Genetic lineage tracing defines myofibroblast origin and function in the injured heart methods
Aim. Evidence-backed execution summary for Genetic lineage tracing defines myofibroblast origin and function in the injured heart methods from Genetic lineage tracing defines myofibroblast origin and function in the injured heart.
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mouse
Subject model for the experiment.
- Use
- confirm full cohort details in the source paper
Periostin-labelled fibroblasts are functional in disease
reagent used in the protocol.
- Use
- To assess the relevance of periostin-labelled myofibroblasts during cardiac injury and fibrosis, we generated compound heterozygote mice carrying the Postn MCM allele and the Rosa26 -DTA (diphtheria toxin A) allele, which generates an inducible Cre-dependent means of killing cells in vivo ( ). Here Postn MCM/+; R26...
Origins of periostin-labelled adult cardiac fibroblasts
reagent used in the protocol.
- Use
- Myofibroblasts in the injured or diseased heart have been suggested to originate and transdifferentiate from many cellular sources, such as an endothelial cells, immune cells, smooth muscle cells, pericytes or epicardial derived resident fibroblasts. However, there is disagreement even amongst these studies each cl...
Origins of periostin-labelled adult cardiac fibroblasts
reagent used in the protocol.
- Use
- As yet another criteria for determining the extent to which select cellular origins contribute to myofibroblasts in the heart, the lineage-tracing analyses with these same Cre lines was compared against a full analysis of antibody markers as presented earlier ( ). Mice were given tamoxifen food for 2 weeks, allowed...
Flow cytometry and cell sorting
reagent used in the protocol.
- Use
- Flow cytometry analysis was performed on isolated cardiac interstitial cells using a BD FACSCanto II running FACSDiva software with the following configuration: 405 nm laser for Alexa405, 633 nm for APC and 488 nm for GFP. Voltages were determined using single-stain and fluorescence minus one (FMO)...
Disscussion
reagent used in the protocol.
- Use
- The conclusion that Tcf21-expressing resident cardiac fibroblasts are the primary source of disease-activated myofibroblasts in the heart is not consistent with previous studies that have suggested alternate lineages. For example, endothelial-to-mesenchymal transition from resident endothelial cells was reported to...
Histology and immunohistochemistry
reagent used in the protocol.
- Use
- Isolated organs were fixed for 3.5 h in freshly diluted 4% PFA at 4 °C, rinsed with PBS and cryoprotected in 30% sucrose/PBS overnight before embedding in OCT (Tissue-Tek). Afterwards, 10 µm cryosections were collected and then blocked for 30 min at room temperature in a blocking so...
Isolation of cardiac fibroblasts
reagent used in the protocol.
- Use
- For FACS analysis whole cardiac ventricles were excised from mice, rinsed with cold sterile HBSS (Fisher Scientific, SH30588.01), and then placed in a 35 mm dishes with 300 µl DMEM (Fisher Scientific, SH30022FS) to prevent drying. For isolating fibroblasts from injury or remote regions, hearts were...
Flow cytometry and cell sorting
reagent used in the protocol.
- Use
- For FACS of lineage-traced cells, injured and uninjured regions of left ventricles were micro-dissected under dissection microscope. Total interstitial cell fractions from these injured or uninjured regions were isolated by enzymatic digestion as described above and cells were stained for surface markers of endothel...
Periostin expression is restricted to myofibroblasts
Next, the identity of the interstitial cell population labelled in Postn MCM/+; R26-eGFP mice was interrogated after MI injury and 2 weeks of tamoxifen treatment ( ). Hearts were harvested and serial cryosections were processed to show fibrosis and coincident eGFP + cell labelling (periostin lineage-traced) along w...
- Use
- Next, the identity of the interstitial cell population labelled in Postn MCM/+; R26-eGFP mice was interrogated after MI injury and 2 weeks of tamoxifen treatment ( ). Hearts were harvested and serial cryosections were processed to show fibrosis and coincident eGFP + cell labelling (periostin lineage-traced) along w...
Periostin-labelled fibroblasts are functional in disease
To assess the relevance of periostin-labelled myofibroblasts during cardiac injury and fibrosis, we generated compound heterozygote mice carrying the Postn MCM allele and the Rosa26 -DTA (diphtheria toxin A) allele, which generates an inducible Cre-dependent means of killing cells in vivo ( ). Here Postn MCM/+; R26...
- Use
- To assess the relevance of periostin-labelled myofibroblasts during cardiac injury and fibrosis, we generated compound heterozygote mice carrying the Postn MCM allele and the Rosa26 -DTA (diphtheria toxin A) allele, which generates an inducible Cre-dependent means of killing cells in vivo ( ). Here Postn MCM/+; R26...
