scholarly journals Trajectory reconstruction identifies dysregulation of perinatal maturation programs in pluripotent stem cell-derived cardiomyocytes

2021 ◽  
Author(s):  
Suraj Kannan ◽  
Matthew Miyamoto ◽  
Brian L. Lin ◽  
Chulan Kwon
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Single Cell ◽  
Pluripotent Stem Cell ◽  
Directed Differentiation ◽  
Trajectory Reconstruction ◽  
Single Cell Rna Sequencing

ABSTRACTA primary limitation in the clinical application of pluripotent stem cell derived cardiomyocytes (PSC-CMs) is the failure of these cells to achieve full functional maturity. In vivo, cardiomyocytes undergo numerous adaptive changes during perinatal maturation. By contrast, PSC-CMs fail to fully undergo these developmental processes, instead remaining arrested at an embryonic stage of maturation. To date, however, the precise mechanisms by which directed differentiation differs from endogenous development, leading to consequent PSC-CM maturation arrest, are unknown. The advent of single cell RNA-sequencing (scRNA-seq) has offered great opportunities for studying CM maturation at single cell resolution. However, perinatal cardiac scRNA-seq has been limited owing to technical difficulties in the isolation of single CMs. Here, we used our previously developed large particle fluorescence-activated cell sorting approach to generate an scRNA-seq reference of mouse in vivo CM maturation with extensive sampling of perinatal time periods. We subsequently generated isogenic embryonic stem cells and created an in vitro scRNA-seq reference of PSC-CM directed differentiation. Through trajectory reconstruction methods, we identified a perinatal maturation program in endogenous CMs that is poorly recapitulated in vitro. By comparison of our trajectories with previously published human datasets, we identified a network of nine transcription factors (TFs) whose targets are consistently dysregulated in PSC-CMs across species. Notably, we demonstrated that these TFs are only partially activated in common ex vivo approaches to engineer PSC-CM maturation. Our study represents the first direct comparison of CM maturation in vivo and in vitro at the single cell level, and can be leveraged towards improving the clinical viability of PSC-CMs.Significance StatementThere is a significant clinical need to generate mature cardiomyocytes from pluripotent stem cells. However, to date, most differentiation protocols yield phenotypically immature cardiomyocytes. The mechanisms underlying this poor maturation state are unknown. Here, we used single cell RNA-sequencing to compare cardiomyocyte maturation pathways in endogenous and pluripotent stem cell-derived cardiomyocytes. We found that in vitro, cardiomyocytes fail to undergo critical perinatal gene expression changes necessary for complete maturation. We found that key transcription factors regulating these changes are poorly expressed in vitro. Our study provides a better understanding of cardiomyocyte maturation both in vivo and in vitro, and may lead to improved approaches for engineering mature cardiomyocytes from stem cells.

scholarly journals Regulation of sarcomagenesis by the empty spiracles homeobox genes EMX1 and EMX2

2021 ◽  
Vol 12 (6) ◽  
Author(s):  
Manuel Pedro Jimenez-García ◽  
Antonio Lucena-Cacace ◽  
Daniel Otero-Albiol ◽  
Amancio Carnero
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Neural Crest ◽  
Tumor Suppressors ◽  
Homeobox Genes ◽  
Neuronal Cell ◽  
Cell Gene Expression

AbstractThe EMX (Empty Spiracles Homeobox) genes EMX1 and EMX2 are two homeodomain gene members of the EMX family of transcription factors involved in the regulation of various biological processes, such as cell proliferation, migration, and differentiation, during brain development and neural crest migration. They play a role in the specification of positional identity, the proliferation of neural stem cells, and the differentiation of certain neuronal cell phenotypes. In general, they act as transcription factors in early embryogenesis and neuroembryogenesis from metazoans to higher vertebrates. The EMX1 and EMX2’s potential as tumor suppressor genes has been suggested in some cancers. Our work showed that EMX1/EMX2 act as tumor suppressors in sarcomas by repressing the activity of stem cell regulatory genes (OCT4, SOX2, KLF4, MYC, NANOG, NES, and PROM1). EMX protein downregulation, therefore, induced the malignance and stemness of cells both in vitro and in vivo. In murine knockout (KO) models lacking Emx genes, 3MC-induced sarcomas were more aggressive and infiltrative, had a greater capacity for tumor self-renewal, and had higher stem cell gene expression and nestin expression than those in wild-type models. These results showing that EMX genes acted as stemness regulators were reproduced in different subtypes of sarcoma. Therefore, it is possible that the EMX genes could have a generalized behavior regulating proliferation of neural crest-derived progenitors. Together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-altering populations or cancer stem cells, acting as tumor suppressors in sarcoma.

