An in vitro limiting-dilution assay of long-term repopulating hematopoietic stem cells in the mouse

Stem cells reside in customized microenvironments (niches) that contribute to their unique ability to divide asymmetrically to give rise to self and to a daughter cell with distinct properties. Notch receptors and their ligands are highly conserved and have been shown to regulate cell-fate decisions in multiple developmental systems through local cell interactions. To assess whether Notch signaling may regulate hematopoiesis to maintain cells in an immature state, we examined the functional role of the recombinant, secreted form of the Notch ligand Jagged-1 during mouse hematopoietic stem cell (HSC) and progenitor cell proliferation and maturation. We found that ligand immobilization on stromal layer or on Sepharose-4B beads is required for the induction of self-renewing divisions of days 28-35 cobblestone area-forming cell. The free, soluble Jagged-1, however, has a dominant-negative effect on self-renewal in the stem-cell compartment. In contrast, free as well as immobilized Jagged-1 promotes growth factor-induced colony formation of committed hematopoietic progenitor cells. Therefore, we propose that differences in Jagged-1 presentation and developmental stage of the Notch receptor-bearing cells influence Notch ligand-binding results toward activation or inhibition of downstream signaling. Moreover, these results suggest potential clinical use of recombinant Notch ligands for expanding human HSC populations in vitro.

Section: Effect Of Sjg1 On the Development Of Cafcsmentioning

confidence: 90%

Section: Cafc Assaymentioning

confidence: 99%

Section: Cafc Assaymentioning

confidence: 99%

See 1 more Smart Citation

Soluble Jagged-1 is able to inhibit the function of its multivalent form to induce hematopoietic stem cell self-renewal in a surrogate in vitro assay

Vas¹,

Szilágyi

Pálóczi

et al. 2004

“…Bone marrow-nucleated cells pooled from two to three animals of each genotype were seeded at twofold dilutions (10 5 -1562 cells/well). Culture medium was gently half-changed with fresh medium weekly, and the CAFCs were scored after 5 weeks [26]. The frequency of CAFC was calculated using the L-CALC software (StemCell Technologies) for limiting dilution analysis.…”

Section: Cobblestone Area-forming Cell (Cafc) Assaymentioning

confidence: 99%

Increased numbers of committed myeloid progenitors but not primitive hematopoietic stem/progenitors in mice lacking STAT6 expression

Bunting

Yu²,

Bradley³

et al. 2004

Signal transducer and activator of transcription-6 (STAT6) plays important roles in cytokine signaling via interleukin-4 and -13 receptors (IL-4R and IL-13R). Mice in which STAT6 has been disrupted by homologous recombination show defects in T helper cell type 2 (Th2) lymphocyte production, resulting in an accumulation of Th1 cells. In addition to defects in differentiation and proliferation of T lymphocytes, STAT6-deficient mice show increased cell-cycle activation and frequency of myeloid progenitors. Although this has been shown to be mediated through Oncostatin M production by T cells, IL-4Ralpha and STAT6 have also recently been found to be enriched for expression in primitive hematopoietic stem cells (HSCs) in gene expression-profiling studies. Therefore, we have investigated whether defects in hematopoietic function in mice lacking STAT6 expression extended into the primitive hematopoietic compartments of the bone marrow. Here, we report that STAT6 deficiency increased bone marrow-committed myeloid progenitors but did not alter the number of cells enriched for HSC/multipotent progenitors, primitive cobblestone area-forming cells assayed in vitro, or bone marrow short-term or long-term repopulating cells assayed in vivo. Therefore, the requirement for STAT6 activation during hematopoiesis is limited, and primitive hematopoietic cell types are insulated against possible effects of cytokine stimulation by Th1 cells.

“…19,20 To establish the potential differences between fetal and adult HSPCs, we first evaluated gene expression in both canonical and noncanonical arms of Wnt signaling by lated from BM and FL as previously published by others 21 and described in Figure 1A. The in vitro functionality of sorted cells was determined by a long-term culture initiating cell (LTC-IC) assay, 17 and we found equivalent frequencies of LTC-ICs in both populations (95% confidence intervals 1/13-1/8 for BM, 1/10-1/5 for FL, P = 0.17; Figure 1B). These frequencies are slightly lower than expected, [22][23][24] and most likely reflect decreased cell viability due to sorting-induced stress.…”

Section: Fetal Hspcs Display Higher Wnt/ -Catenin Activity Than Adultmentioning

confidence: 99%

Frontline Science: Wnt/β-catenin pathway promotes early engraftment of fetal hematopoietic stem/progenitor cells

Kwarteng

Hétu-Arbour

Heinonen

2018

The switch from fetal to adult hematopoietic stem/progenitor cells (HSPCs) is associated with profound changes in several genetic programs. Although HSPC ageing corresponds to alterations in Wnt signaling, relatively little is known about the relative roles of different Wnt signaling pathways in HSPC ontogeny. We hypothesized that proliferating fetal HSPCs would be more dependent on canonical β-catenin-dependent Wnt signaling when compared to quiescent adult bone marrow HSPCs. We have compared here Wnt signaling activities in murine fetal and adult HSPCs and demonstrate a shift from Wnt/β-catenin-dependent signaling in fetal liver HSPCs to more predominantly noncanonical Wnt/polarity signaling in adult HSPCs. β-Catenin was selectively required for fetal HSPC competitiveness shortly after transplant, and protected cells from oxidative stress. Our results emphasize the complexity of Wnt signaling dynamics in HSPC maintenance and function.