Flow Cytometry and Cell Sorting Using Hematopoietic Progenitor Cells

Vadakke‐Madathil, Sangeetha; Limaye, Lalita; Kale, Vaijayanti; Chaudhry, Hina W.

doi:10.1007/978-1-4939-9631-5_18

Cited by 7 publications

(4 citation statements)

References 13 publications

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“…The characteristics of this specific population could suggest a humoral system origin, due to features such as granulation and cell size, which are associated with immune cells in invertebrates. Previous studies have indeed shown subpopulations of cells containing granules, probably from the hematopoietic line of the heterogenous population of embryonic cells (Vadakke-Madathil et al, 2019). We note that the crustacean hematopoietic lineage consists primarily of semigranular and granular cells (Söderhäll, 2016).…”

Section: Discussionsupporting

confidence: 56%

Enhanced proliferation in a prawn embryonic primary cell culture ectopically expressing mutated Ras

Sudarshan

Weil²,

Rotem-Dai³

et al. 2023

Front. Mar. Sci.

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Crustacean cell line immortalization has gained a great deal of attention in recent decades for both scientific and applied reasons. Our goal in this study was to advance the state of art towards establishing an immortalized cell line by improving the proliferation rates of primary cells isolated from embryos of the giant freshwater prawn Macrobrachium rosenbergii by using a lentivirus expressing the Ras oncogene. The choice of Ras derived from its involvement in various cellular pathways, such as cell growth, differentiation, and survival, and its use as a tool for in-vitro immortalization, e.g., a specific mutated Ras (RasV12) was used to generate an arthropod cell line. Complementarily, in-silico screening of M. rosenbergii transcriptomic libraries for Ras expression indicated that Ras is already expressed at very early stages of embryo development. In the current study, we transduced primary M. rosenbergii embryonic cells with a lentivirus expressing RasV12 by using the white spot syndrome virus (WSSV IE1) promoter. Expression and sequencing (as followed by sequencing cDNA, confocal microscopy and FACS analysis) of the mutated Ras in the transduced cells confirmed that the lentivirus was successfully integrated into the genome. The lenti-MrRas transduction rate was 23% in the total primary cell population and more than 80% in a sub-population of cells with high granularity. Proliferation of lenti-MrRas transfected cells was enhanced to almost 1200% of the seeding density by the end of our experiment (18 days), which was double that of the control. We were thus successful in enhancing the longevity of embryonic primary cell cultures by ectopic expression of the mutated Ras protein, but the improvement was not sufficient for immortalization.

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Section: Discussionsupporting

confidence: 56%

Enhanced proliferation in a prawn embryonic primary cell culture ectopically expressing mutated Ras

Sudarshan

Weil²,

Rotem-Dai³

et al. 2023

Front. Mar. Sci.

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“…FSC and SSC have been useful to distinguish various cell types. 38 Owing to a general quiescent state of the adult pancreatic cells, we hypothesized that adult pancreatic progenitor cells may be small in size with a high nuclear-to-cytoplasm ratio, similar to adult hematopoietic stem cells. 39 , 40 To test this hypothesis, we fractioned according to FSC and SSC parameters on the ductal CD133 high CD71 low population, which was previously identified as enriched for progenitor-like cells.…”

Section: Resultsmentioning

confidence: 99%

Rare, Tightly-Bound, Multi-Cellular Clusters in the Pancreatic Ducts of Adult Mice Function Like Progenitor Cells and Survive and Proliferate After Acinar Cell Injury

Tremblay,

Ortiz,

Quijano

et al. 2024

Stem Cells

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Pancreatic ductal progenitor cells have been proposed to contribute to adult tissue maintenance and regeneration after injury, but the identity of such ductal cells remains elusive. Here, from adult mice, we identify a near homogenous population of ductal progenitor-like clusters, with an average of 8 cells per cluster. They are a rare subpopulation, about 0.1% of the total pancreatic cells, and can be sorted using a fluorescence-activated cell sorter with the CD133highCD71lowFSCmid-high phenotype. They exhibit properties in self-renewal and tri-lineage differentiation (including endocrine-like cells) in a unique 3-dimensional colony assay system. An in vitro lineage tracing experiment, using a novel HprtDsRed/+ mouse model, demonstrates that a single cell from a cluster clonally gives rise to a colony. Droplet RNAseq analysis demonstrates that these ductal clusters express embryonic multipotent progenitor cell markers Sox9, Pdx1, and Nkx6-1, and genes involved in actin cytoskeleton regulation, inflammation responses, organ development and cancer. Surprisingly, these ductal clusters resist prolonged trypsin digestion in vitro, preferentially survive in vivo after a severe acinar cell injury and become proliferative within 14 days post-injury. Thus, the ductal clusters are the fundamental units of progenitor-like cells in the adult murine pancreas with implications in diabetes treatment and tumorigenicity.

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“…Flow cytometry can be used to distinguish individual cells or small clusters based on light scattering at different angles: the detected light beam in line with the originating beam is defined as forward scatter (FSC), and that in perpendicular as side scatter (SSC). FSC is indicative of particle size while SSC complexity or granularity, and the combination of these two parameters has been useful to distinguish various cell types 19 . We hypothesized that adult pancreatic progenitor cells may be small in size with a high nuclear-to-cytoplasm ratio, similar to adult hematopoietic stem cells 20,21 , owing to a generally quiescent state in homeostasis 22 .…”

Section: Resultsmentioning

confidence: 99%

Identification of a distinct ductal subpopulation with self-renewal and differentiation potential from the adult murine pancreas

Tremblay

Ortíz

Quijano

et al. 2022

Preprint

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Pancreatic ducts function to deliver digestive enzymes into the intestines. Upon injury, ducts can become proliferative and contribute to tissue regeneration; however, the identity of the ductal cells that contribute to these processes is unknown. We combined fluorescence-activated cell sorting, a methylcellulose-containing 3-dimensional culture, droplet RNA-sequencing, and a clonal lineage tracing tool to identify and isolate a distinct subpopulation of pancreatic ductal cells that exhibit progenitor cell properties. These ductal cells are unique in that they form tightly-bound clusters (termed FSCmid-high), with an average of 8 cells per cluster. FSCmid-high clusters comprise only about 0.1% of the total pancreas, are tri-potent for duct, acinar and endocrine lineages, and self-renew robustly in vitro. Transcriptomic analysis of FSCmid-high clusters reveals enrichment for genes involved in cell-cell interactions, organ development, and cancer pathways. FSCmid-high clusters express embryonic pancreatic progenitor markers Sox9, Pdx1, and Nkx6-1 at both transcription and protein levels. FSCmid-high clusters are resistant to enzymatic dissociation and survive severe in vivo acinar injury, which induces formation of ductal rosettes that become proliferative within 14 days. Thus, FSCmid-high clusters represent a small subset of ductal cells with progenitor cell properties. These rare progenitor-like duct cell clusters have implications in pancreas regeneration and tumor initiation/progression.

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Flow Cytometry and Cell Sorting Using Hematopoietic Progenitor Cells

Cited by 7 publications

References 13 publications

Enhanced proliferation in a prawn embryonic primary cell culture ectopically expressing mutated Ras

Enhanced proliferation in a prawn embryonic primary cell culture ectopically expressing mutated Ras

Rare, Tightly-Bound, Multi-Cellular Clusters in the Pancreatic Ducts of Adult Mice Function Like Progenitor Cells and Survive and Proliferate After Acinar Cell Injury

Identification of a distinct ductal subpopulation with self-renewal and differentiation potential from the adult murine pancreas

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