Single-cell molecular profiling provides a high-resolution map of basophil and mast cell differentiation

AbstractDifferentiation of hematopoietic stem and progenitor cells ensure a continuous supply of mature blood cells. Recent models of differentiation are represented as a landscape, in which individual progenitors traverse a continuum of multipotent cell states before reaching an entry point that marks lineage commitment. Basophils and mast cells have received little attention in these models and their differentiation trajectories are yet to be explored. Here, we have performed multicolor flow cytometry and high-coverage single-cell RNA sequencing analyses to chart the differentiation of hematopoietic progenitors into basophils and mast cells in mouse. Analysis of flow cytometry data reconstructed a detailed map of the differentiation, including a bifurcation of progenitors into two specific trajectories. Molecular profiling and pseudotime ordering of the single cells revealed gene expression changes during differentiation, with temporally separated regulation of mast cell protease genes. We validate that basophil and mast cell signature genes increased along the trajectories into their respective lineage, and we demonstrate how genes critical for each respective lineage are upregulated during the formation of the mature cells. Cell fate assays showed that multicolor flow cytometry and transcriptional profiling successfully predict the bipotent phenotype of a previously uncharacterized population of basophil-mast cell progenitor-like cells in mouse peritoneum. Taken together, we provide a detailed roadmap of basophil and mast cell development through a combination of molecular and functional profiling.

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Comprehensive analysis of single-cell RNA sequencing data from healthy human marrow hematopoietic cells

BMC Research Notes ◽

10.1186/s13104-020-05357-y ◽

2020 ◽

Vol 13 (1) ◽

Author(s):

Xin Zhao ◽

Shouguo Gao ◽

Sachiko Kajigaya ◽

Qingguo Liu ◽

Zhijie Wu ◽

...

Keyword(s):

Single Cell ◽

Rna Sequencing ◽

Cell Fate ◽

Noncoding Rna ◽

Single Cells ◽

Regulation Of Gene Expression ◽

Sequencing Data ◽

Healthy Human ◽

Hematopoietic Stem ◽

Single Cell Rna Sequencing

Abstract Objective Single cell methodology enables detection and quantification of transcriptional changes and unravelling dynamic aspects of the transcriptional heterogeneity not accessible using bulk sequencing approaches. We have applied single-cell RNA-sequencing (scRNA-seq) to fresh human bone marrow CD34+ cells and profiled 391 single hematopoietic stem/progenitor cells (HSPCs) from healthy donors to characterize lineage- and stage-specific transcription during hematopoiesis. Results Cells clustered into six distinct groups, which could be assigned to known HSPC subpopulations based on lineage specific genes. Reconstruction of differentiation trajectories in single cells revealed four committed lineages derived from HSCs, as well as dynamic expression changes underlying cell fate during early erythroid-megakaryocytic, lymphoid, and granulocyte-monocyte differentiation. A similar non-hierarchical pattern of hematopoiesis could be derived from analysis of published single-cell assay for transposase-accessible chromatin sequencing (scATAC-seq), consistent with a sequential relationship between chromatin dynamics and regulation of gene expression during lineage commitment (first, altered chromatin conformation, then mRNA transcription). Computationally, we have reconstructed molecular trajectories connecting HSCs directly to four hematopoietic lineages. Integration of long noncoding RNA (lncRNA) expression from the same cells demonstrated mRNA transcriptome, lncRNA, and the epigenome were highly homologous in their pattern of gene activation and suppression during hematopoietic cell differentiation.

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The Single-Cell Transcriptional Landscape of Human Hematopoietic Progenitors

Blood ◽

10.1182/blood-2018-99-116327 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1266-1266

Author(s):

Mariana Loperfido ◽

Danilo Pellin ◽

Cristina Baricordi ◽

Samuel Louis Wolock ◽

Allon Moshe Klein ◽

...