Origins of periostin-labelled adult cardiac fibroblasts
Since resident Tcf21 lineage-traced cells were the overwhelming source of periostin-expressing myofibroblasts in the infarct region of the heart, a more elaborate investigation of Tcf21 expression and lineage-traced cells was undertaken using Tcf21 MCM/+; R26-eGFP mice ( ). Here, we first began with uninjured mice...
- Use
- Since resident Tcf21 lineage-traced cells were the overwhelming source of periostin-expressing myofibroblasts in the infarct region of the heart, a more elaborate investigation of Tcf21 expression and lineage-traced cells was undertaken using Tcf21 MCM/+; R26-eGFP mice ( ). Here, we first began with uninjured mice...
Flow cytometry and cell sorting
Flow cytometry analysis was performed on isolated cardiac interstitial cells using a BD FACSCanto II running FACSDiva software with the following configuration: 405 nm laser for Alexa405, 633 nm for APC and 488 nm for GFP. Voltages were determined using single-stain and fluorescence minus one (FMO)...
- Use
- Flow cytometry analysis was performed on isolated cardiac interstitial cells using a BD FACSCanto II running FACSDiva software with the following configuration: 405 nm laser for Alexa405, 633 nm for APC and 488 nm for GFP. Voltages were determined using single-stain and fluorescence minus one (FMO)...
Periostin + myofibroblasts can be partially inactivated
To more carefully assess the identity of these persistent periostin lineage-traced (eGFP + ) cells, FACS was used for cellular purification followed by RNAseq analysis ( ). Compared with the RNAseq profile of currently activated periostin + myofibroblasts taken right after 2 weeks of Ang/PE infusion, the 'reco...
- Use
- To more carefully assess the identity of these persistent periostin lineage-traced (eGFP + ) cells, FACS was used for cellular purification followed by RNAseq analysis ( ). Compared with the RNAseq profile of currently activated periostin + myofibroblasts taken right after 2 weeks of Ang/PE infusion, the 'reco...
Disscussion
The conclusion that Tcf21-expressing resident cardiac fibroblasts are the primary source of disease-activated myofibroblasts in the heart is not consistent with previous studies that have suggested alternate lineages. For example, endothelial-to-mesenchymal transition from resident endothelial cells was reported to...
- Use
- The conclusion that Tcf21-expressing resident cardiac fibroblasts are the primary source of disease-activated myofibroblasts in the heart is not consistent with previous studies that have suggested alternate lineages. For example, endothelial-to-mesenchymal transition from resident endothelial cells was reported to...
Animal procedures
Tamoxifen citrate containing mouse chow at a treatment dosage of 400 mg kg -1 (Harlan laboratories) was used to activate the inducible MerCreMer protein or the CreERT2 protein, thereby inducing Cre recombinase activity. The duration of treatment is indicated within each experiment. MI was induced i...
- Use
- Tamoxifen citrate containing mouse chow at a treatment dosage of 400 mg kg -1 (Harlan laboratories) was used to activate the inducible MerCreMer protein or the CreERT2 protein, thereby inducing Cre recombinase activity. The duration of treatment is indicated within each experiment. MI was induced i...
Histology and immunohistochemistry
Isolated organs were fixed for 3.5 h in freshly diluted 4% PFA at 4 °C, rinsed with PBS and cryoprotected in 30% sucrose/PBS overnight before embedding in OCT (Tissue-Tek). Afterwards, 10 µm cryosections were collected and then blocked for 30 min at room temperature in a blocking so...
- Use
- Isolated organs were fixed for 3.5 h in freshly diluted 4% PFA at 4 °C, rinsed with PBS and cryoprotected in 30% sucrose/PBS overnight before embedding in OCT (Tissue-Tek). Afterwards, 10 µm cryosections were collected and then blocked for 30 min at room temperature in a blocking so...
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Periostin expression is restricted to myofibroblasts
Next, the identity of the interstitial cell population labelled in Postn MCM/+; R26-eGFP mice was interrogated after MI injury and 2 weeks of tamoxifen treatment ( ). Hearts were harvested and serial cryosections were processed to show fibrosis and coincident eGFP + cell labelling (periostin lineage-traced) along with antibodies against vimentin, PDGFRα, αSMA, CD31, CD45 and FSP1 to identify fibroblasts or other interstitial cell types ( ). Approximately 98% of the periostin lineage-traced cells were vimentin positive, while more than half were PDGFRα-positive and ∼80% were αSMA-positive but almost none were CD31, CD45 or FSP1 reactive ( ). Consistent with these results, florescent-activated cell sorting (FACS) analysis of enzymatically isolated eGFP + cells from injured Postn MCM/+; R26-eGFP mouse hearts quantitatively confirmed the histological results, w...
