Hematopoietic stem cells (HSCs) are a self-renewing population of bone marrow cells that replenish the cellular elements of blood throughout life. HSCs represent a paradigm for the study of stem-cell biology, because robust methods for prospective isolation of HSCs have facilitated rigorous characterization of these cells. Recently, a new isolation method was reported, using the SLAM family of cell-surface markers, including CD150 (SlamF1), to offer potential advantages over established protocols. We examined the overlap between SLAM family member expression with an established isolation scheme based on Hoechst dye efflux (side population; SP) in conjunction with canonical HSC cell-surface markers (Sca-1, c-Kit, and lineage markers). Importantly, we find that stringent gating of SLAM markers is essential to achieving purity in HSC isolation and that the inclusion of canonical HSC markers in the SLAM scheme can greatly augment HSC purity. Furthermore, we observe that both CD150(+) and CD150(-) cells can be found within the SP population and that both populations can contribute to long-term multilineage reconstitution. Thus, using SLAM family markers to isolate HSCs excludes a substantial fraction of the marrow HSC compartment. Interestingly, these 2 subpopulations are functionally distinct, with respect to lineage output as well as proliferative status.
"That category of HSC was reportedly less likely to generate lymphocytes than CD150Lo/− HSC . Similarly, Goodell and colleagues found lymphopoietic potential was lowest among HSC that strongly exclude Hoechst dye . These “lower side population” HSC were the most increased by JAK2V617F (Fig. 2B). "
[Show abstract][Hide abstract] ABSTRACT: Although extremely rare, hematopoietic stem cells (HSCs) are divisible into subsets that differ with respect to differentiation potential and cell surface marker expression. For example, we recently found that CD86- CD150+ CD48- HSCs have limited potential for lymphocyte production. This could be an important new tool for studying hematological abnormalities. Here, we analyzed HSC subsets with a series of stem cell markers in JAK2V617F transgenic (Tg) mice, where the mutation is sufficient to cause myeloproliferative neoplasia with lymphocyte deficiency. Total numbers of HSC were elevated 3 to 20 fold in bone marrow of JAK2V617F mice. Careful analysis suggested the accumulation involved multiple HSC subsets, but particularly those characterized as CD150HI CD86- CD18L°CD41+ and excluding Hoechst dye. Real-Time PCR analysis of their HSC revealed that the erythropoiesis associated gene transcripts Gata1, Klf1 and Epor were particularly high. Flow cytometry analyses based on two differentiation schemes for multipotent progenitors (MPP) also suggested alteration by JAK2 signals. The low CD86 on HSC and multipotent progenitors paralleled the large reductions we found in lymphoid progenitors, but the few that were produced functioned normally when sorted and placed in culture. Either of two HSC subsets conferred disease when transplanted. Thus, flow cytometry can be used to observe the influence of abnormal JAK2 signaling on stem and progenitor subsets. Markers that similarly distinguish categories of human HSCs might be very valuable for monitoring such conditions. They could also serve as indicators of HSC fitness and suitability for transplantation.
PLoS ONE 04/2014; 9(4):e93643. DOI:10.1371/journal.pone.0093643 · 3.23 Impact Factor
"An attractive feature of this study was that the process of stem cell isolation was based not on biomarkers of mesenchymal stem cells, but rather on their biological function. The ability of stem cells to efflux Hoechst 33342 has been demonstrated in a wide range of stem cell populations, including hematopoietic stem cells , cancer stem cells  or adult stem cells (including DPCs) . In this study, we performed a miRNA array analysis on two different cell populations (DPCs and PDLCs), and despite the differences in their stem cell characteristics, we obtained similar miRNA signatures for each cell. "
[Show abstract][Hide abstract] ABSTRACT: Dental pulp cells (DPCs) are known to be enriched in stem/progenitor cells but not well characterized yet. Small non-coding microRNAs (miRNAs) have been identified to control protein translation, mRNA stability and transcription, and have been reported to play important roles in stem cell biology, related to cell reprogramming, maintenance of stemness and regulation of cell differentiation. In order to characterize dental pulp stem/progenitor cells and its mechanism of differentiation, we herein sorted stem-cell-enriched side population (SP) cells from human DPCs and periodontal ligament cells (PDLCs), and performed a locked nucleic acid (LNA)-based miRNA array. As a result, miR-720 was highly expressed in the differentiated main population (MP) cells compared to that in SP cells. In silico analysis and a reporter assay showed that miR-720 targets the stem cell marker NANOG, indicating that miR-720 could promote differentiation of dental pulp stem/progenitor cells by repressing NANOG. Indeed, gain-and loss-of-function analyses showed that miR-720 controls NANOG transcript and protein levels. Moreover, transfection of miR-720 significantly decreased the number of cells positive for the early stem cell marker SSEA-4. Concomitantly, mRNA levels of DNA methyltransferases (DNMTs), which are known to play crucial factors during stem cell differentiation, were also increased by miR-720 through unknown mechanism. Finally, miR-720 decreased DPC proliferation as determined by immunocytochemical analysis against ki-67, and promoted odontogenic differentiation as demonstrated by alizarin red staining, as well as alkaline phosphatase and osteopontin mRNA levels. Our findings identify miR-720 as a novel miRNA regulating the differentiation of DPCs.
PLoS ONE 12/2013; 8(12):e83545. DOI:10.1371/journal.pone.0083545 · 3.23 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cancer progression is often paralleled by a decline in bone mass, raising risk of fracture. Concerns persist regarding anabolic interventions for skeletal protection, as these may inadvertently exacerbate neoplastic tissue expansion. Given bone's inherent mechanosensitivity, low intensity vibration (LIV), a mechanical signal that encourages osteoblastogenesis, could possibly slow cancer-associated bone loss, but this goal must be achieved without fostering disease progression. Seventy 12w female F1-SWRxSWXJ-9 mice, a strain prone to developing granulosa cell tumors, were randomized into baseline control (BC: n=10), age-matched control (AC: n=30), and LIV (n=30), which received mechanical signals (90Hz @ 0.3g) for 15m/day, 5 day/w over the course of 1 year. Survival curves for AC (10 died) and LIV (8 died) followed similar trends (p=0.62), indicating longevity was unperturbed by LIV. At 1 year, bone volume of proximal tibiae in LIV mice was 25% greater than AC (p<0.02), while bone volume of L5 vertebrae was 16% higher in LIV over AC (p<0.02). Primary lesions and peripheral metastases were apparent in both LIV and AC; however, overall tumor incidence was approximately 30% less in LIV (p=0.27) and, when disease was evident, involved fewer organ systems (p=0.09). Marrow-derived mesenchymal stem cells (MSC) were 52% lower (p<0.01) in LIV, and 31% lower (p=0.08) in mice lacking pathology, suggesting higher MSC levels in this model of cancer susceptibility may have contributed to tumor progression. These experiments indicate that LIV helps protect bone mass in mice inherently susceptible to cancer without compromising life expectancy, perhaps through mechanical control of stem cell fate. Further, these data reflect the numerous system-level benefits of exercise in general, and mechanical signals in particular, in the preservation of bone density and the suppression of cancer progression.
Bone 05/2012; 51(3):570-7. DOI:10.1016/j.bone.2012.05.004 · 3.97 Impact Factor
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