt responses, that induce some early multipotent NKX2-1+ progenitors to lose lung fate as they clonally diverge into a variety of non-lung endodermal lineages. Finally, we find that this initial developmental plasticity is regulatable via Wnt modulation, and subsides over time, ultimately resulting in iPSC-derived AEC2s that exhibit a stable phenotype and nearly limitless self-renewal capacity in vitro. Our methods and computational approaches can be widely applied to study and control directed differentiation, producing an inexhaustible supply of mature lineages, exemplified here by the generation of AEC2s.

scholarly journals Comparative Analyses of Single-Cell Transcriptomic Profiles between In Vitro Totipotent Blastomere-like Cells and In Vivo Early Mouse Embryonic Cells

Cells ◽  
2021 ◽  
Vol 10 (11) ◽  
pp. 3111
Author(s):  
Po-Yu Lin ◽  
Denny Yang ◽  
Chi-Hsuan Chuang ◽  
Hsuan Lin ◽  
Wei-Ju Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Embryonic Cells ◽  
Cell Stage ◽  
Developmental Potential ◽  
Functional Features ◽  
Early Mouse ◽  
Extraembryonic Tissues

The developmental potential within pluripotent cells in the canonical model is restricted to embryonic tissues, whereas totipotent cells can differentiate into both embryonic and extraembryonic tissues. Currently, the ability to culture in vitro totipotent cells possessing molecular and functional features like those of an early embryo in vivo has been a challenge. Recently, it was reported that treatment with a single spliceosome inhibitor, pladienolide B (plaB), can successfully reprogram mouse pluripotent stem cells into totipotent blastomere-like cells (TBLCs) in vitro. The TBLCs exhibited totipotency transcriptionally and acquired expanded developmental potential with the ability to yield various embryonic and extraembryonic tissues that may be employed as novel mouse developmental cell models. However, it is disputed whether TBLCs are ‘true’ totipotent stem cells equivalent to in vivo two-cell stage embryos. To address this question, single-cell RNA sequencing was applied to TBLCs and cells from early mouse embryonic developmental stages and the data were integrated using canonical correlation analyses. Differential expression analyses were performed between TBLCs and multi-embryonic cell stages to identify differentially expressed genes. Remarkably, a subpopulation within the TBLCs population expressed a high level of the totipotent-related genes Zscan4s and displayed transcriptomic features similar to mouse two-cell stage embryonic cells. This study underscores the subtle differences between in vitro derived TBLCs and in vivo mouse early developmental cell stages at the single-cell transcriptomic level. Our study has identified a new experimental model for stem cell biology, namely ‘cluster 3’, as a subpopulation of TBLCs that can be molecularly defined as near totipotent cells.

scholarly journals Analysis of cardiac differentiation at single cell resolution reveals a requirement of hypertrophic signaling for HOPX transcription

2017 ◽  
Author(s):  
Clayton E Friedman ◽  
Quan Nguyen ◽  
Samuel W Lukowski ◽  
Han Sheng Chiu ◽  
Abbigail Helfer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Cell Fate ◽  
Heart Development ◽  
Time Course ◽  
Cardiac Differentiation ◽  
Transcriptional Networks ◽  
Hypertrophic Signaling

AbstractDifferentiation into diverse cell lineages requires the orchestration of gene regulatory networks guiding diverse cell fate choices. Utilizing human pluripotent stem cells, we measured expression dynamics of 17,718 genes from 43,168 cells across five time points over a thirty day time-course of in vitro cardiac-directed differentiation. Unsupervised clustering and lineage prediction algorithms were used to map fate choices and transcriptional networks underlying cardiac differentiation. We leveraged this resource to identify strategies for controlling in vitro differentiation as it occurs in vivo. HOPX, a non-DNA binding homeodomain protein essential for heart development in vivo was identified as dys-regulated in in vitro derived cardiomyocytes. Utilizing genetic gain and loss of function approaches, we dissect the transcriptional complexity of the HOPX locus and identify the requirement of hypertrophic signaling for HOPX transcription in hPSC-derived cardiomyocytes. This work provides a single cell dissection of the transcriptional landscape of cardiac differentiation for broad applications of stem cells in cardiovascular biology.

scholarly journals Understanding cell fate control by continuous single-cell quantification

Blood ◽  
2019 ◽  
Vol 133 (13) ◽  
pp. 1406-1414 ◽  
Author(s):  
Dirk Loeffler ◽  
Timm Schroeder
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Cell Imaging ◽  
Population Based ◽  
Molecular Processes ◽  
Cell Fate Decisions ◽  
Single Cell Imaging