scholarly journals Design Principles for Pluripotent Stem Cell-Derived Organoid Engineering

2019 ◽  
Vol 2019 ◽  
pp. 1-17 ◽  
Author(s):  
Teresa P. Silva ◽  
João P. Cotovio ◽  
Evguenia Bekman ◽  
Maria Carmo-Fonseca ◽  
Joaquim M. S. Cabral ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cell ◽  
The Self ◽  
Self Organization ◽  
Complex Process ◽  
Reliable Source ◽  
Human Disorders

Human morphogenesis is a complex process involving distinct microenvironmental and physical signals that are manipulated in space and time to give rise to complex tissues and organs. Advances in pluripotent stem cell (PSC) technology have promoted the in vitro recreation of processes involved in human morphogenesis. The development of organoids from human PSCs represents one reliable source for modeling a large spectrum of human disorders, as well as a promising approach for drug screening and toxicological tests. Based on the “self-organization” capacity of stem cells, different PSC-derived organoids have been created; however, considerable differences between in vitro-generated PSC-derived organoids and their in vivo counterparts have been reported. Advances in the bioengineering field have allowed the manipulation of different components, including cellular and noncellular factors, to better mimic the in vivo microenvironment. In this review, we focus on different examples of bioengineering approaches used to promote the self-organization of stem cells, including assembly, patterning, and morphogenesis in vitro, contributing to tissue-like structure formation.

Pluripotent Stem Cell Modeling of Normal and Abnormal Megakaryocyte Development and Platelet Production

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1276-1276
Author(s):  
Brenden W Smith ◽  
Darrell N Kotton ◽  
Gustavo Mostoslavsky ◽  
George J. Murphy
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pluripotent Stem Cells ◽  
Peripheral Blood ◽  
Patient Specific ◽  
Directed Differentiation ◽  
Platelet Production ◽  
Blood Disorder

Abstract Abstract 1276 Thrombocytopenia is a multi-factorial blood disorder characterized by an abnormally low number of circulating platelets that can have devastating effects upon a wide swath of patients independent of age, race, or socioeconomic group. The two major reasons for thrombocytopenia are increased turnover in immune thrombocytopenia purpura (ITP) and decreased production due to bone marrow failure as a result of chemotherapy, aging, or drugs. Even in ITP, there is some evidence that decreased production may play a role in the etiology of the disease. Thus, patients not making enough platelets are usually treated with platelet transfusions, which carry risks of allergic reactions, infections, and eventually sensitization to allo-antigens making patients refractory to transfusions. With these facts in mind, there is a clear need for the development of novel, autologous sources of mature platelets, and the ability to produce patient-specific megakaryocytes from pluripotent stem cells would have a potential therapeutic role. We have developed a novel, excisable reprogramming vector (STEMCCA) capable of generating ‘clinical grade’ induced Pluripotent Stem Cells (iPSC) free of any residual reprogramming transgenes, and have employed this vector in the derivation of both normal and megakaryocyte disease-specific cell lines. To develop a novel source of platelet precursors for hematopoietic and cell-based therapy studies, we have established conditions for the efficient directed differentiation of these lines into a virtually unlimited supply of functional megakaryocyte-lineage cells that express a constellation of accepted megakaryocyte markers, appropriate Wright-Giemsa stained morphology, expected polyploidy via endoreduplication, and both normal and aberrant platelet production. iPSC-derived megakaryocytes were subsequently tagged with viral vectors expressing fluorescent proteins (for quantification of platelet contribution in peripheral blood) and/or luciferase (for in vivo imaging studies) and administered to mouse models via the retro orbital sinus. Transplanted mice were monitored for the presence of the transferred megakaryocytes and resulting platelets via Ly 5.1/5.2 chimerism as well as for the presence of GFP positive cells using FACS analysis. Peripheral blood from these mice was screened at 1 day post transplantation for chimerism and expression of GFP, and at subsequent 2 day time periods when GFP positive cells were noted in order to track the continued viability or death of the megakaryocyte-lineage cells and resulting platelets. Following these cell transfer experiments, the presence of green platelets in the peripheral blood of these mice indicated that the megakaryocyte-lineage cells produced from the directed differentiation of iPSC are indeed viable in vivo and are capable of the production of platelets. The duration of reconstitution and the functionality of the platelets derived from the iPSC generated megakaryocytes as well as those generated from embryonic stem cell (ESC) controls are currently being assessed by quantifying the labeled platelets over time, and carrying out tests of platelet function in vivo (bleeding time) and in vitro (platelet aggregation studies). Our current work focuses on the hypothesis that an iPSC-based system is capable of producing sufficient numbers of fully functional megakaryocytes to ameliorate thrombocytopenia in vivo. The implications of successfully testing this hypothesis are profound, for they suggest that early megakaryocyte and platelet development can be directly evaluated in vitro and, moreover, that megakaryocyte-lineage cells produced from patient-specific, directly differentiated iPSC lines can become a potent source for transfusion studies and regenerative medicine. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mapping the biogenesis of forward programmed megakaryocytes from induced pluripotent stem cells