Keyword(s):

Bone Marrow ◽

Dendritic Cell ◽

Mast Cells ◽

Single Cell ◽

Cell Fate ◽

Blood Cells ◽

Single Cells ◽

Hematopoietic Cell ◽

Hematopoietic Progenitors ◽

Cd34 Cells

Abstract In humans, hematopoietic stem/progenitor cells (HSPCs) constitute a heterogeneous population in the bone marrow (BM) endowed with the potential of generating and maintaining an adequate and diverse pool of blood cells lifelong. However, the nature of hematopoietic cell fate choice remains unresolved and is still highly contentious. In fact, despite the recent efforts and the advent of single-cell technologies, conflicting classical, early-split or continuous models for the generation of human blood cells still coexist in the literature as of today. We here used single-cell RNA-Seq (scRNA-Seq) to provide a detailed map of the transcriptional states of hematopoietic progenitors in the human BM. We believe that this work provides unprecedented insights into the structure of early cell-fate choices of the human hematopoietic system. Single-cell analysis of human HSPCs is a well-worn path, but all previous studies aimed at investigating the shape of early cell fate choices human hematopoiesis have been based on the use of immune-selected CD34+ cells. We too started by separating CD34+ cells purified by magnetic beads selection into seven subpopulations, marking cells of differing fate potential and we successfully tagged and sequenced the transcriptome of 6,011 single cells. Our scRNA-Seq map of CD34+ subpopulations shows that HSPCs do not undergo a single-step transition from HSPCs to unilineage states. Instead, they form a clear hierarchy. The earliest fate split separates erythroid-megakaryocyte progenitors from lymphoid-myeloid progenitors, which separate further into lymphoid, dendritic cell and granulocytic progenitors. We noted, however, that branches towards basophils/eosinophils/mast cells and monocytes commitments were missing. Indeed, many cells negative for mature lineage markers in human BM are CD34low/- and could account for additional transitional states at which the CD34 expression is rapidly downregulated, therefore greatly reducing their probability of capture. Thus, differently from previous works, we also extended our analysis to the whole bone marrow fraction lacking the main markers of terminal differentiation (Lin- cells). In doing so we used a novel graded FACS-sorting strategy that corrects for expansion of cells as they differentiated, and thus allows examining early states alongside later ones that comprise the vast majority of Lin- progenitors. In fractionating the cells by maturity, we made use of a cell surface marker, GeneA, which we identified from the initial data set as capturing cells that are multipotent until just beyond the first branch-point.The result of this analysis was a high resolution landscape from the transcriptome of 15,401 single cells, showing multiple branch points, and a topology that remarkably similar to what we reported in mice (Tusi et al. Nature 2018). This map defines the early branching of dendritic cell vs monocyte precursors and supports the novel concept that basophils/eosinophils/mast cells do not derive from common granulocytes/monocytes progenitors but originate instead from what are currently defined as megakaryo/erythroid progenitors.By the in depth analysis of our single-cell maps we generated a number of information of value for the broader hematology community: * We put the "classical" CD34+ FACS-based cell subpopulations into the context of the newly generated BM Lin- population structure (valuable for transplantation research) * We investigated the expression of transcription factors along differentiation trajectories and branching points (valuable for in vitro reprogramming efforts) * We analyzed the expression of several blood cancer genes over the main differentiation hubs and hematopoietic branches (valuable for the study, diagnosis and therapy of hematological tumors) * We provided a detailed comparison of the expression of gene orthologues in human vs mouse hematopoiesis (valuable for the modeling of blood cell disorders in the mouse) * We identified and validated GeneA as a reliable marker for the earliest branches of HSPCs specification in transplantation cell products (valuable for transplantation research and gene therapy) Overall, our study provides a detailed view of the early hematopoietic cell state hierarchy in humans and a valuable resource for HSPC biology. Disclosures No relevant conflicts of interest to declare.

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GATA2-Dependent Developmental and Regenerative Networks

Blood ◽

10.1182/blood-2019-126875 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1182-1182

Author(s):

Alexandra Soukup ◽

Kirby D Johnson ◽

Daniel J Conn ◽

Evgenia Shishkova ◽

Koichi Ricardo Katsumura ◽

...

