A Platform for Studying of the Three-Dimensional Migration of Hematopoietic Stem/Progenitor Cells

With the aim of establishing an effective method to expand hematopoietic stem/progenitor cells for application in hematopoietic stem cell transplantation, we performed ex vivo expansion of hematopoietic stem/progenitor cells derived from mouse fetal liver cells in three-dimensional cocultures with stromal cells. In these cocultures, stromal cells were first cultured within three-dimensional scaffolds to form stromal layers and then fetal liver cells containing hematopoietic cells were seeded on these scaffolds to expand the hematopoietic cells over the 2 weeks of coculture in a serum-containing medium without the addition of cytokines. Prior to coculture, stromal cell growth was suppressed by treatment with the DNA synthesis inhibitor mitomycin C, and its effect on hematopoietic stem/progenitor cell expansion was compared with that in control cocultures in which fetal liver cells were cocultured with three-dimensional freeze-thawed stromal cells. After coculture with mitomycin C-treated stromal cells, we achieved a several-fold expansion of the primitive hematopoietic cells (c-kit+hematopoietic progenitor cells >7.8-fold, and CD34+hematopoietic stem/progenitor cells >3.5-fold). Compared with control cocultures, expansion of hematopoietic stem/progenitor cells tended to be lower, although that of hematopoietic progenitor cells was comparable. Thus, our results suggest that three-dimensional freeze-thawed stromal cells have higher potential to expand hematopoietic stem/progenitor cells compared with mitomycin C-treated stromal cells.

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Evaluating Interaction of Cord Blood Hematopoietic Stem/Progenitor Cells with Functionally Integrated Three-Dimensional Microenvironments

Stem Cells Translational Medicine ◽

10.1002/sctm.17-0157 ◽

2018 ◽

Vol 7 (3) ◽

pp. 271-282 ◽

Cited By ~ 5

Author(s):

Saloomeh Mokhtari ◽

Pedro M. Baptista ◽

Dipen A. Vyas ◽

Charles Jordan Freeman ◽

Emma Moran ◽

...

Keyword(s):

Cord Blood ◽

Progenitor Cells ◽

Three Dimensional ◽

Hematopoietic Stem

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Lincrna Hotset Regulates Hox Gene Transcription and Promotes Early Hematopoietic Fates

Blood ◽

10.1182/blood.v124.21.1573.1573 ◽

2014 ◽

Vol 124 (21) ◽

pp. 1573-1573

Author(s):

Changwang Deng ◽

Ying Li ◽

Besabeh Tusi ◽

Wei Jian ◽

Bhavita Patel ◽

...

Keyword(s):

Gene Expression ◽

Embryonic Development ◽

Progenitor Cells ◽

Gene Transcription ◽

Three Dimensional ◽

Embryoid Bodies ◽

Hematopoietic Differentiation ◽

Hematopoietic Stem ◽

Hematopoietic Lineage ◽

Stem And Progenitor Cells

Abstract Mammalian genomes encode tens of thousands of long intergenic noncoding RNAs (lincRNAs). Accumulated evidence suggests that many of lincRNAs are expressed in a development- or tissue-specific manner. However, it remains largely unknown how lincRNAs reprogram lineage-specific gene expression and regulate lineage-specific differentiation processes during early embryonic development. It is also important to identify specific lincRNA(s) that is involved in early hematopoietic lineage formation and differentiation. Here, we reported a HoxB locus-associated lincRNA HOTSET that is conservatively expressed in both mouse and human hematopoietic stem and progenitor cells (HS/PCs). During embryonic development, HOTSET RNA is dramatically induced upon embryoid body (EB) hematopoietic differentiation. Expression of HOTSET RNA is required for development of the mesoderm-derived Flk1+ bipotential precursors of blood and endothelium lineages and CD41+/c-Kit+ hematopoietic stem and progenitor cells. We demonstrated that HOTSET RNA directly interacts with the SET domains of Setd1a and MLL1 histone methyltransferases in a sense-strand dependent manner. HOTSET RNA directly binds to the HoxB1-6 gene promoters and recruits the Setd1a/MLL1 complexes to the anterior HoxB locus upon differentiation. Furthermore, HOTSET RNA mediated H3K4 methylation is essential for long-range chromatin loops that bring anterior HoxB genes into a close proximity with HOTSET RNA locus and for subsequent activation of anterior HoxB gene expression. In addition, knockdown of HOTSET RNA disrupts the recruitment of the Setd1a/MLL1 complexes, H3K4me3 levels, the three-dimensional chromatin architecture, and HoxB gene transcription at the HoxB gene cluster. Depletion of HOTSET RNA also impairs differentiation of the CD41+/c-Kit+ HS/PC population and hematopoietic transcription factor and signaling networks required for early hematopoietic differentiation. Importantly, re-expression of HoxB2-4 genes in the HOTSET-depleted embryoid bodies rescues the expression of core hematopoietic transcription factors as well as the Flk1+ mesoderm-derived precursors and CD41+/c-Kit+ HS/PCs that undergo hematopoietic differentiation. Thus, HOTSET plays an important role in early hematopoietic lineage commitment by shaping the three-dimensional chromatin landscape in the HoxB locus to activate anterior HoxB genes that subsequently modulate lineage-specific transcription networks during hematopoietic development. Disclosures No relevant conflicts of interest to declare.