Periostin expression is restricted to myofibroblasts
A recent study suggested that the type of cardiac injury might induce different populations of cells to become myofibroblasts. Hence, in addition to MI injury we also performed cardiac pressure overload by transverse aortic constriction (TAC) and infusion of the profibrotic neuroendocrine agonists, angiotensin II and phenylephrine (Ang/PE), as additional models of cardiac fibrosis. Both disease stimulations were conducted for 2 weeks in Postn MCM/+; R26-eGFP mice, concurrent with tamoxifen treatment ( and ). Both stimuli generated a large induction of periostin lineage-traced eGFP + cells throughout the heart ( ), which by immunohistochemistry were again defined as myofibroblasts ( ). One unique aspect of the pressure overload response is that eGFP + cells were concentrated within the left ventricular free wall, septum and left atria, regions of the heart that are particularly stret...
Periostin-labelled fibroblasts are functional in disease
To assess the relevance of periostin-labelled myofibroblasts during cardiac injury and fibrosis, we generated compound heterozygote mice carrying the Postn MCM allele and the Rosa26 -DTA (diphtheria toxin A) allele, which generates an inducible Cre-dependent means of killing cells in vivo ( ). Here Postn MCM/+; R26-DTA mice were given tamoxifen continuously after MI surgery and hearts from surviving mice were harvested 2 weeks afterwards ( ). Ablation of periostin + cells was verified by western blot, which showed a dramatic reduction in periostin protein in hearts of Postn MCM/+; R26-DTA mice after tamoxifen, compared with Postn MCM control mice not containing the R26-DTA allele ( ). Postn -/- heart-protein extract was used as a control. Most importantly, Postn MCM/+; R26-DTA mice subjected to MI injury showed much greater lethality in the first few days due to ventric...
Periostin lineage-tracing labels all myofibroblasts
To more definitively examine the extent to which the periostin + cells account for all of the collagen-producing myofibroblasts in the heart we also crossed the lineage-tracing Postn MCM/+; R26-tdTomato mouse with transgenic mice expressing GFP under the control of the collagen1a1 chimeric promoter ( ). The collagen1a1 promoter used to make the GFP transgene is a composite of a proximal promoter and an upstream DNase I hypersensitivity (HS4,5) region that conveys unique properties to the transgene, such that GFP is only expressed in tissue-resident fibroblasts and myofibroblasts but no other cell types ( ). The data show that nearly all periostin lineage-traced cells analysed 7 days post-MI in the injury region of the heart were collagen1a1-GFP expressing ( and ). To further determine if periostin lineage-traced cells from Postn MCM/+; R26-eGFP mice accounted for all myofibroblasts...
Origins of periostin-labelled adult cardiac fibroblasts
Myofibroblasts in the injured or diseased heart have been suggested to originate and transdifferentiate from many cellular sources, such as an endothelial cells, immune cells, smooth muscle cells, pericytes or epicardial derived resident fibroblasts. However, there is disagreement even amongst these studies each claiming that one of these cell sources is dominant. Here we attempted to quantify the cellular sources for periostin-traced myofibroblasts in the mouse heart using lineage tracing with four independent genetic loci together with concurrent periostin expression ( ). We used Rosa26 nLacZ reporter mice carrying either Tcf21 MCM (resident fibroblasts), LysM Cre (macrophages), Cdh5 Cre (endothelial cells) and Myh11 CreERT2 (smooth muscle cells) along with a periostin promoter transgene-driving ZsGreen ( ). For the lineage-tracing component, mice with inducible Cre alleles were...
Origins of periostin-labelled adult cardiac fibroblasts
As yet another criteria for determining the extent to which select cellular origins contribute to myofibroblasts in the heart, the lineage-tracing analyses with these same Cre lines was compared against a full analysis of antibody markers as presented earlier ( ). Mice were given tamoxifen food for 2 weeks, allowed 3 days off, then infarcted at 10 weeks of age and harvested 1 week later ( ). LysM Cre labelled cells primarily gave rise to CD45 and FSP1 expressing cells, but they lacked markers of fibroblasts ( and ). The Myh11 CreERT2 traced cells lacked vimentin and histological analysis showed a localization pattern to the media of vessels in the heart where smooth muscle cells reside, but not within the infarct region that would be characteristic of myofibroblasts ( and ). Endothelial cells labelled with the Cdh5 Cre allele were mostly CD31 positive and only a small portion were co-...
Origins of periostin-labelled adult cardiac fibroblasts
Since resident Tcf21 lineage-traced cells were the overwhelming source of periostin-expressing myofibroblasts in the infarct region of the heart, a more elaborate investigation of Tcf21 expression and lineage-traced cells was undertaken using Tcf21 MCM/+; R26-eGFP mice ( ). Here, we first began with uninjured mice since Tcf21 is highly expressed in tissue-resident fibroblasts at baseline within the heart. Tcf21 MCM/+; R26-eGFP mice were given tamoxifen for 2 weeks and then harvested ( ). The lineage-tracing strategy labelled large numbers of resident fibroblasts throughout the uninjured adult heart, which were positive for vimentin and PDGFRα, but not αSMA, CD31 or FSP1 ( ). These results were confirmed by quantitative FACS analysis, which again showed that all resting Tcf21 lineage-traced fibroblasts from the heart expressed vimentin but not CD31, CD45 or FSP1 ( ).
