scholarly journals A Novel FACS-Based Workflow for Simultaneous Assessment of RedOx Status, Cellular Phenotype, and Mitochondrial Genome Stability

BioChem ◽  
2021 ◽  
Vol 1 (1) ◽  
pp. 1-18
Author(s):  
Patrick M. McTernan ◽  
Paige S. Katz ◽  
Constance Porretta ◽  
David A. Welsh ◽  
Robert W. Siggins
Keyword(s):  
Mitochondrial Dna ◽  
Genome Stability ◽  
Mitochondrial Dynamics ◽  
Cell Types ◽  
Redox Status ◽  
Pharmacological Interventions ◽  
Hematopoietic Stem ◽  
Mtdna Damage ◽  
Stem And Progenitor Cells ◽  
Mitochondrial Genome Stability

Intracellular reduction-oxidation (RedOx) status mediates a myriad of critical biological processes. Importantly, RedOx status regulates the differentiation of hematopoietic stem and progenitor cells (HSPCs), mesenchymal stromal cells (MSCs) and maturation of CD8+ T Lymphocytes. In most cells, mitochondria are the greatest contributors of intracellular reactive oxygen species (ROS). Excess ROS leads to mitochondrial DNA (mtDNA) damage and protein depletion. We have developed a fluorescence-activated cell sorting (FACS)-based protocol to simultaneously analyze RedOx status and mtDNA integrity. This simultaneous analysis includes measurements of ROS (reduced glutathione (GSH)), ATP5H (nuclear encoded protein), MTCO1 (mitochondrial DNA encoded protein), and cell surface markers to allow discrimination of different cell populations. Using the ratio of MTCO1 to ATP5H median fluorescence intensity (MFI), we can gain an understanding of mtDNA genomic stability, since MTCO1 levels are decreased when mtDNA becomes significantly damaged. Furthermore, this workflow can be optimized for sorting cells, using any of the above parameters, allowing for downstream quantification of mtDNA genome copies/nucleus by quantitative PCR (qPCR). This unique methodology can be used to enhance analyses of the impacts of pharmacological interventions, as well as physiological and pathophysiological processes on RedOx status along with mitochondrial dynamics in most cell types.

scholarly journals Mitochondrial DNA Heteroplasmy as an Informational Reservoir Dynamically Linked to Metabolic and Immunological Processes Associated with COVID-19 Neurological Disorders

2021 ◽  
Author(s):  
George B. Stefano ◽  
Richard M. Kream
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Function ◽  
Neurological Disorders ◽  
Nuclear Dna ◽  
Mitochondrial Dynamics ◽  
Cell Types ◽  
Tissue Specific ◽  
Copy Numbers ◽  
The Central Nervous System ◽  
Mitochondrial Dna Heteroplasmy

AbstractMitochondrial DNA (mtDNA) heteroplasmy is the dynamically determined co-expression of wild type (WT) inherited polymorphisms and collective time-dependent somatic mutations within individual mtDNA genomes. The temporal expression and distribution of cell-specific and tissue-specific mtDNA heteroplasmy in healthy individuals may be functionally associated with intracellular mitochondrial signaling pathways and nuclear DNA gene expression. The maintenance of endogenously regulated tissue-specific copy numbers of heteroplasmic mtDNA may represent a sensitive biomarker of homeostasis of mitochondrial dynamics, metabolic integrity, and immune competence. Myeloid cells, monocytes, macrophages, and antigen-presenting dendritic cells undergo programmed changes in mitochondrial metabolism according to innate and adaptive immunological processes. In the central nervous system (CNS), the polarization of activated microglial cells is dependent on strategically programmed changes in mitochondrial function. Therefore, variations in heteroplasmic mtDNA copy numbers may have functional consequences in metabolically competent mitochondria in innate and adaptive immune processes involving the CNS. Recently, altered mitochondrial function has been demonstrated in the progression of coronavirus disease 2019 (COVID-19) due to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Accordingly, our review is organized to present convergent lines of empirical evidence that potentially link expression of mtDNA heteroplasmy by functionally interactive CNS cell types to the extent and severity of acute and chronic post-COVID-19 neurological disorders.

scholarly journals Adult blood stem cell localization reflects the abundance of reported bone marrow niche cell types and their combinations

Blood ◽  
2020 ◽  
Vol 136 (20) ◽  
pp. 2296-2307 ◽  
Author(s):  
Konstantinos D. Kokkaliaris ◽  
Leo Kunz ◽  
Nina Cabezas-Wallscheid ◽  
Constantina Christodoulou ◽  
Simon Renders ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cell Types ◽  
Bone Marrow Niche ◽  
Functional Heterogeneity ◽  
Hematopoietic Stem ◽  
Native Bone ◽  
Cell Localization ◽  
Exact Localization ◽  
Stem And Progenitor Cells ◽  
Multiple Cell

