Single-Cell Phenotyping of Complex Heterogeneous Tissue

Kraus, Petra; Li, Kangning; Sipes, Darren; Varden, Lara; Yerden, Rachel; Henderson, Althea; Sur, Shantanu; Lufkin, Thomas

doi:10.1007/978-981-10-4857-9_16-1

Cited by 8 publications

(12 citation statements)

References 65 publications

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“…Larger and smaller cell bodies are seen side-by-side indicating morphological heterogeneity. Often long, bulging cell protrusions are visible in vivo and in vitro (Figure 1 and [2,20]). Cells in the AF are elongated, resembling more the shape of fibroblasts (Figure 1).…”

Section: Resultsmentioning

confidence: 99%

“…We have frequently isolated and propagated bovine cells from the outer AF and the NP through non-enzymatic explant outgrowth culture [20,77]. These cells exhibit some criteria established for mesenchymal stem cells, but continue to display heterogeneity when phenotyped by a selection of transcripts, by morphology or by cell movement [2,20,77,78,79]. In this context others and we find morphological, molecular and “behavioral” differences in vivo and in vitro pointing to at least two cell populations within the outer AF and NP [2,3,61,68,73].…”

Section: Discussionmentioning

confidence: 99%

“…In this field of regenerative medicine, strategies are pursued to refurbish ailing IVDs with autologous or allogenic cells, derived either from the disc itself or other sources, with special interest in mesenchymal stem cells (MSC) derived from bone marrow, articular cartilage or adipose tissue. Alternatively, strategies to stimulate intrinsic disc cells through the injection of growth factors or the use of gene therapy to replace degenerating extracellular matrix (ECM) molecules are envisioned as ways to “rejuvenate” IVD function as a shock absorber in the spinal joints [2,3,4,5,6]. On first glance the IVD might appear as an anatomically simple structure, comprised of concentric rings in the annulus fibrosus (AF) and gradually transitioning towards a central nucleus pulposus (NP) [1,7,8].…”

Section: Introductionmentioning

confidence: 99%

“…However, compared to other vertebrate organs, the IVD is unique in many ways: The avascular nature of the IVD is reflected in a lack of coloration (see Figure 1A in [3]) and the few remaining cells in the mature IVD are embedded in a vast amount of ECM, likely experiencing only minor cell-cell contact [3]. These few resident cells within the outer AF and NP of mature IVDs display a heterogeneous profile when assessed on a single-cell level for their transcriptional activities [2,3]. Given several ongoing clinical trials [5,9,10,11] and an alarming emergence of stem cell tourism worldwide [12] there remains a need to further characterize these cells on a molecular level, should they be the subject or target of regenerative treatments [3,13,14,15].…”

Section: Introductionmentioning

confidence: 99%

“…Analyzing these genes with RNA in situ hybridization (RISH), we point to heterogeneity among cells within the outer AF or NP, which is typically not accounted for by methods involving cell pooling for RNA extraction, such as qRT-PCR, microarray expression profiling or non-single-cell RNA sequencing [2,3,38]. Here, we also explore the use of fluorescent (FL) transcript tagging to allow for transcript quantification of proposed biomarkers through both population averaging and single-cell analysis and we propose that this analysis based on FL values enables further evaluation of cellular heterogeneity within the population of cells actively transcribing a biomarker.…”

Section: Introductionmentioning

confidence: 99%

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Quantitative Single-Cell Transcript Assessment of Biomarkers Supports Cellular Heterogeneity in the Bovine IVD