Origins of periostin-labelled adult cardiac fibroblasts
Next we performed lineage tracing after MI injury in Tcf21 MCM/+; R26-eGFP mice. Tamoxifen was given for 2 weeks before MI (with 3 days no treatment before injury), followed by harvesting of hearts 2 weeks later for analysis ( ). The results showed a 10-fold increase in total Tcf21-labelled fibroblasts in the infarct region and associated border zone, reminiscent of how periostin-labelled myofibroblasts similarly expand ( ). Immunohistochemistry-based quantification of all Tcf21 lineage-traced (eGFP + ) fibroblasts also showed a profile consistent with periostin-labelled myofibroblasts within the infarct, in that they were positive for vimentin, αSMA and PDGFRα ( ). To further characterize Tcf21-expressing cells in a similar mouse model of ischaemia-reperfusion (I/R) injury to the heart, a Tcf21 LacZ knock-in allele was used to mark currently expressing fibroblasts ( ). An...
Measurement outputs
What raw and processed outputs should exist?
Next, the identity of the interstitial cell population labelled in Postn MCM/+; R26-eGFP mice was interrogated after MI injury and 2 weeks of tamoxifen treatment ( ). Hearts we...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
A recent study suggested that the type of cardiac injury might induce different populations of cells to become myofibroblasts. Hence, in addition to MI injury we also performed...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
To assess the relevance of periostin-labelled myofibroblasts during cardiac injury and fibrosis, we generated compound heterozygote mice carrying the Postn MCM allele and the Ro...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
To more definitively examine the extent to which the periostin + cells account for all of the collagen-producing myofibroblasts in the heart we also crossed the lineage-tracing...
- Raw artifact
- Per-sample or per-animal endpoint measurements collected during the experiment
- Processed artifact
- Structured table with cleaned measurements ready for comparison
- Reported as
- Summary statistics and between-group or across-timepoint comparisons
Analysis plan
How should the outputs become interpretable results?
Acquisition
Collect raw experimental outputs with enough metadata to preserve sample identity, condition, and timing.
inferred from protocolPreprocessing / cleaning
The conclusion that Tcf21-expressing resident cardiac fibroblasts are the primary source of disease-activated myofibroblasts in the heart is not consistent with previous studies that have suggested alternate lineages.
from paperScoring or quantification
Quantify the primary readouts for this experiment: Next, the identity of the interstitial cell population labelled in Postn MCM/+; R26-eGFP mice was interrogated after MI injury and 2 weeks of tamoxifen treatment ( ). Hearts we...; A recent study suggested that the type of cardiac injury might induce different populations of cells to become myofibroblasts. Hence, in addition to MI injury we also performed...; To assess the relevance of periostin-labelled myofibroblasts during cardiac injury and fibrosis, we generated compound heterozygote mice carrying the Postn MCM allele and the Ro...; To more definitively examine the extent to which the periostin + cells account for all of the collagen-producing myofibroblasts in the heart we also crossed the lineage-tracing....
from paperStatistical comparison
The conclusion that Tcf21-expressing resident cardiac fibroblasts are the primary source of disease-activated myofibroblasts in the heart is not consistent with previous studies...; For studies involving cardiac injury such as MI, group sizes were determined based on previously observed post-operative mortality rates for this procedure. No experimental anim...
from paperReporting output
Report representative outputs alongside summary comparisons for Next, the identity of the interstitial cell population labelled in Postn MCM/+; R26-eGFP mice was interrogated after MI injury and 2 weeks of tamoxifen treatment ( ). Hearts we..., A recent study suggested that the type of cardiac injury might induce different populations of cells to become myofibroblasts. Hence, in addition to MI injury we also performed..., To assess the relevance of periostin-labelled myofibroblasts during cardiac injury and fibrosis, we generated compound heterozygote mice carrying the Postn MCM allele and the Ro..., To more definitively examine the extent to which the periostin + cells account for all of the collagen-producing myofibroblasts in the heart we also crossed the lineage-tracing....
inferred from protocolStructured statistical methods
The conclusion that Tcf21-expressing resident cardiac fibroblasts are the primary source of disease-activated myofibroblasts in the heart is not consistent with previous studies...; For studies involving cardiac injury such as MI, group sizes were determined based on previously observed post-operative mortality rates for this procedure. No experimental anim...