2021 ◽  
Author(s):  
Moyra Lawrence ◽  
Arash Shahsavari ◽  
Susanne Bornelöv ◽  
Thomas Moreau ◽  
Katarzyna Kania ◽  
...  
Keyword(s):  
Stem Cells ◽  
Single Cell ◽  
Pluripotent Stem Cells ◽  
Surface Markers ◽  
Single Cell Rna Sequencing ◽  
Induced Pluripotent ◽  
Insight Into ◽  
Megakaryocyte Progenitors

SummaryPlatelet deficiency, known as thrombocytopenia, can cause haemorrhage and is treated with platelet transfusions. We developed a system for the production of platelet precursor cells, megakaryocytes, from pluripotent stem cells. These cultures can be maintained for >100 days, implying culture renewal by megakaryocyte progenitors (MKPs). However, it is unclear whether the MKP state in vitro mirrors the state in vivo, and MKPs cannot be purified using conventional surface markers. We performed single cell RNA sequencing throughout in vitro differentiation and mapped each state to its equivalent in vivo. This enabled the identification of 5 surface markers which reproducibly purify MKPs, allowing us an insight into their transcriptional and epigenetic profiles. Finally, we performed culture optimisation, increasing MKP production. Altogether, this study has mapped parallels between the MKP states in vivo and in vitro and allowed the purification of MKPs, accelerating the progress of in vitro-derived transfusion products towards the clinic.

scholarly journals Cryopreservation of Induced Pluripotent Stem Cell-Derived Dopaminergic Neurospheres for Clinical Application

2021 ◽  
pp. 1-14
Author(s):  
Satoe Hiramatsu ◽  
Asuka Morizane ◽  
Tetsuhiro Kikuchi ◽  
Daisuke Doi ◽  
Kenji Yoshida ◽  
...  
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Clinical Application ◽  
Pluripotent Stem Cell ◽  
3D Structure ◽  
Induced Pluripotent Stem Cell ◽  
Cell Aggregates ◽  
Induced Pluripotent