Abstract The exact localization of hematopoietic stem cells (HSCs) in their native bone marrow (BM) microenvironment remains controversial, because multiple cell types have been reported to physically associate with HSCs. In this study, we comprehensively quantified HSC localization with up to 4 simultaneous (9 total) BM components in 152 full-bone sections from different bone types and 3 HSC reporter lines. We found adult femoral α-catulin-GFP+ or Mds1GFP/+Flt3Cre HSCs proximal to sinusoids, Cxcl12 stroma, megakaryocytes, and different combinations of those populations, but not proximal to bone, adipocyte, periarteriolar, or Schwann cells. Despite microanatomical differences in femurs and sterna, their adult α-catulin-GFP+ HSCs had similar distributions. Importantly, their microenvironmental localizations were not different from those of random dots, reflecting the relative abundance of imaged BM populations rather than active enrichment. Despite their functional heterogeneity, dormant label-retaining (LR) and non-LR hematopoietic stem and progenitor cells both had indistinguishable localization from α-catulin-GFP+ HSCs. In contrast, cycling juvenile BM HSCs preferentially located close to Cxcl12 stroma and farther from sinusoids/megakaryocytes. We expect our study to help resolve existing confusion regarding the exact localization of different HSC types, their physical association with described BM populations, and their tissue-wide combinations.

The influence of mitochondrial dynamics on mitochondrial genome stability

2017 ◽  
Vol 64 (1) ◽  
pp. 199-214 ◽  
Author(s):  
Christopher T. Prevost ◽  
Nicole Peris ◽  
Christina Seger ◽  
Deanna R. Pedeville ◽  
Kathryn Wershing ◽  
...  
Keyword(s):  
Mitochondrial Genome ◽  
Genome Stability ◽  
Mitochondrial Dynamics ◽  
Mitochondrial Genome Stability

scholarly journals Simultaneous tracking of division and differentiation from individual hematopoietic stem and progenitor cells reveals within-family homogeneity despite population heterogeneity

2019 ◽  
Author(s):  
Tamar Tak ◽  
Giulio Prevedello ◽  
Gaël Simon ◽  
Noémie Paillon ◽  
Ken R. Duffy ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
High Throughput ◽  
Common Ancestor ◽  
Single Cells ◽  
Population Level ◽  
Cell Types ◽  
Culture Conditions ◽  
Population Heterogeneity ◽  
Hematopoietic Stem ◽  
Stem And Progenitor Cells

AbstractThe advent of high throughput single cell methods such as scRNA-seq has uncovered substantial heterogeneity in the pool of hematopoietic stem and progenitor cells (HSPCs). A significant issue is how to reconcile those findings with the standard model of hematopoietic development, and a fundamental question is how much instruction is inherited by offspring from their ancestors. To address this, we further developed a high-throughput method that enables simultaneously determination of common ancestor, generation, and differentiation status of a large collection of single cells. Data from it revealed that while there is substantial population-level heterogeneity, cells that derived from a common ancestor were highly concordant in their division progression and share similar differentiation outcomes, revealing significant familial effects on both division and differentiation. Although each family diversifies to some extent, the overall collection of cell types observed in a population is largely composed of homogeneous families from heterogeneous ancestors. Heterogeneity between families could be explained, in part, by differences in ancestral expression of cell-surface markers that are used for phenotypic HSPC identification: CD48, SCA-1, c-kit and Flt3. These data call for a revision of the fundamental model of haematopoiesis from a single tree to an ensemble of trees from distinct ancestors where common ancestor effect must be considered. As HSPCs are cultured in the clinic before bone marrow transplantation, our results suggest that the broad range of engraftment and proliferation capacities of HSPCs could be consequences of the heterogeneity in their engrafted families, and altered culture conditions might reduce heterogeneity between families, possibly improving transplantation outcomes.

scholarly journals A Cell type-specific Class of Chromatin Loops Anchored at Large DNA Methylation Nadirs

2017 ◽  
Author(s):  
Mira Jeong ◽  
Xingfan Huang ◽  
Xiaotian Zhang ◽  
Jianzhong Su ◽  
Muhammad S. Shamim ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Cell Function ◽  
Cell Types ◽  
Specific Class ◽  
Hematopoietic Stem ◽  
Cell State ◽  
Chromatin Loops ◽  
Stem And Progenitor Cells ◽  
A Cell ◽  
Cell Type Specific

