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Zebrafish (Danio rerio) has emerged as a powerful genetic model for the study of vertebrate hematopoiesis. However, methods for detection and isolation of hematopoietic stem cells (HSCs) have not yet been reported. In mammals, the combination of Hoechst 33342 staining with flow cytometry can be used for separation of a bone marrow side population (SP), which is highly enriched for HSCs. We applied a similar procedure to hematopoietic kidney marrow cells from adult zebrafish, and identified a segregated cohort of SP cells, which demonstrate a set of features typical of stem cells. SP cells show extremely low scatter characteristics, and are small in size with a minimum of cytoplasm. Treatment of zebrafish kidney marrow cells with reserpine or fumitremorgin C, which inhibit the ABCG2 transporter responsible for Hoechst 33342 efflux, caused a clear reduction in the number of SP cells. Consistent with the quiescent state of HSCs, the SP cells are strongly resistant to the myelosuppressive agent 5-fluorouracil. In addition, SP cells specifically demonstrate higher expression of genes known to be markers of HSCs of mammals. Hence, our results show that the SP phenotype is conserved between mammals and teleosts, and the properties of the zebrafish SP cells indicate a significant enrichment for HSCs. These rapid flow cytometric methods for purification of HSCs from zebrafish may greatly facilitate genetic analysis of stem cells using the advantages of this vertebrate model.

Objective: Clock genes are known to mediate circadian rhythms in the central nervous system and peripheral organs. Although they are expressed in mouse hematopoietic progenitor and stem cells, it is unknown if they are related to circadian rhythms in these cells. We therefore investigated the 24-hour patterns in the activity of several clock genes in the bone marrow (BM) side population (SP) primitive stem cells, and compared these 24-hour patterns to clock gene variations in the whole BM and liver., Methods: Cells were obtained from 84 B6D2F(1) mice in three replicate experiments on the second day after release into constant darkness from a standardizing light-dark schedule. mRNA expression of clock genes was measured with quantitative reverse transcriptase polymerase chain reaction., Results: mPer2 displayed circadian rhythms in SP cells, whole BM, and liver cells. mPer1 and mRev-erb alpha showed a circadian rhythm in whole BM and liver, but not SP cells. mBmal1 was not expressed rhythmically in SP cells, nor in the whole BM, contrary to rhythms observed in the liver., Conclusions: With the exception of mPer2, most clock genes studied in primitive hematopoietic SP stem cells were not oscillating in a fully organized circadian manner, which is similar to immature cells in rapidly proliferating organs, such as the testis and thymus. These findings indicate that circadian clock gene expression variations in BM are developmentally regulated.

Objective: Circadian genes have recently been characterized in many tissues, but not in hematopoietic stem cells. These cells are rare in the bone marrow (BM), which makes it difficult to collect enough cells for detailed molecular analysis in a short period of time without reduced RNA quality. The aim was to improve methodology and reliability of clock gene expression analysis in purified mouse hematopoietic stem cells., Methods: Stem cells were highly enriched by high-speed flow cytometric cell sorting of the side population (SP) cells from Hoechst 33342 (Hoechst)-stained mouse BM. Total RNA was isolated from sorted SP and whole BM cells and exposed to DNase treatment. The relative mRNA levels of major clock genes mPer1, mPer2, mBmal1, mCry1, mClock, and mRev-erb alpha were measured with real-time quantitative reverse transcription polymerase chain reaction (Q-RT-PCR) and normalized to m36B4, used as a reference gene. The clonogenity of sorted SP cells and whole BM; cells taken before and after sorting, were tested in colony-formation assay., Results: Clock gene activity in sorted SP cells showed pronounced relative differences compared with whole BM for mPer1 and mCry1. The high-speed sorting procedure did not influence clock gene expression or cell clonogenity, even when this was performed with a delay period up to 24 hours., Conclusions: We demonstrated expression of six clock genes in mouse hematopoietic stem cells. A combination of high-speed flow cytometric sorting and Q-RT-PCR was shown to be useful and reliable for analysis of clock gene activity in small stem cell fractions.

Circadian clocks have been shown to operate developmentally in mouse and human hematopoietic stem and progenitor cells in vivo, but little is known about their possible oscillations in vitro. Here, we show that repeated circadian oscillations could be induced in both cultured bone marrow-derived mesenchymal- and adipose-derived stem cells (MSCs and ASCs, respectively) by serum shock. In particular, the novel finding of rhythmic clock gene expression induced by cAMP analogs showed similarities as well as differences to serum-induced oscillations. Rhythmic PER1 expression was found in serum-shocked MSCs, suggesting the phosphorylation status of PER1 is important for its activity in circadian rhythms. Furthermore, immunofluoresent staining showed that the localization of PER1 was dependent on the level of PER1 expression. These inducible self-sustained circadian clocks in primary cultures of human MSCs in vitro with rhythmic changes in expression levels, phosphorylation, and localization of clock protein, PER1, may be of importance for maintaining the induced oscillations in stem cells. Therefore, the established cell models described here appear to be valuable for studying the molecular mechanism driving and coordinating the circadian network between stem and stromal cells. [ABSTRACT FROM AUTHOR]