Background: Pluripotent stem cell (PSC)-derived dopaminergic (DA) neurons are an expected source of cell therapy for Parkinson’s disease. The transplantation of cell aggregates or neurospheres, instead of a single cell suspension has several advantages, such as keeping the 3D structure of the donor cells and ease of handling. For this PSC-based therapy to become a widely available treatment, cryopreservation of the final product is critical in the manufacturing process. However, cryopreserving cell aggregates is more complicated than cryopreserving single cell suspensions. Previous studies showed poor survival of the DA neurons after the transplantation of cryopreserved fetal ventral-mesencephalic tissues. Objective: To achieve the cryopreservation of induced pluripotent stem cell (iPSC)-derived DA neurospheres toward clinical application. Methods: We cryopreserved iPSC-derived DA neurospheres in various clinically applicable cryopreservation media and freezing protocols and assessed viability and neurite extension. We evaluated the population and neuronal function of cryopreserved cells by the selected method in vitro. We also injected the cells into 6-hydroxydopamine (6-OHDA) lesioned rats, and assessed their survival, maturation and function in vivo. Results: The iPSC-derived DA neurospheres cryopreserved by Proton Freezer in the cryopreservation medium Bambanker hRM (BBK) showed favorable viability after thawing and had equivalent expression of DA-specific markers, dopamine secretion, and electrophysiological activity as fresh spheres. When transplanted into 6-OHDA-lesioned rats, the cryopreserved cells survived and differentiated into mature DA neurons, resulting in improved abnormal rotational behavior. Conclusion: These results show that the combination of BBK and Proton Freezer is suitable for the cryopreservation of iPSC-derived DA neurospheres.

scholarly journals Empty spiracles homeobox genes EMX1 and EMX2 regulate WNT pathway activation in sarcomagenesis

2021 ◽  
Vol 40 (1) ◽  
Author(s):  
Manuel Pedro Jimenez-García ◽  
Antonio Lucena-Cacace ◽  
Daniel Otero-Albiol ◽  
Amancio Carnero
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Transcription Factors ◽  
Cell Biology ◽  
Wnt Pathway ◽  
Ectopic Expression ◽  
Homeobox Genes ◽  
Cell Phenotype

Abstract Background Sarcomas are a very heterogeneous group of tumors with intrinsic developmental programs derived from the cell of origin. This implies a functional hierarchy inside tumors governed by sarcoma stem cells. Therefore, genetic and/or epigenetic changes profoundly affect the biology of sarcoma tumor stem cells. EMX genes are proposed to be transcription factors that are involved in the sarcomagenesis process, regardless of the neural or mesodermal embryological sarcoma origin. It has been shown that EMX1 or EMX2 overexpression reduces tumorigenic properties, while reducing the levels of these genes enhances these properties. Furthermore, it has been shown that EMX genes decrease the expression of stem cell regulatory genes and the stem cell phenotype. Taken together, these results indicate that the EMX1 and EMX2 genes negatively regulate these tumor-remodeling populations or sarcoma stem cells, acting as tumor suppressors in sarcoma. Methods Bioinformatic analysis, quantitative mRNA and protein expression analysis, cell models of sarcoma by ectopic expression of EMX genes. By cell biology methods we measured tumorigenesis and populations enriched on stem cell phenotypes, either in vitro or in vivo. Results In this work, we showed that the canonical Wnt pathway is one of the mechanisms that explains the relationships of EMX1/EMX2 and stem cell genes in sarcoma. The Wnt-EMX1/EMX2 relationship was validated in silico with sarcoma patient datasets, in vitro in primary derived sarcoma cell lines, and in vivo. EMX expression was found to negatively regulate the Wnt pathway. In addition, the constitutive activation of the Wnt pathway revers to a more aggressive phenotype with stem cell properties, and stemness gene transcription increased even in the presence of EMX1 and/or EMX2 overexpression, establishing the relationship among the Wnt pathway, stem cell genes and the EMX transcription factors. Conclusions Our data showed that Empty Spiracles Homeobox Genes EMX1 and EMX2 represses WNT signalling and activation of WNT pathway bypass EMX-dependent stemness repression and induces sarcomagenesis. These results also suggest the relevance of the Wnt/b-catenin/stemness axis as a therapeutic target in sarcoma.

scholarly journals Single-Cell RNA Sequencing Reveals Novel Markers of Male Pituitary Stem Cells and Hormone-Producing Cell Types

Endocrinology ◽  
2018 ◽  
Vol 159 (12) ◽  
pp. 3910-3924 ◽  
Author(s):  
Leonard Y M Cheung ◽  
Akima S George ◽  
Stacey R McGee ◽  
Alexandre Z Daly ◽  
Michelle L Brinkmeier ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Pituitary Gland ◽  
Single Cell ◽  
Rna Sequencing ◽  
Clustering Algorithms ◽  
White Blood Cells ◽  
Cell Types ◽  
Single Cell Rna Sequencing