Kapper

Mondal

et al. 2019

Veterinary Sciences

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Severe and chronic low back pain is often associated with intervertebral disc (IVD) degeneration. While imposing a considerable socio-economic burden worldwide, IVD degeneration is also severely impacting on the quality of life of affected individuals. Cell-based regenerative medicine approaches have moved into clinical trials, yet IVD cell identities in the mature disc remain to be fully elucidated and tissue heterogeneity exists, requiring a better characterization of IVD cells. The bovine coccygeal IVD is an accepted research model to study IVD mechano-biology and disc homeostasis. Recently, we identified novel IVD biomarkers in the outer annulus fibrosus (AF) and nucleus pulposus (NP) of the mature bovine coccygeal IVD through RNA in situ hybridization (AP-RISH) and z-proportion test. Here we follow up on Lam1, Thy1, Gli1, Gli3, Noto, Ptprc, Scx, Sox2 and Zscan10 with fluorescent RNA in situ hybridization (FL-RISH) and confocal microscopy. This permits sub-cellular transcript localization and the addition of quantitative single-cell derived values of mRNA expression levels to our previous analysis. Lastly, we used a Gaussian mixture modeling approach for the exploratory analysis of IVD cells. This work complements our earlier cell population proportion-based study, confirms the previously proposed biomarkers and indicates even further heterogeneity of cells in the outer AF and NP of a mature IVD.

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Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Quantitative Single-Cell Transcript Assessment of Biomarkers Supports Cellular Heterogeneity in the Bovine IVD

Kapper

Mondal

et al. 2019

Veterinary Sciences

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Small Extracellular Vesicles by Nucleus Pulposus Cells Maintain Niche and Cell Homeostasis via Receptor Shuffling and Metabolic Enzyme Supplements

Samanta,

Yoo,

Koh

et al. 2024

Preprint

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Background: Small extracellular vesicles (sEV) are a conserved mean of communication across the domains of life and lately gained more interest in mammalian non-cancerous work as non-cellular, biological therapeutic with encouraging results in recent studies of chronic degenerative diseases. The nucleus pulposus (NP) is the avascular and aneural center of an intervertebral disc (IVD), home to unique niche conditions and affected in IVD degeneration. We investigated the proteome of autologous and mesenchymal stem cell (MSC) sEVs for their potential to contribute to cell and tissue homeostasis in this niche. Methods: We aimed to identify characteristics of NP sEVs via mass spectrometric proteome and functional enrichment analysis. We explored the proteome profile of sEVs generated by autologous parent cell lines from bovine coccygeal IVD and adipose tissue, with a focus on NP cells, using adult and fetal donors. We compared these findings to published sEV databases and MSC sEV data. Findings: We propose several mechanisms associated with NP sEVs: 1. Membrane receptor trafficking to modify signal responses promoting niche homeostasis. 2. Cell homeostasis via proteasome delivery; 3. Redox and energy homeostasis via metabolic enzymes delivery. 4. Immunomodulation beyond an association with a serum protein corona. Conclusion: The proteome signature of sEVs generated by NP parent cells is similar to previously published sEV data, yet with a focus on supplementing anaerobic metabolism and redox balance while contributing to the maintenance of an aneural and avascular microniche.

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Stem cells in intervertebral disc regeneration–more talk than action?

Kraus¹,

Samanta²,

Lufkin³

et al. 2022

Biocell

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Pain and lifestyle changes are common consequences of intervertebral disc degeneration (IVDD) and affect a large part of the aging population. The stemness of cells is exploited in the field of regenerative medicine as key to treat degenerative diseases. Transplanted cells however often face delivery and survival challenges, especially in tissues with a naturally harsh microniche environment such as the intervertebral disc. Recent interest in the secretome of stem cells, especially cargo protected from microniche-related decay as frequently present in degenerating tissues, provides new means of rejuvenating ailing cells and tissues. Exosomes, a type of extracellular vesicles with purposeful cargo gained particular interest in conveying stem cell related attributes of rejuvenation, which will be discussed here in the context of IVDD.

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Single-Cell Phenotyping of Complex Heterogeneous Tissue

Cited by 8 publications

References 65 publications

Quantitative Single-Cell Transcript Assessment of Biomarkers Supports Cellular Heterogeneity in the Bovine IVD

Quantitative Single-Cell Transcript Assessment of Biomarkers Supports Cellular Heterogeneity in the Bovine IVD

Small Extracellular Vesicles by Nucleus Pulposus Cells Maintain Niche and Cell Homeostasis via Receptor Shuffling and Metabolic Enzyme Supplements

Stem cells in intervertebral disc regeneration–more talk than action?

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