Keyword(s):

Single Cell ◽

Cell Fate ◽

Progenitor Cell ◽

Fetal Liver ◽

Single Cells ◽

Wild Type ◽

Monocytic Differentiation ◽

Hematopoietic Stem ◽

Altered Expression ◽

Myeloid Progenitor

Coding and regulatory human GATA2 mutations that deregulate protein expression and/or function cause immunodeficiency that often progresses to MDS/AML (McReynolds et al., 2018). In the mouse, decreased GATA2 expression impairs hematopoietic stem/progenitor cell (HSPC) genesis and function (de Pater et al., 2013; Gao et al., 2013; Tsai et al., 1994). While prior studies demonstrated Gata2 +9.5 and -77 enhancers are essential for HSC emergence (+9.5) and/or progenitor cell fate (+9.5 and -77) (Johnson et al., 2012; Johnson et al., 2015; Mehta et al., 2017) and hematopoietic regeneration (+9.5) (Soukup et al., 2019), the mechanisms mediating these processes are not completely established. The -77 enhancer is required for fetal liver progenitors to undergo erythroid, megakaryocytic, granulocytic and monocytic differentiation. By contrast, progenitors with a -77 homozygous deletion (-77-/-) exhibit a predominant monocytic cell fate and generate macrophages ex vivo (Johnson et al., 2015). Using multiomic and single-cell strategies, we asked how this enhancer orchestrates a balance between fate-promoting and -suppressing circuitry in cell populations and single cells. Quantitative proteomics was conducted to discover the -77-regulated protein ensemble conferring multiple fates in a myeloid progenitor population [Common Myeloid Progenitor (CMP) and Granulocyte-Monocyte Progenitor (GMP)] from E14.5 fetal liver of -77+/+ and -77-/- mouse embryos. -77-/- progenitors exhibited decreased levels of GATA2 (4.7-fold) and proteins generated from GATA2 target genes (GATA1: 51-fold; HDC: 52-fold). The 202 proteins upregulated in -77-/- progenitors highlighted immune and inflammatory mechanisms, while the 232 downregulated proteins were linked to erythroid, megakaryocyte and granulocyte biology, indicative of loss of these fate potentials. Innate immune machinery was upregulated in -77-/- vs. -77+/+ progenitors, including interferon (IFN) signaling pathway components such as the IFN-inducible transcription factor and critical monocytic differentiation determinant Interferon Regulatory Factor 8 (IRF8; 2.7 fold higher) (Kurotaki et al., 2013) and diverse pathogen sensors. Expressing GATA2 at physiological levels in -77-/- progenitors normalized the aberrant transcriptome. Since -77 deletion downregulated Gata2 and upregulated Irf8, we tested whether this opposing expression pattern occurs in distinct and/or identical cells in the population using single cell transcriptomics. -77 deletion decreased Gata2 expression, which was anti-correlative with Irf8, and detailed single cell analyses indicated that -77 loss downregulates GATA2, corrupting the transcriptome/proteome, Irf8 expression increases, and IRF8 enables or drives the predominant monocytic differentiation. To determine how GATA2-dependent mechanisms governing progenitor fate relate to those guiding HSPC expansion and differentiation during regeneration, we utilized our +9.5 human disease Ets motif mutation that abrogates myeloablation-dependent GATA2 induction and hematopoietic regeneration in bone marrow (Soukup et al., 2019). While population RNA-seq with Lin-Sca1+c-Kit+ (LSK) cells revealed little to no perturbations in the steady-state, 5-FU treatment of mutants led to altered expression of only 14% of the transcripts regulated in regenerating wild type cells (423/2974). Gene Ontology analysis of differentially expressed genes indicated that genes dysregulated by the Ets motif mutation included those linked to cell cycle regulation and cellular proliferation. As LSKs consist of LT-HSCs, ST-HSCs, and MPPs, we used single-cell transcriptomics to elucidate defective regenerative circuits in individual cells. Analysis of 17000-20000 wild type and mutant LSK cells revealed GATA2-dependent mechanisms distinct from those mediating progenitor cell fate control in the fetal liver. These studies have revealed context-dependent GATA2 mechanisms governing developmental and regenerative hematopoiesis, which will enable the development of strategies to detect, diagnose, and treat GATA2-linked blood diseases. Disclosures No relevant conflicts of interest to declare.