AbstractHigher order chromatin structure and DNA methylation are implicated in multiple developmental processes, but their relationship to cell state is unknown. Here, we found that large (~10kb) DNA methylation nadirs can form long loops connecting anchor loci that may be dozens of megabases apart, as well as interchromosomal links. The interacting loci comprise ~3.5Mb of the human genome. The data are more consistent with the formation of these loops by phase separation of the interacting loci to form a genomic subcompartment, rather than with CTCF-mediated extrusion. Interestingly, unlike previously characterized genomic subcompartments, this subcompartment is only present in particular cell types, such as stem and progenitor cells. Further, we identify one particular loop anchor that is functionally associated with maintenance of the hematopoietic stem cell state. Our work reveals that H3K27me3-marked large DNA methylation nadirs represent a novel set of very long-range loops and links associated with cellular identity.SummaryHi-C and DNA methylation analyses reveal novel chromatin loops between distant sites implicated in stem and progenitor cell function.

scholarly journals Ultraviolet B, melanin and mitochondrial DNA: Photo-damage in human epidermal keratinocytes and melanocytes modulated by alpha-melanocyte-stimulating hormone

F1000Research ◽  
2016 ◽  
Vol 5 ◽  
pp. 881 ◽  
Author(s):  
Markus Böhm ◽  
Helene Z. Hill
Keyword(s):  
Mitochondrial Dna ◽  
Copy Number ◽  
Cell Types ◽  
Ultraviolet B ◽  
Epidermal Keratinocytes ◽  
Human Epidermal Keratinocytes ◽  
Mtdna Damage ◽  
Melanocyte Stimulating Hormone ◽  
Copy Numbers ◽  
Stimulating Hormone

Alpha-melanocyte-stimulating hormone (alpha-MSH) increases melanogenesis and protects from UV-induced DNA damage. However, its effect on mitochondrial DNA (mtDNA) damage is unknown. We have addressed this issue in a pilot study using human epidermal keratinocytes and melanocytes incubated with alpha-MSH and irradiated with UVB. Real-time touchdown PCR was used to quantify total and deleted mtDNA. The deletion detected encompassed the common deletion but was more sensitive to detection. There were 4.4 times more mtDNA copies in keratinocytes than in melanocytes. Irradiation alone did not affect copy numbers. Alpha-MSH slightly increased copy numbers in both cell types in the absence of UVB and caused a similar small decrease in copy number with dose in both cell types. Deleted copies were nearly twice as frequent in keratinocytes as in melanocytes. Alpha-MSH reduced the frequency of deleted copies by half in keratinocytes but not in melanocytes. UVB dose dependently led to an increase in the deleted copy number in alpha-MSH-treated melanocytes. UVB irradiation had little effect on deleted copy number in alpha-MSH-treated keratinocytes. In summary, alpha-MSH enhances mtDNA damage in melanocytes presumably by increased melanogenesis, while α-MSH is protective in keratinocytes, the more so in the absence of irradiation.

scholarly journals Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit

2021 ◽  
Vol 6 (1) ◽  
Author(s):  
Elad Jacoby ◽  
Moriya Ben Yakir-Blumkin ◽  
Shiri Blumenfeld-Kan ◽  
Yehuda Brody ◽  
Amilia Meir ◽  
...  
Keyword(s):  
Mitochondrial Dna ◽  
Mouse Model ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Functional Benefit ◽  
Syngeneic Mouse Model ◽  
Multisystemic Disease ◽  
Stem And Progenitor Cells ◽  
Disease Modifying

AbstractMitochondria are cellular organelles critical for numerous cellular processes and harboring their own circular mitochondrial DNA (mtDNA). Most mtDNA associated disorders (either deletions, mutations, or depletion) lead to multisystemic disease, often severe at a young age, with no disease-modifying therapies. Mitochondria have a capacity to enter eukaryotic cells and to be transported between cells. We describe a method of ex vivo augmentation of hematopoietic stem and progenitor cells (HSPCs) with normal exogenous mitochondria, termed mitochondrial augmentation therapy (MAT). Here, we show that MAT is feasible and dose dependent, and improves mitochondrial content and oxygen consumption of healthy and diseased HSPCs. Ex vivo mitochondrial augmentation of HSPCs from a patient with a mtDNA disorder leads to superior human engraftment in a non-conditioned NSGS mouse model. Using a syngeneic mouse model of accumulating mitochondrial dysfunction (Polg), we show durable engraftment in non-conditioned animals, with in vivo transfer of mitochondria to recipient hematopoietic cells. Taken together, this study supports MAT as a potential disease-modifying therapy for mtDNA disorders.

scholarly journals A cell proliferation-dependent multiprotein complex NC-3A positively regulates the CD34 promoter via a TCATTT-containing element