Abstract Cells and the molecular processes underlying their behavior are highly dynamic. Understanding these dynamic biological processes requires noninvasive continuous quantitative single-cell observations, instead of population-based average or single-cell snapshot analysis. Ideally, single-cell dynamics are measured long-term in vivo; however, despite progress in recent years, technical limitations still prevent such studies. On the other hand, in vitro studies have proven to be useful for answering long-standing questions. Although technically still demanding, long-term single-cell imaging and tracking in vitro have become valuable tools to elucidate dynamic molecular processes and mechanisms, especially in rare and heterogeneous populations. Here, we review how continuous quantitative single-cell imaging of hematopoietic cells has been used to solve decades-long controversies. Because aberrant cell fate decisions are at the heart of tissue degeneration and disease, we argue that studying their molecular dynamics using quantitative single-cell imaging will also improve our understanding of these processes and lead to new strategies for therapies.

Single-Cell Analysis of Lymphoid-Primed Multipotent Progenitors (LMPPs) Reveal Alterations in Lineage Commitment during Aging

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 244-244
Author(s):  
Sneha Borikar ◽  
Vivek Philip ◽  
Jennifer J. Trowbridge
Keyword(s):  
Single Cell ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Progenitor Cell ◽  
Conflicts Of Interest ◽  
Single Cells ◽  
Differentiation Potential ◽  
Increased Risk

Abstract During aging, the hematopoietic compartment undergoes lineage skewing, biased toward myeloid differentiation at the expense of lymphoid differentiation. This skewing clinically presents as impaired adaptive immunity and an increased risk of myeloproliferative disorders. However, little is known of the regulatory mechanisms underlying these changes in differentiation potential due in part to the inadequacy of current analytic techniques to evaluate lineage potency of individual progenitor cells. Recent demonstration that long-lived hematopoietic progenitor cells drive steady-state hematopoiesis has shifted focus onto the progenitor cell compartment to understand clonal dynamics of native hematopoiesis. Here, we critically assess the functional and molecular alterations in the multipotent progenitor cell pool with aging at the single-cell level. We developed novel in vitro and in vivo assays to define the heterogeneity of the LMPP population and test cell-fate potential from single cells. Our results demonstrate, for the first time, distinct, intrinsic lineage potential of single in vitro LMPPs at the cellular and molecular level. We find that clonal alterations in the lymphoid-primed multipotent progenitor (LMPP) compartment contributes to the functional alterations in hematopoiesis observed during aging. Unbiased single-cell transcriptome analysis reveals that true multipotential clones and lymphoid-restricted clones are reduced with aging, while bipotential and myeloid-restricted clones are modestly expanded. Furthermore, myeloid-restricted clones gain myc driver signatures, molecularly identifying clones emerging during aging that are susceptible to transformation. Our study reveals that aging alters the clonal composition of multipotential progenitor cells, directly contributing to the global loss of the lymphoid compartment and increased susceptibility to myeloid transformation. Disclosures No relevant conflicts of interest to declare.

scholarly journals Oscillation between B-lymphoid and myeloid lineages in Myc-induced hematopoietic tumors following spontaneous silencing/reactivation of the EBF/Pax5 pathway

Blood ◽  
2003 ◽  
Vol 101 (5) ◽  
pp. 1950-1955 ◽  
Author(s):  
Duonan Yu ◽  
David Allman ◽  
Michael H. Goldschmidt ◽  
Michael L. Atchison ◽  
John G. Monroe ◽  
...  
Keyword(s):  
B Cell ◽  
Single Cell ◽  
Target Genes ◽  
Myeloid Differentiation ◽  
Target Cells ◽  
Cell Clones ◽  
B Lymphoid ◽  
Secondary Lymphoma

B lymphomagenesis is an uncontrolled expansion of immature precursors that fail to complete their differentiation program. This failure could be at least partly explained by inappropriate expression of several oncogenic transcription factors, such as Pax5 and Myc. Both Pax5 and c-Myc are implicated in the pathogenesis of non-Hodgkin lymphomas. To address their role in lymphomagenesis, we analyzed B-cell lymphomas derived from p53-null bone marrow progenitors infected in vivo by a Myc-encoding retrovirus. All Myc-induced lymphomas invariably maintained expression of Pax5, which is thought to be incompatible with terminal differentiation. However, upon culturing in vitro, several cell lines spontaneously down-regulated Pax5 and its target genes CD19, N-Myc, and MB1. Unexpectedly, other B-cell markers (eg, CD45R) were also down-regulated, and markers of myeloid lineage (CD11b and F4/80 antigen) were acquired instead. Moreover, cells assumed the morphology reminiscent of myeloid cells. A pool of F4/80-positive cells as well as several single-cell clones were obtained and reinjected into syngeneic mice. Remarkably, pooled cells rapidly re-expressed Pax5 and formed tumors of relatively mature lymphoid phenotype, with surface immunoglobulins being abundantly expressed. Approximately half of tumorigenic single-cell clones also abandoned myeloid differentiation and gave rise to B lymphomas. However, when secondary lymphoma cells were returned to in vitro conditions, they once again switched to myeloid differentiation. This process could be curbed via enforced expression of retrovirally encoded Pax5. Our data demonstrate that some Myc target cells are bipotent B-lymphoid/myeloid progenitors with the astonishing capacity to undergo successive rounds of lineage switching.