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Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions

10.1101/2020.10.15.339226 ◽

2020 ◽

Author(s):

Haiqing Xiong ◽

Yingjie Luo ◽

Qianhao Wang ◽

Xianhong Yu ◽

Aibin He

Keyword(s):

Transcriptional Regulation ◽

Single Cell ◽

Cell Fate ◽

Single Cells ◽

Embryonic Stem ◽

Joint Detection ◽

Hematopoietic Stem ◽

Cell Fate Decisions ◽

Higher Dimensional ◽

Context Specific

SUMMARYDeciphering mechanisms in cell fate decisions requires single-cell holistic reconstructions of multi-dimensional epigenome in transcriptional regulation. Here we develop CoTECH, a combinatorial barcoding method allowing for high-throughput single-cell joint detection of chromatin occupancy and transcriptome. First, we used CoTECH to examine bivalent histone marks (H3K4me3 and H3K27me3) with transcription from naïve to primed mouse embryonic stem cells. Concurrent bivalent marks in pseudo-single cells linked via transcriptome were computationally derived, resolving pseudotemporal bivalency trajectories and disentangling a context-specific interplay between H3K4me3/H3K27me3 and transcription level. Next, CoTECH with H3K27ac, an active enhancer marker, revealed the regulatory basis of endothelial-to-hematopoietic transition in two waves of hematopoietic cells and distinctive enhancer-gene linking schemes guiding hemogenic endothelial cell (HEC) emergence, indicating a unique epigenetic control of transcriptional regulation for hematopoietic stem cell priming. Together, CoTECH provides an efficient framework for single-cell co-assay of chromatin occupancy and transcription, thus, enabling higher-dimensional epigenomic reconstructions.

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Mast Cell Involvement in Fibrosis in Chronic Graft-Versus-Host Disease

International Journal of Molecular Sciences ◽

10.3390/ijms22052385 ◽

2021 ◽

Vol 22 (5) ◽

pp. 2385

Author(s):

Ethan Strattan ◽

Gerhard Carl Hildebrandt

Keyword(s):

Wound Healing ◽

Mast Cell ◽

Mast Cells ◽

Graft Versus Host Disease ◽

Chronic Gvhd ◽

Fibrotic Disease ◽

Acute Leukemias ◽

Hematopoietic Stem ◽

Host Disease ◽

Graft Versus Host

Allogeneic hematopoietic stem cell transplantation (HSCT) is most commonly a treatment for inborn defects of hematopoiesis or acute leukemias. Widespread use of HSCT, a potentially curative therapy, is hampered by onset of graft-versus-host disease (GVHD), classified as either acute or chronic GVHD. While the pathology of acute GVHD is better understood, factors driving GVHD at the cellular and molecular level are less clear. Mast cells are an arm of the immune system that are known for atopic disease. However, studies have demonstrated that they can play important roles in tissue homeostasis and wound healing, and mast cell dysregulation can lead to fibrotic disease. Interestingly, in chronic GVHD, aberrant wound healing mechanisms lead to pathological fibrosis, but the cellular etiology driving this is not well-understood, although some studies have implicated mast cells. Given this novel role, we here review the literature for studies of mast cell involvement in the context of chronic GVHD. While there are few publications on this topic, the papers excellently characterized a niche for mast cells in chronic GVHD. These findings may be extended to other fibrosing diseases in order to better target mast cells or their mediators for treatment of fibrotic disease.

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Mast Cell-Specific Expression of Human Siglec-8 in Conditional Knock-in Mice

International Journal of Molecular Sciences ◽

10.3390/ijms20010019 ◽

2018 ◽

Vol 20 (1) ◽

pp. 19 ◽

Cited By ~ 13

Author(s):

Yadong Wei ◽

Krishan Chhiba ◽

Fengrui Zhang ◽

Xujun Ye ◽

Lihui Wang ◽

...

Keyword(s):