Abstract Transcription factors and signaling pathways that regulate stem cells and specialized hormone-producing cells in the pituitary gland have been the subject of intense study and have yielded a mechanistic understanding of pituitary organogenesis and disease. However, the regulation of stem cell proliferation and differentiation, the heterogeneity among specialized hormone-producing cells, and the role of nonendocrine cells in the gland remain important, unanswered questions. Recent advances in single-cell RNA sequencing (scRNAseq) technologies provide new avenues to address these questions. We performed scRNAseq on ∼13,663 cells pooled from six whole pituitary glands of 7-week-old C57BL/6 male mice. We identified pituitary endocrine and stem cells in silico, as well as other support cell types such as endothelia, connective tissue, and red and white blood cells. Differential gene expression analyses identify known and novel markers of pituitary endocrine and stem cell populations. We demonstrate the value of scRNAseq by in vivo validation of a novel gonadotrope-enriched marker, Foxp2. We present novel scRNAseq data of in vivo pituitary tissue, including data from agnostic clustering algorithms that suggest the presence of a somatotrope subpopulation enriched in sterol/cholesterol synthesis genes. Additionally, we show that incomplete transcriptome annotation can cause false negatives on some scRNAseq platforms that only generate 3′ transcript end sequences, and we use in vivo data to recover reads of the pituitary transcription factor Prop1. Ultimately, scRNAseq technologies represent a significant opportunity to address long-standing questions regarding the development and function of the different populations of the pituitary gland throughout life.

scholarly journals Transcriptomic entropy benchmarks stem cell-derived cardiomyocyte maturation against endogenous tissue at single cell level

2020 ◽  
Author(s):  
Suraj Kannan ◽  
Michael Farid ◽  
Brian L. Lin ◽  
Matthew Miyamoto ◽  
Chulan Kwon
Keyword(s):  
Stem Cell ◽  
Single Cell ◽  
Rna Sequencing ◽  
Pluripotent Stem Cell ◽  
Developmental Trajectory ◽  
Single Cell Level ◽  
Batch Effects ◽  
Cell Level ◽  
Single Cell Rna Sequencing

The immaturity of pluripotent stem cell (PSC)-derived tissues has emerged as a universal problem for their biomedical applications. While efforts have been made to generate adult-like cells from PSCs, direct benchmarking of PSC-derived tissues against in vivo development has not been established. Thus, maturation status is often assessed on an ad-hoc basis. Single cell RNA-sequencing (scRNA-seq) offers a promising solution, though cross-study comparison is limited by dataset-specific batch effects. Here, we developed a novel approach to quantify PSC-derived cardiomyocyte (CM) maturation through transcriptomic entropy. Transcriptomic entropy is robust across datasets regardless of differences in isolation protocols, library preparation, and other potential batch effects. With this new model, we analyzed over 45 scRNA-seq datasets and over 52,000 CMs, and established a cross-study, cross-species CM maturation reference. This reference enabled us to directly compare PSC-CMs with the in vivo developmental trajectory and thereby to quantify PSC-CM maturation status. We further found that our entropy-based approach can be used for other cell types, including pancreatic beta cells and hepatocytes. Our study presents a biologically relevant and interpretable metric for quantifying PSC-derived tissue maturation, and is extensible to numerous tissue engineering contexts.Significance StatementThere is significant interest in generating mature cardiomyocytes from pluripotent stem cells. However, there are currently few effective metrics to quantify the maturation status of a single cardiomyocyte. We developed a new metric for measuring cardiomyocyte maturation using single cell RNA-sequencing data. This metric, called entropy score, uses the gene distribution to estimate maturation at the single cell level. Entropy score enables comparing pluripotent stem cell-derived cardiomyocytes directly against endogenously-isolated cardiomyocytes. Thus, entropy score can better assist in development of approaches to improve the maturation of pluripotent stem cell-derived cardiomyocytes.

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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