High-Throughput Single-Cell Analysis for Wound Healing Applications

Januszyk, Michael; Gurtner, Geoffrey C.

doi:10.1089/wound.2012.0395

Cited by 19 publications

(13 citation statements)

References 61 publications

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“…The resolution afforded by these assays is insufficient to capture the complex relationships in heterogeneous samples, and is unable to detect differential expression among cell subgroups. 67,68 Single cells within seemingly homogeneous populations exhibit differences not only in their gene expression, but also in protein levels and phenotypic output, shifting functional consequences of the population. Although methods such as flow cytometry have been able to interrogate protein expression on single cells, the rate-limiting factor is the number of proteins that can be tested at a time.…”

Section: Single-cell Studies Reveal Progenitor Cell Subpopulation Defmentioning

confidence: 99%

“…Fundamentally, enrichment of subpopulations is accomplished through the inclusion of surface marker genes in the transcriptional query, followed by linear discriminate analysis to determine the surface markers most closely mirroring expression of critical subpopulation defining genes. 68,103 The feasibility of this approach has been demonstrated by transplanting a subpopulation of human ASCs enriched for genes related to osteogenesis, for reconstruction of bone. 73 Similarly, a subpopulation of murine ASCs with enhanced wound healing potential has been transplanted for treating diabetic wounds in mice.…”

Section: Enriched Progenitor Cell Subpopulation Therapymentioning

confidence: 99%

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Progenitor Cell Dysfunctions Underlie Some Diabetic Complications

Rodrigues

Wong

Rennert

et al. 2015

The American Journal of Pathology

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Stem cells and progenitor cells are integral to tissue homeostasis and repair. They contribute to health through their ability to self-renew and commit to specialized effector cells. Recently, defects in a variety of progenitor cell populations have been described in both preclinical and human diabetes. These deficits affect multiple aspects of stem cell biology, including quiescence, renewal, and differentiation, as well as homing, cytokine production, and neovascularization, through mechanisms that are still unclear. More important, stem cell aberrations resulting from diabetes have direct implications on tissue function and seem to persist even after return to normoglycemia. Understanding how diabetes alters stem cell signaling and homeostasis is critical for understanding the complex pathophysiology of many diabetic complications. Moreover, the success of cell-based therapies will depend on a more comprehensive understanding of these deficiencies. This review has three goals: to analyze stem cell pathways dysregulated during diabetes, to highlight the effects of hyperglycemic memory on stem cells, and to define ways of using stem cell therapy to overcome diabetic complications. Diabetes is characterized by insulin resistance and hyperglycemia, and affects a diverse array of cells, leading to a myriad of tissue complications. These include, but are not limited to, cardiac arrest, stroke, nephropathy, retinopathy, and non-traumatic lower limb amputations.1 Results from randomized clinical trials indicate that adequate glycemic control in diabetic patients reduces the risk of developing one or several of these complications. 2e4 The Diabetes Control and Complications Trial reports a reduction in the development or progression of diabetic nephropathy (50% reduction), neuropathy (60% reduction), and retinopathy (76% reduction) after intensive glycemic control. However, 33% of Americans with diabetes remain undiagnosed, approximately 12% of US adults with diabetes exhibit poor glycemic control, and different medical organizations recommend different glycemic targets, increasing the occurrence of diabetic complications.

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Section: Single-cell Studies Reveal Progenitor Cell Subpopulation Defmentioning

confidence: 99%

Section: Enriched Progenitor Cell Subpopulation Therapymentioning

confidence: 99%

Progenitor Cell Dysfunctions Underlie Some Diabetic Complications

Rodrigues

Wong

Rennert

et al. 2015

The American Journal of Pathology

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“…Only in the last few years have high-resolution measurement tools evolved to interrogate heterogeneous cell populations like mesenchymal progenitors with sufficient precision to detect potentially subtle population shifts 15 . Our laboratory has recently developed a novel method that combines high throughput single cell gene expression analysis with complex mathematical modeling to identify critical perturbations in cell subpopulations 16 .…”