source structuredSource and audit
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Evidence quotes (8)
Next, the identity of the interstitial cell population labelled in Postn MCM/+; R26-eGFP mice was interrogated after MI injury and 2 weeks of tamoxifen treatment ( ). Hearts were harvested and serial cryosections were processed to show fibrosis and coincident eGFP + cell labelling (periostin lineage-traced) along with antibodies against vimentin, PDGFRα, αSMA, CD31, CD45 and FSP1 to identify fibroblasts or other interstitial cell types ( ). Approximately 98% of the periostin lineage-traced cells were vimentin positive, while more than half were PDGFRα-positive and ∼80% were αSMA-positive but almost none were CD31, CD45 or FSP1 reactive ( ). Consistent with these results, florescent-activated cell sorting (FACS) analysis of enzymatically isolated eGFP + cells from injured Postn MCM/+; R26-eGFP mouse hearts quantitatively confirmed the histological results, with nearly an identical marker profile ( ). Thy1 was also uniquely used in the FACS analysis since it is a surface marker and it has been reported to identify fibroblasts ( ). Since the presence of vimentin reactivity and absence of CD31 and CD45 reactivity is a criterion for total fibroblast identi...
A recent study suggested that the type of cardiac injury might induce different populations of cells to become myofibroblasts. Hence, in addition to MI injury we also performed cardiac pressure overload by transverse aortic constriction (TAC) and infusion of the profibrotic neuroendocrine agonists, angiotensin II and phenylephrine (Ang/PE), as additional models of cardiac fibrosis. Both disease stimulations were conducted for 2 weeks in Postn MCM/+; R26-eGFP mice, concurrent with tamoxifen treatment ( and ). Both stimuli generated a large induction of periostin lineage-traced eGFP + cells throughout the heart ( ), which by immunohistochemistry were again defined as myofibroblasts ( ). One unique aspect of the pressure overload response is that eGFP + cells were concentrated within the left ventricular free wall, septum and left atria, regions of the heart that are particularly stretched in response to pressure overload, but less so in the right ventricle and right atria ( ). In contrast, eGFP + cellular distribution was more uniform throughout the entire heart with 2 weeks of Ang/PE stimulation ( ). Taken together, these data suggest that periostin lineage-tracing identifies m...
To assess the relevance of periostin-labelled myofibroblasts during cardiac injury and fibrosis, we generated compound heterozygote mice carrying the Postn MCM allele and the Rosa26 -DTA (diphtheria toxin A) allele, which generates an inducible Cre-dependent means of killing cells in vivo ( ). Here Postn MCM/+; R26-DTA mice were given tamoxifen continuously after MI surgery and hearts from surviving mice were harvested 2 weeks afterwards ( ). Ablation of periostin + cells was verified by western blot, which showed a dramatic reduction in periostin protein in hearts of Postn MCM/+; R26-DTA mice after tamoxifen, compared with Postn MCM control mice not containing the R26-DTA allele ( ). Postn -/- heart-protein extract was used as a control. Most importantly, Postn MCM/+; R26-DTA mice subjected to MI injury showed much greater lethality in the first few days due to ventricular wall rupture, which is consistent with lethality in Postn -/- mice subject to the same injury due to a defect in the formation of a protective scar, or in a subset of developing embryos when collagen-dependent structural regions are formed in the cardiovascular system in and around...
To more definitively examine the extent to which the periostin + cells account for all of the collagen-producing myofibroblasts in the heart we also crossed the lineage-tracing Postn MCM/+; R26-tdTomato mouse with transgenic mice expressing GFP under the control of the collagen1a1 chimeric promoter ( ). The collagen1a1 promoter used to make the GFP transgene is a composite of a proximal promoter and an upstream DNase I hypersensitivity (HS4,5) region that conveys unique properties to the transgene, such that GFP is only expressed in tissue-resident fibroblasts and myofibroblasts but no other cell types ( ). The data show that nearly all periostin lineage-traced cells analysed 7 days post-MI in the injury region of the heart were collagen1a1-GFP expressing ( and ). To further determine if periostin lineage-traced cells from Postn MCM/+; R26-eGFP mice accounted for all myofibroblasts in the heart we performed single-cell sorting by Fluidigm followed by RNAseq analysis after MI injury, versus uninjured hearts. Analysis of the transcriptome from 185 individual cells that passed quality assurance (see Methods) was performed from the hearts of MI injured and sham mice, and 63 of the...