scholarly journals In Vivo Pre-Instructed HSCs Robustly Execute Asymmetric Cell Divisions In Vitro

2020 ◽  
Vol 21 (21) ◽  
pp. 8225
Author(s):  
Mukul Girotra ◽  
Vincent Trachsel ◽  
Aline Roch ◽  
Matthias P. Lutolf
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Asymmetric Distribution ◽  
Hematopoietic Stem ◽  
Asymmetric Cell Divisions ◽  
Native Bone ◽  
Daughter Cells ◽  
Cell Divisions

Hematopoietic stem cells (HSCs) are responsible for life-long production of all mature blood cells. Under homeostasis, HSCs in their native bone marrow niches are believed to undergo asymmetric cell divisions (ACDs), with one daughter cell maintaining HSC identity and the other committing to differentiate into various mature blood cell types. Due to the lack of key niche signals, in vitro HSCs differentiate rapidly, making it challenging to capture and study ACD. To overcome this bottleneck, in this study, we used interferon alpha (IFNα) treatment to ”pre-instruct” HSC fate directly in their native niche, and then systematically studied the fate of dividing HSCs in vitro at the single cell level via time-lapse analysis, as well as multigene and protein expression analysis. Triggering HSCs’ exit from dormancy via IFNα was found to significantly increase the frequency of asynchronous divisions in paired daughter cells (PDCs). Using single-cell gene expression analyses, we identified 12 asymmetrically expressed genes in PDCs. Subsequent immunocytochemistry analysis showed that at least three of the candidates, i.e., Glut1, JAM3 and HK2, were asymmetrically distributed in PDCs. Functional validation of these observations by colony formation assays highlighted the implication of asymmetric distribution of these markers as hallmarks of HSCs, for example, to reliably discriminate committed and self-renewing daughter cells in dividing HSCs. Our data provided evidence for the importance of in vivo instructions in guiding HSC fate, especially ACD, and shed light on putative molecular players involved in this process. Understanding the mechanisms of cell fate decision making should enable the development of improved HSC expansion protocols for therapeutic applications.

scholarly journals Hypoxia promotes a perinatal-like progenitor state in the adult murine epicardium

2021 ◽  
Author(s):  
Angeliqua Sayed ◽  
Szimonetta Turoczi ◽  
Francisca Soares-da-Silva ◽  
Giovanna Marazzi ◽  
Jean-Sebastien Hulot ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Fate ◽  
Transcriptional Profiling ◽  
Differentiation Potential ◽  
Mammalian Heart ◽  
Epicardial Cells ◽  
Cell Culture Systems ◽  
Vitro Model

Abstract The epicardium is a reservoir of progenitors that give rise to coronary vasculature and stroma during development and mediates cardiac vascular repair in lower vertebrates. However, its role as a source of progenitors in the adult mammalian heart remains unclear due to lack of clear lineage markers and single-cell culture systems to elucidate epicardial progeny cell fate. We found that in vivo exposure of mice to physiological hypoxia induced adult epicardial cells to re-enter the cell cycle and to express a subset of developmental genes. Multiplex single cell transcriptional profiling revealed a lineage relationship between epicardial cells and smooth muscle, stromal, and endothelial fates, and that physiological hypoxia promoted an endothelial cell fate. In vitro clonal analyses of purified epicardial cells showed that cell growth and subsequent differentiation is dependent upon hypoxia, and that resident epicardial cells retain progenitor identity in the adult mammalian heart with self-renewal and multilineage differentiation potential. These results point to a source of progenitor cells in the adult heart that can promote heart revascularization, providing an invaluable in vitro model for further studies.