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confidence: 99%

Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells

Duscher

Rennert

Januszyk

et al. 2014

Sci Rep

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Advanced age is associated with an increased risk of vascular morbidity, attributable in part to impairments in new blood vessel formation. Mesenchymal stem cells (MSCs) have previously been shown to play an important role in neovascularization and deficiencies in these cells have been described in aged patients. Here we utilize single cell transcriptional analysis to determine the effect of aging on MSC population dynamics. We identify an age-related depletion of a subpopulation of MSCs characterized by a pro-vascular transcriptional profile. Supporting this finding, we demonstrate that aged MSCs are also significantly compromised in their ability to support vascular network formation in vitro and in vivo. Finally, aged MSCs are unable to rescue age-associated impairments in cutaneous wound healing. Taken together, these data suggest that age-related changes in MSC population dynamics result in impaired therapeutic potential of aged progenitor cells. These findings have critical implications for therapeutic cell source decisions (autologous versus allogeneic) and indicate the necessity of strategies to improve functionality of aged MSCs.

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“…Recent studies have highlighted aberrant changes to the cellular ecology in chronic wounds which critically alter healing capacity [ 15 ]. To fully elucidate the mechanisms leading to impaired wound healing in diabetes, it is necessary to delineate the cellular and molecular perturbations driving this abnormal tissue state, including changes to the nature and number of critical cell populations such as stem/progenitor cells [ 15 , 16 ].…”

Section: Introductionmentioning

confidence: 99%

Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing

et al. 2020

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Background: Recent advances in high-throughput single-cell sequencing technologies have led to their increasingly widespread adoption for clinical applications. However, challenges associated with tissue viability, cell yield, and delayed time-to-capture have created unique obstacles for data processing. Chronic wounds, in particular, represent some of the most difficult target specimens, due to the significant amount of fibrinous debris, extracellular matrix components, and non-viable cells inherent in tissue routinely obtained from debridement. Methods: Here, we examined the feasibility of single cell RNA sequencing (scRNA-seq) analysis to evaluate human chronic wound samples acquired in the clinic, subjected to prolonged cold ischemia time, and processed without FACS sorting. Wound tissue from human diabetic and non-diabetic plantar foot ulcers were evaluated using an optimized 10X Genomics scRNA-seq platform and analyzed using a modified data pipeline designed for low-yield specimens. Cell subtypes were identified informatically and their distributions and transcriptional programs were compared between diabetic and non-diabetic tissue. Results: 139,000 diabetic and non-diabetic wound cells were delivered for 10X capture after either 90 or 180 min of cold ischemia time. cDNA library concentrations were 858.7 and 364.7 pg/µL, respectively, prior to sequencing. Among all barcoded fragments, we found that 83.5% successfully aligned to the human transcriptome and 68% met the minimum cell viability threshold. The average mitochondrial mRNA fraction was 8.5% for diabetic cells and 6.6% for non-diabetic cells, correlating with differences in cold ischemia time. A total of 384 individual cells were of sufficient quality for subsequent analyses; from this cell pool, we identified transcriptionally-distinct cell clusters whose gene expression profiles corresponded to fibroblasts, keratinocytes, neutrophils, monocytes, and endothelial cells. Fibroblast subpopulations with differing fibrotic potentials were identified, and their distributions were found to be altered in diabetic vs. non-diabetic cells. Conclusions: scRNA-seq of clinical wound samples can be achieved using minor modifications to standard processing protocols and data analysis methods. This simple approach can capture widespread transcriptional differences between diabetic and non-diabetic tissue obtained from matched wound locations.

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High-Throughput Single-Cell Analysis for Wound Healing Applications

Cited by 19 publications

References 61 publications

Progenitor Cell Dysfunctions Underlie Some Diabetic Complications

Progenitor Cell Dysfunctions Underlie Some Diabetic Complications

Aging disrupts cell subpopulation dynamics and diminishes the function of mesenchymal stem cells

Characterization of Diabetic and Non-Diabetic Foot Ulcers Using Single-Cell RNA-Sequencing

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