Myofibroblasts in the injured or diseased heart have been suggested to originate and transdifferentiate from many cellular sources, such as an endothelial cells, immune cells, smooth muscle cells, pericytes or epicardial derived resident fibroblasts. However, there is disagreement even amongst these studies each claiming that one of these cell sources is dominant. Here we attempted to quantify the cellular sources for periostin-traced myofibroblasts in the mouse heart using lineage tracing with four independent genetic loci together with concurrent periostin expression ( ). We used Rosa26 nLacZ reporter mice carrying either Tcf21 MCM (resident fibroblasts), LysM Cre (macrophages), Cdh5 Cre (endothelial cells) and Myh11 CreERT2 (smooth muscle cells) along with a periostin promoter transgene-driving ZsGreen ( ). For the lineage-tracing component, mice with inducible Cre alleles were given tamoxifen for 2 weeks, then given MI injury 3 days later, while the two other mouse lines had constitutive and non-regulated Cre alleles and thus had continuous labelling ( ). Hearts were processed for antibody detection of nuclear localized LacZ (β-galactosidase) versus ZsGreen expressio...
As yet another criteria for determining the extent to which select cellular origins contribute to myofibroblasts in the heart, the lineage-tracing analyses with these same Cre lines was compared against a full analysis of antibody markers as presented earlier ( ). Mice were given tamoxifen food for 2 weeks, allowed 3 days off, then infarcted at 10 weeks of age and harvested 1 week later ( ). LysM Cre labelled cells primarily gave rise to CD45 and FSP1 expressing cells, but they lacked markers of fibroblasts ( and ). The Myh11 CreERT2 traced cells lacked vimentin and histological analysis showed a localization pattern to the media of vessels in the heart where smooth muscle cells reside, but not within the infarct region that would be characteristic of myofibroblasts ( and ). Endothelial cells labelled with the Cdh5 Cre allele were mostly CD31 positive and only a small portion were co-labelled for vimentin, while none were αSMA expressing further suggesting that endogenous endothelial cell lineages do not generate myofibroblasts in the heart with MI injury ( and ). Thus, endothelial cells, smooth muscle cells and immune cells are negligible sources for generating myofibrobla...
Since resident Tcf21 lineage-traced cells were the overwhelming source of periostin-expressing myofibroblasts in the infarct region of the heart, a more elaborate investigation of Tcf21 expression and lineage-traced cells was undertaken using Tcf21 MCM/+; R26-eGFP mice ( ). Here, we first began with uninjured mice since Tcf21 is highly expressed in tissue-resident fibroblasts at baseline within the heart. Tcf21 MCM/+; R26-eGFP mice were given tamoxifen for 2 weeks and then harvested ( ). The lineage-tracing strategy labelled large numbers of resident fibroblasts throughout the uninjured adult heart, which were positive for vimentin and PDGFRα, but not αSMA, CD31 or FSP1 ( ). These results were confirmed by quantitative FACS analysis, which again showed that all resting Tcf21 lineage-traced fibroblasts from the heart expressed vimentin but not CD31, CD45 or FSP1 ( ).
Next we performed lineage tracing after MI injury in Tcf21 MCM/+; R26-eGFP mice. Tamoxifen was given for 2 weeks before MI (with 3 days no treatment before injury), followed by harvesting of hearts 2 weeks later for analysis ( ). The results showed a 10-fold increase in total Tcf21-labelled fibroblasts in the infarct region and associated border zone, reminiscent of how periostin-labelled myofibroblasts similarly expand ( ). Immunohistochemistry-based quantification of all Tcf21 lineage-traced (eGFP + ) fibroblasts also showed a profile consistent with periostin-labelled myofibroblasts within the infarct, in that they were positive for vimentin, αSMA and PDGFRα ( ). To further characterize Tcf21-expressing cells in a similar mouse model of ischaemia-reperfusion (I/R) injury to the heart, a Tcf21 LacZ knock-in allele was used to mark currently expressing fibroblasts ( ). An I/R model often generates a smaller injury area compared with MI so that expansion can be better examined. While the uninjured heart again showed expression in tissue-resident fibroblasts throughout the heart (corresponding to ∼10% of the total cell number in the heart), areas of direct injury...
Machine-readable layer
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"name": "Genetic lineage tracing defines myofibroblast origin and function in the injured heart methods",
"description": "Evidence-backed execution summary for Genetic lineage tracing defines myofibroblast origin and function in the injured heart methods from Genetic lineage tracing defines myofibroblast origin and function in the injured heart.",
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{
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"name": "Periostin expression is restricted to myofibroblasts",
"text": "Next, the identity of the interstitial cell population labelled in Postn MCM/+; R26-eGFP mice was interrogated after MI injury and 2 weeks of tamoxifen treatment ( ). Hearts were harvested and serial cryosections were processed to show fibrosis and coincident eGFP + cell labelling (periostin lineage-traced) along with antibodies against vimentin, PDGFRα, αSMA, CD31, CD45 and FSP1 to identify fibroblasts or other interstitial cell types ( ). Approximately 98% of the periostin lineage-traced cells were vimentin positive, while more than half were PDGFRα-positive and ∼80% were αSMA-positive but almost none were CD31, CD45 or FSP1 reactive ( ). Consistent with these results, florescent-activated cell sorting (FACS) analysis of enzymatically isolated eGFP + cells from injured Postn MCM/+; R26-eGFP mouse hearts quantitatively confirmed the histological results, w..."