The Development of Homologous (Canine/Anti-Canine) Antibodies in Dogs with Haemophilia A (Factor VIII Deficiency): A Ten-Year Longitudinal Study

1993 ◽  
Vol 69 (01) ◽  
pp. 021-024 ◽  
Author(s):  
Shawn Tinlin ◽  
Sandra Webster ◽  
Alan R Giles
Keyword(s):  
Factor Viii ◽  
Canine Model ◽  
Significant Inhibition ◽  
Human Condition ◽  
Haemophilia A ◽  
Variable Expression ◽  
The Family ◽  
Over Time

SummaryThe development of inhibitors to factor VIII in patients with haemophilia A remains as a serious complication of replacement therapy. An apparently analogous condition has been described in a canine model of haemophilia A (Giles et al., Blood 1984; 63:451). These animals and their relatives have now been followed for 10 years. The observation that the propensity for inhibitor development was not related to the ancestral factor VIII gene has been confirmed by the demonstration of vertical transmission through three generations of the segment of the family related to a normal (non-carrier) female that was introduced for breeding purposes. Haemophilic animals unrelated to this animal have not developed functionally significant factor VIII inhibitors despite intensive factor VIII replacement. Two animals have shown occasional laboratory evidence of factor VIII inhibition but this has not been translated into clinical significant inhibition in vivo as assessed by clinical response and F.VIII recovery and survival characteristics. Substantial heterogeneity of inhibitor expression both in vitro and in vivo has been observed between animals and in individual animals over time. Spontaneous loss of inhibitors has been observed without any therapies designed to induce tolerance, etc., being instituted. There is also phenotypic evidence of polyclonality of the immune response with variable expression over time in a given animal. These observations may have relevance to the human condition both in determining the pathogenetic factors involved in this condition and in highlighting the heterogeneity of its expression which suggests the need for caution in the interpretation of the outcome of interventions designed to modulate inhibitor activity.

Non-Viral Vectors for Gene Delivery

2018 ◽  
Vol 9 (1) ◽  
pp. 4-11 ◽  
Author(s):  
Aparna Bansal ◽  
Himanshu
Keyword(s):  
Gene Therapy ◽  
Viral Vectors ◽  
Transfection Efficiency ◽  
Gene Transfection ◽  
Expression System ◽  
Target Cells ◽  
Specific Expression ◽  
Naked Dna

Introduction: Gene therapy has emerged out as a promising therapeutic pave for the treatment of genetic and acquired diseases. Gene transfection into target cells using naked DNA is a simple and safe approach which has been further improved by combining vectors or gene carriers. Both viral and non-viral approaches have achieved a milestone to establish this technique, but non-viral approaches have attained a significant attention because of their favourable properties like less immunotoxicity and biosafety, easy to produce with versatile surface modifications, etc. Literature is rich in evidences which revealed that undoubtedly, non–viral vectors have acquired a unique place in gene therapy but still there are number of challenges which are to be overcome to increase their effectiveness and prove them ideal gene vectors. Conclusion: To date, tissue specific expression, long lasting gene expression system, enhanced gene transfection efficiency has been achieved with improvement in delivery methods using non-viral vectors. This review mainly summarizes the various physical and chemical methods for gene transfer in vitro and in vivo.

scholarly journals Single-cell RNA sequencing reveals ex vivo signatures of SARS-CoV-2-reactive T cells through ‘reverse phenotyping’

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
David S. Fischer ◽  
Meshal Ansari ◽  
Karolin I. Wagner ◽  
Sebastian Jarosch ◽  
Yiqi Huang ◽  
...  
Keyword(s):  
T Cells ◽  
Single Cell ◽  
Rna Sequencing ◽  
Ex Vivo ◽  
Severe Disease ◽  
Human Leukocyte ◽  
Cell Receptor ◽  
Single Cell Rna Sequencing

AbstractThe in vivo phenotypic profile of T cells reactive to severe acute respiratory syndrome (SARS)-CoV-2 antigens remains poorly understood. Conventional methods to detect antigen-reactive T cells require in vitro antigenic re-stimulation or highly individualized peptide-human leukocyte antigen (pHLA) multimers. Here, we use single-cell RNA sequencing to identify and profile SARS-CoV-2-reactive T cells from Coronavirus Disease 2019 (COVID-19) patients. To do so, we induce transcriptional shifts by antigenic stimulation in vitro and take advantage of natural T cell receptor (TCR) sequences of clonally expanded T cells as barcodes for ‘reverse phenotyping’. This allows identification of SARS-CoV-2-reactive TCRs and reveals phenotypic effects introduced by antigen-specific stimulation. We characterize transcriptional signatures of currently and previously activated SARS-CoV-2-reactive T cells, and show correspondence with phenotypes of T cells from the respiratory tract of patients with severe disease in the presence or absence of virus in independent cohorts. Reverse phenotyping is a powerful tool to provide an integrated insight into cellular states of SARS-CoV-2-reactive T cells across tissues and activation states.

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