Flow Cytometry ◽

Mast Cell ◽

Mast Cells ◽

Es Cells ◽

Embryonic Stem ◽

Surface Protein ◽

Cell Types ◽

Mouse Strains ◽

Specific Expression

Sialic acid-binding Ig-like lectin 8 (Siglec-8) is expressed on the surface of human eosinophils, mast cells, and basophils—cells that participate in allergic and other diseases. Ligation of Siglec-8 by specific glycan ligands or antibodies triggers eosinophil death and inhibits mast cell degranulation; consequences that could be leveraged as treatment. However, Siglec-8 is not expressed in murine and most other species, thus limiting preclinical studies in vivo. Based on a ROSA26 knock-in vector, a construct was generated that contains the CAG promoter, a LoxP-floxed-Neo-STOP fragment, and full-length Siglec-8 cDNA. Through homologous recombination, this Siglec-8 construct was targeted into the mouse genome of C57BL/6 embryonic stem (ES) cells, and chimeric mice carrying the ROSA26-Siglec-8 gene were generated. After cross-breeding to mast cell-selective Cre-recombinase transgenic lines (CPA3-Cre, and Mcpt5-Cre), the expression of Siglec-8 in different cell types was determined by RT-PCR and flow cytometry. Peritoneal mast cells (dual FcεRI+ and c-Kit+) showed the strongest levels of surface Siglec-8 expression by multicolor flow cytometry compared to expression levels on tissue-derived mast cells. Siglec-8 was seen on a small percentage of peritoneal basophils, but not other leukocytes from CPA3-Siglec-8 mice. Siglec-8 mRNA and surface protein were also detected on bone marrow-derived mast cells. Transgenic expression of Siglec-8 in mice did not affect endogenous numbers of mast cells when quantified from multiple tissues. Thus, we generated two novel mouse strains, in which human Siglec-8 is selectively expressed on mast cells. These mice may enable the study of Siglec-8 biology in mast cells and its therapeutic targeting in vivo.

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Leveraging high-powered RNA-Seq datasets to improve inference of regulatory activity in single-cell RNA-Seq data

10.1101/553040 ◽

2019 ◽

Cited By ~ 1

Author(s):

Ning Wang ◽

Andrew E. Teschendorff

Keyword(s):

Transcription Factors ◽

Single Cell ◽

Cell Fate ◽

Regulatory Networks ◽

Large Scale ◽

Single Cells ◽

Differential Expression Analysis ◽

Dropout Rate ◽

Rna Seq ◽

Regulatory Activity

AbstractInferring the activity of transcription factors in single cells is a key task to improve our understanding of development and complex genetic diseases. This task is, however, challenging due to the relatively large dropout rate and noisy nature of single-cell RNA-Seq data. Here we present a novel statistical inference framework called SCIRA (Single Cell Inference of Regulatory Activity), which leverages the power of large-scale bulk RNA-Seq datasets to infer high-quality tissue-specific regulatory networks, from which regulatory activity estimates in single cells can be subsequently obtained. We show that SCIRA can correctly infer regulatory activity of transcription factors affected by high technical dropouts. In particular, SCIRA can improve sensitivity by as much as 70% compared to differential expression analysis and current state-of-the-art methods. Importantly, SCIRA can reveal novel regulators of cell-fate in tissue-development, even for cell-types that only make up 5% of the tissue, and can identify key novel tumor suppressor genes in cancer at single cell resolution. In summary, SCIRA will be an invaluable tool for single-cell studies aiming to accurately map activity patterns of key transcription factors during development, and how these are altered in disease.

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Development of both human connective tissue-type and mucosal-type mast cells in mice from hematopoietic stem cells with identical distribution pattern to human body

Blood ◽

10.1182/blood-2003-04-1160 ◽

2004 ◽

Vol 103 (3) ◽

pp. 860-867 ◽

Cited By ~ 39

Author(s):

Naotomo Kambe ◽

Hidefumi Hiramatsu ◽

Mika Shimonaka ◽

Hisanori Fujino ◽

Ryuta Nishikomori ◽

...

Keyword(s):

Stem Cells ◽

Mast Cell ◽

Mast Cells ◽

Hematopoietic Stem Cells ◽

Cell Development ◽

Tissue Type ◽

Human Mast Cell ◽

Hematopoietic Stem ◽

Nog Mice

Abstract The transplantation of primitive human cells into sublethally irradiated immune-deficient mice is the well-established in vivo system for the investigation of human hematopoietic stem cell function. Although mast cells are the progeny of hematopoietic stem cells, human mast cell development in mice that underwent human hematopoietic stem cell transplantation has not been reported. Here we report on human mast cell development after xenotransplantation of human hematopoietic stem cells into nonobese diabetic severe combined immunodeficient \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \((\mathrm{NOD{/}SCID}){/}{\gamma}_{\mathrm{c}}^{null}\) \end{document} (NOG) mice with severe combined immunodeficiency and interleukin 2 (IL-2) receptor γ-chain allelic mutation. Supported by the murine environment, human mast cell clusters developed in mouse dermis, but they required more time than other forms of human cell reconstitution. In lung and gastric tract, mucosal-type mast cells containing tryptase but lacking chymase located on gastric mucosa and in alveoli, whereas connective tissue-type mast cells containing both tryptase and chymase located on gastric submucosa and around major airways, as in the human body. Mast cell development was also observed in lymph nodes, spleen, and peritoneal cavity but not in the peripheral blood. Xenotransplantation of human hematopoietic stem cells into NOG mice can be expected to result in a highly effective model for the investigation of human mast cell development and function in vivo.