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{
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"name": "Periostin expression is restricted to myofibroblasts",
"text": "A recent study suggested that the type of cardiac injury might induce different populations of cells to become myofibroblasts. Hence, in addition to MI injury we also performed cardiac pressure overload by transverse aortic constriction (TAC) and infusion of the profibrotic neuroendocrine agonists, angiotensin II and phenylephrine (Ang/PE), as additional models of cardiac fibrosis. Both disease stimulations were conducted for 2 weeks in Postn MCM/+; R26-eGFP mice, concurrent with tamoxifen treatment ( and ). Both stimuli generated a large induction of periostin lineage-traced eGFP + cells throughout the heart ( ), which by immunohistochemistry were again defined as myofibroblasts ( ). One unique aspect of the pressure overload response is that eGFP + cells were concentrated within the left ventricular free wall, septum and left atria, regions of the heart that are particularly stret..."
},
{
"@type": "HowToStep",
"position": 3,
"name": "Periostin-labelled fibroblasts are functional in disease",
"text": "To assess the relevance of periostin-labelled myofibroblasts during cardiac injury and fibrosis, we generated compound heterozygote mice carrying the Postn MCM allele and the Rosa26 -DTA (diphtheria toxin A) allele, which generates an inducible Cre-dependent means of killing cells in vivo ( ). Here Postn MCM/+; R26-DTA mice were given tamoxifen continuously after MI surgery and hearts from surviving mice were harvested 2 weeks afterwards ( ). Ablation of periostin + cells was verified by western blot, which showed a dramatic reduction in periostin protein in hearts of Postn MCM/+; R26-DTA mice after tamoxifen, compared with Postn MCM control mice not containing the R26-DTA allele ( ). Postn -/- heart-protein extract was used as a control. Most importantly, Postn MCM/+; R26-DTA mice subjected to MI injury showed much greater lethality in the first few days due to ventric..."
},
{
"@type": "HowToStep",
"position": 4,
"name": "Periostin lineage-tracing labels all myofibroblasts",
"text": "To more definitively examine the extent to which the periostin + cells account for all of the collagen-producing myofibroblasts in the heart we also crossed the lineage-tracing Postn MCM/+; R26-tdTomato mouse with transgenic mice expressing GFP under the control of the collagen1a1 chimeric promoter ( ). The collagen1a1 promoter used to make the GFP transgene is a composite of a proximal promoter and an upstream DNase I hypersensitivity (HS4,5) region that conveys unique properties to the transgene, such that GFP is only expressed in tissue-resident fibroblasts and myofibroblasts but no other cell types ( ). The data show that nearly all periostin lineage-traced cells analysed 7 days post-MI in the injury region of the heart were collagen1a1-GFP expressing ( and ). To further determine if periostin lineage-traced cells from Postn MCM/+; R26-eGFP mice accounted for all myofibroblasts..."
},
{
"@type": "HowToStep",
"position": 5,
"name": "Origins of periostin-labelled adult cardiac fibroblasts",
"text": "Myofibroblasts in the injured or diseased heart have been suggested to originate and transdifferentiate from many cellular sources, such as an endothelial cells, immune cells, smooth muscle cells, pericytes or epicardial derived resident fibroblasts. However, there is disagreement even amongst these studies each claiming that one of these cell sources is dominant. Here we attempted to quantify the cellular sources for periostin-traced myofibroblasts in the mouse heart using lineage tracing with four independent genetic loci together with concurrent periostin expression ( ). We used Rosa26 nLacZ reporter mice carrying either Tcf21 MCM (resident fibroblasts), LysM Cre (macrophages), Cdh5 Cre (endothelial cells) and Myh11 CreERT2 (smooth muscle cells) along with a periostin promoter transgene-driving ZsGreen ( ). For the lineage-tracing component, mice with inducible Cre alleles were..."
},
{
"@type": "HowToStep",
"position": 6,
"name": "Origins of periostin-labelled adult cardiac fibroblasts",
"text": "As yet another criteria for determining the extent to which select cellular origins contribute to myofibroblasts in the heart, the lineage-tracing analyses with these same Cre lines was compared against a full analysis of antibody markers as presented earlier ( ). Mice were given tamoxifen food for 2 weeks, allowed 3 days off, then infarcted at 10 weeks of age and harvested 1 week later ( ). LysM Cre labelled cells primarily gave rise to CD45 and FSP1 expressing cells, but they lacked markers of fibroblasts ( and ). The Myh11 CreERT2 traced cells lacked vimentin and histological analysis showed a localization pattern to the media of vessels in the heart where smooth muscle cells reside, but not within the infarct region that would be characteristic of myofibroblasts ( and ). Endothelial cells labelled with the Cdh5 Cre allele were mostly CD31 positive and only a small portion were co-..."