Blood ◽  
1996 ◽  
Vol 88 (9) ◽  
pp. 3336-3348 ◽  
Author(s):  
D Perrotti ◽  
T Bellon ◽  
R Trotta ◽  
R Martinez ◽  
B Calabretta
Keyword(s):  
Promoter Activity ◽  
Hematopoietic Cells ◽  
Cell Types ◽  
Multiprotein Complex ◽  
Mobility Shift ◽  
Hematopoietic Stem ◽  
Enhancer Element ◽  
Human Cd34 ◽  
Stem And Progenitor Cells ◽  
Mobility Shift Assay

The CD34 cell surface antigen is a glycoprotein expressed by hematopoietic stem and progenitor cells and also by certain nonhematopoietic cell-types. Because CD34 expression is regulated both at the transcriptional and the posttranscriptional level, we attempted to identify factors that, by interacting with the 5′ flanking region of the human CD34 gene, may regulate its promoter activity in proliferating hematopoietic cells. By electrophoretic mobility shift assay, UV cross-linking and DNase I footprinting analyses, we identified a multiprotein complex, designated NC-3A, that specifically interacts with the CD34 promoter region from nucleotides -375 to -351. Sequence analysis of this region revealed the presence of a distinct motif, TCATTT. Chloramphenicol acetyl-transferase assays used to assess promoter activity in transiently transfected cells showed that this TCATTT-containing element, which is conserved in both the human and the murine CD34 genes, mediates positive regulatory activity in hematopoietic and nonhematopoietic cells, and acts as an enhancer when placed upstream of a heterologous promoter. Moreover, loss of CD34 promoter activity was caused by mutation of the TCATTT motif. In addition, the interaction of the nuclear multiprotein complex NC-3A with this enhancer element is proliferation-dependent. These data indicate that, although not cell-type specific, the formation of a multiprotein complex NC-3A interacting with the region from nucleotides -375 to 351 plays an important role in controlling CD34 promoter activity in proliferating hematopoietic cells.

scholarly journals Mitochondrial DNA Integrity: Role in Health and Disease

Cells ◽  
2019 ◽  
Vol 8 (2) ◽  
pp. 100 ◽  
Author(s):  
Priyanka Sharma ◽  
Harini Sampath
Keyword(s):  
Mitochondrial Dna ◽  
Mitochondrial Genome ◽  
Genome Stability ◽  
Dna Lesions ◽  
Genome Integrity ◽  
Dna Integrity ◽  
Age Related ◽  
Genome Damage ◽  
Health And Disease ◽  
Mitochondrial Genome Stability

As the primary cellular location for respiration and energy production, mitochondria serve in a critical capacity to the cell. Yet, by virtue of this very function of respiration, mitochondria are subject to constant oxidative stress that can damage one of the unique features of this organelle, its distinct genome. Damage to mitochondrial DNA (mtDNA) and loss of mitochondrial genome integrity is increasingly understood to play a role in the development of both severe early-onset maladies and chronic age-related diseases. In this article, we review the processes by which mtDNA integrity is maintained, with an emphasis on the repair of oxidative DNA lesions, and the cellular consequences of diminished mitochondrial genome stability.

scholarly journals The hemogenic endothelium: a critical source for the generation of PSC-derived hematopoietic stem and progenitor cells

2021 ◽  
Author(s):  
Lucas Lange ◽  
Michael Morgan ◽  
Axel Schambach
Keyword(s):  
Stem Cells ◽  
De Novo ◽  
Cell Types ◽  
Hemogenic Endothelium ◽  
Hematopoietic Stem ◽  
Autologous Cell ◽  
Stem And Progenitor Cells ◽  
Bona Fide

AbstractIn vitro generation of hematopoietic cells and especially hematopoietic stem cells (HSCs) from human pluripotent stem cells (PSCs) are subject to intensive research in recent decades, as these cells hold great potential for regenerative medicine and autologous cell replacement therapies. Despite many attempts, in vitro, de novo generation of bona fide HSCs remains challenging, and we are still far away from their clinical use, due to insufficient functionality and quantity of the produced HSCs. The challenges of generating PSC-derived HSCs are already apparent in early stages of hemato-endothelial specification with the limitation of recapitulating complex, dynamic processes of embryonic hematopoietic ontogeny in vitro. Further, these current shortcomings imply the incompleteness of our understanding of human ontogenetic processes from embryonic mesoderm over an intermediate, specialized hemogenic endothelium (HE) to their immediate progeny, the HSCs. In this review, we examine the recent investigations of hemato-endothelial ontogeny and recently reported progress for the conversion of PSCs and other promising somatic cell types towards HSCs with the focus on the crucial and inevitable role of the HE to achieve the long-standing goal—to generate therapeutically applicable PSC-derived HSCs in vitro.

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