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SCELLECTOR: ranking amplification bias in single cells using shallow sequencing

BMC Bioinformatics ◽

10.1186/s12859-020-03858-y ◽

2020 ◽

Vol 21 (1) ◽

Author(s):

Vivekananda Sarangi ◽

Alexandre Jourdon ◽

Taejeong Bae ◽

Arijit Panda ◽

Flora Vaccarino ◽

...

Keyword(s):

Single Cell ◽

Multiple Displacement Amplification ◽

Single Cells ◽

Sequencing Data ◽

High Coverage ◽

Amplification Bias ◽

Single Cell Profiling ◽

High Concordance ◽

Human Neuronal Cells

Abstract Background The study of mosaic mutation is important since it has been linked to cancer and various disorders. Single cell sequencing has become a powerful tool to study the genome of individual cells for the detection of mosaic mutations. The amount of DNA in a single cell needs to be amplified before sequencing and multiple displacement amplification (MDA) is widely used owing to its low error rate and long fragment length of amplified DNA. However, the phi29 polymerase used in MDA is sensitive to template fragmentation and presence of sites with DNA damage that can lead to biases such as allelic imbalance, uneven coverage and over representation of C to T mutations. It is therefore important to select cells with uniform amplification to decrease false positives and increase sensitivity for mosaic mutation detection. Results We propose a method, Scellector (single cell selector), which uses haplotype information to detect amplification quality in shallow coverage sequencing data. We tested Scellector on single human neuronal cells, obtained in vitro and amplified by MDA. Qualities were estimated from shallow sequencing with coverage as low as 0.3× per cell and then confirmed using 30× deep coverage sequencing. The high concordance between shallow and high coverage data validated the method. Conclusion Scellector can potentially be used to rank amplifications obtained from single cell platforms relying on a MDA-like amplification step, such as Chromium Single Cell profiling solution.

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Mechanism of mast cell deficiency in mutant mice of mi/mi genotype: an analysis by co-culture of mast cells and fibroblasts

Blood ◽

10.1182/blood.v75.6.1247.1247 ◽

1990 ◽

Vol 75 (6) ◽

pp. 1247-1251 ◽

Cited By ~ 54

Author(s):

Y Ebi ◽

T Kasugai ◽

Y Seino ◽

H Onoue ◽

T Kanemoto ◽

...

Keyword(s):

Mast Cell ◽

Mast Cells ◽

Culture Medium ◽

Fibroblast Cell ◽

Mutant Mice ◽

Pokeweed Mitogen ◽

Spleen Cells ◽

Hematopoietic Stem ◽

Nih 3T3 ◽

Cell Conditioned Medium

Abstract Mutant mice of mi/mi genotype are osteopetrotic and are deficient in mast cells. The osteopetrosis of mi/mi mice can be cured by bone marrow transplantation from congenic normal (+/+) mice, and therefore, the cause of the osteopetrosis is attributed to a defect of osteoclasts. Since both osteoclasts and mast cells are the progeny of multipotential hematopoietic stem cells, we examined whether mast cells were defective in mi/mi mice. In spite of the deficiency of mast cells in tissues of mi/mi mice, mast cells did develop when spleen cells of mi/mi mice were cultured with pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM). The proliferative response of cultured mast cells (CMC) derived from mi/mi mice to PWM-SCM was comparable with that of CMC from +/+ mice. In contrast, when CMC were co-cultured with the NIH/3T3 fibroblast cell line in culture medium lacking PWM-SCM, only +/+ CMC entered into the S phase of the cell cycle and were maintained; mi/mi CMC gradually disappeared. Moreover, fibroblasts derived from the skin of mi/mi mice normally supported the proliferation of +/+ CMC. Thus, the mast cell deficiency of mi/mi mice appears to be due to the inability of mi/mi mast cells to respond to the proliferative stimulus presented by fibroblasts.

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