},
{
"@type": "HowToStep",
"position": 7,
"name": "Origins of periostin-labelled adult cardiac fibroblasts",
"text": "Since resident Tcf21 lineage-traced cells were the overwhelming source of periostin-expressing myofibroblasts in the infarct region of the heart, a more elaborate investigation of Tcf21 expression and lineage-traced cells was undertaken using Tcf21 MCM/+; R26-eGFP mice ( ). Here, we first began with uninjured mice since Tcf21 is highly expressed in tissue-resident fibroblasts at baseline within the heart. Tcf21 MCM/+; R26-eGFP mice were given tamoxifen for 2 weeks and then harvested ( ). The lineage-tracing strategy labelled large numbers of resident fibroblasts throughout the uninjured adult heart, which were positive for vimentin and PDGFRα, but not αSMA, CD31 or FSP1 ( ). These results were confirmed by quantitative FACS analysis, which again showed that all resting Tcf21 lineage-traced fibroblasts from the heart expressed vimentin but not CD31, CD45 or FSP1 ( )."
},
{
"@type": "HowToStep",
"position": 8,
"name": "Origins of periostin-labelled adult cardiac fibroblasts",
"text": "Next we performed lineage tracing after MI injury in Tcf21 MCM/+; R26-eGFP mice. Tamoxifen was given for 2 weeks before MI (with 3 days no treatment before injury), followed by harvesting of hearts 2 weeks later for analysis ( ). The results showed a 10-fold increase in total Tcf21-labelled fibroblasts in the infarct region and associated border zone, reminiscent of how periostin-labelled myofibroblasts similarly expand ( ). Immunohistochemistry-based quantification of all Tcf21 lineage-traced (eGFP + ) fibroblasts also showed a profile consistent with periostin-labelled myofibroblasts within the infarct, in that they were positive for vimentin, αSMA and PDGFRα ( ). To further characterize Tcf21-expressing cells in a similar mouse model of ischaemia-reperfusion (I/R) injury to the heart, a Tcf21 LacZ knock-in allele was used to mark currently expressing fibroblasts ( ). An..."
}
],
"tool": [
{
"@type": "HowToTool",
"name": "Periostin expression is restricted to myofibroblasts"
},
{
"@type": "HowToTool",
"name": "Periostin-labelled fibroblasts are functional in disease"
},
{
"@type": "HowToTool",
"name": "Origins of periostin-labelled adult cardiac fibroblasts"
},
{
"@type": "HowToTool",
"name": "Flow cytometry and cell sorting"
},
{
"@type": "HowToTool",
"name": "Periostin + myofibroblasts can be partially inactivated"
},
{
"@type": "HowToTool",
"name": "Disscussion"
},
{
"@type": "HowToTool",
"name": "Animal procedures"
},
{
"@type": "HowToTool",
"name": "Histology and immunohistochemistry"
}
],
"supply": [
{
"@type": "HowToSupply",
"name": "Periostin-labelled fibroblasts are functional in disease"
},
{
"@type": "HowToSupply",
"name": "Origins of periostin-labelled adult cardiac fibroblasts"
},
{
"@type": "HowToSupply",
"name": "Origins of periostin-labelled adult cardiac fibroblasts"
},
{
"@type": "HowToSupply",
"name": "Flow cytometry and cell sorting"
},
{
"@type": "HowToSupply",
"name": "Disscussion"
},
{
"@type": "HowToSupply",
"name": "Histology and immunohistochemistry"
},
{
"@type": "HowToSupply",
"name": "Isolation of cardiac fibroblasts"
},
{
"@type": "HowToSupply",
"name": "Flow cytometry and cell sorting"
}
],
"isBasedOn": {
"@type": "ScholarlyArticle",
"headline": "Genetic lineage tracing defines myofibroblast origin and function in the injured heart",
"datePublished": "2016",
"author": [
{
"@type": "Person",
"name": "Onur Kanisicak"
},
{
"@type": "Person",
"name": "Hadi Khalil"
},
{
"@type": "Person",
"name": "Malina J. Ivey"
},
{
"@type": "Person",
"name": "Jason Karch"
},
{
"@type": "Person",
"name": "Bryan D. Maliken"
},
{
"@type": "Person",
"name": "Robert N. Correll"
},
{
"@type": "Person",
"name": "Matthew J. Brody"
},
{
"@type": "Person",
"name": "Suh-Chin J. Lin"
},
{
"@type": "Person",
"name": "Bruce J. Aronow"
},
{
"@type": "Person",
"name": "Michelle D. Tallquist"
},
{
"@type": "Person",
"name": "Jeffery D. Molkentin"
}
],
"identifier": "10.1038/ncomms12260"
}
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