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Ex vivo amplification of human hematopoietic stem and progenitor cells in an alginate three-dimensional culture system

International Journal of Laboratory Hematology ◽

10.1111/j.1751-553x.2011.01324.x ◽

2011 ◽

pp. no-no ◽

Cited By ~ 1

Author(s):

Y. YUAN ◽

K.-T. TSE ◽

F. W.-Y. SIN ◽

B. XUE ◽

H.-H. FAN ◽

...

Keyword(s):

Progenitor Cells ◽

Culture System ◽

Ex Vivo ◽

Three Dimensional ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells ◽

Three Dimensional Culture

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Cytokine combinations differentially influence the SDF-1α-dependent migratory activity of cultivated murine hematopoietic stem and progenitor cells

Biological Chemistry ◽

10.1515/bc.2008.099 ◽

2008 ◽

Vol 389 (7) ◽

Cited By ~ 13

Author(s):

Susannah H. Kassmer ◽

Bernd Niggemann ◽

Michael Punzel ◽

Christine Mieck ◽

Kurt S. Zänker ◽

...

Keyword(s):

Progenitor Cells ◽

Intracellular Signaling ◽

Three Dimensional ◽

Collagen Matrix ◽

Time Lapse ◽

Hematopoietic Stem ◽

Migratory Activity ◽

Stem And Progenitor Cells ◽

Stromal Cell Derived Factor

AbstractStromal cell-derived factor-1α (SDF-1α) is a strong migratory stimulant for hematopoietic stem and progenitor cells (HSPCs). The hematopoietic cytokines thrombopoietin (TPO), Flt3-ligand (FL), stem cell factor (SCF) and interleukin 11 (IL-11) are able to stimulate amplification of primitive murine hematopoietic stem cells (HSCs)in vitro. The effects of these cytokines on SDF-1α-induced migratory activity of murine Lin-c-kit+HSPC were analyzed by cultivation of these cells in the presence of 12 combinations of FL, TPO, SCF and IL-11. Migratory activity was measured in a three-dimensional collagen matrix using time-lapse video microscopy. Each cytokine combination had a distinct effect on SDF-1α-stimulated migratory activity. For instance, FL- and SCF-cultivated cells showed a high migratory SDF-1α response, while cells cultivated with SCF, TPO and IL-11 did not react to SDF-1α stimulation with an elevated migration rate. Our data indicate that the differences in the migratory SDF-1α response are not related to different CXCR4 expression levels, but rather to the differential engagement of the CXCR4-dependent MAPKp42/44and PI3K signal transduction pathways. This indicates that hematopoietic cytokines can have a significant impact on SDF-1α-stimulated migratory activity and the underlying intracellular signaling processes in cultivated HSPCs.

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Genome-wide analysis of focal DNA hypermethylation in IDH-mutant AML samples

10.1101/2021.03.03.433799 ◽

2021 ◽

Author(s):

Elisabeth R. Wilson ◽

Nichole Helton ◽

Sharon E. Heath ◽

Robert S. Fulton ◽

Christopher A. Miller ◽

...

Keyword(s):

Dna Methylation ◽

Progenitor Cells ◽

Cpg Island ◽

Three Dimensional ◽

Dna Hypermethylation ◽

Hematopoietic Stem ◽

Highly Active ◽

Highly Expressed Genes ◽

Significant Enrichment ◽

Genome Interactions

AbstractAltered DNA methylation is a common feature of acute myeloid leukemia (AML) and is thought to play a significant role in disease pathogenesis. Gain of function mutations in IDH1 or IDH2 result in widespread but highly focal regions of hypermethylation across the genome that occurs due to the production of 2-hydroxyglutarate that inhibits TET-mediated demethylation. We used whole-genome bisulfite sequencing to identify canonical regions of DNA hypermethylation that are associated specifically with IDH1 and IDH2 mutations in primary AML samples. Consistent with previous reports, IDH mutant (IDHmut) AMLs were the most hypermethylated among all mutationally-defined AML categories analyzed. We observed notable differences in the degree of hypermethylation associated with IDH mutation type, with IDH1mut AMLs having more profound hypermethylation at specific regions than IDH2mut samples. AMLs with biallelic inactivating mutations in TET2 displayed more modest DNA methylation changes compared to normal hematopoietic stem/progenitor cells, but methylation in these samples was increased in the IDHmut-specific regions, providing further support that these mutations act on the same TET-mediated demethylation pathway. Focal hypermethylated regions in IDHmut AML samples tended to occur in regions with low steady state methylation levels in normal stem/progenitor cells, which implies that both DNA methylation and demethylation pathways are active at these loci. Indeed, analysis of AML samples containing mutations in both IDH1 or IDH2 and DNMT3AR882 were less hypermethylated, providing evidence that focal IDHmut-associated hypermethylation is mediated by DNMT3A. IDHmut-specific regions of hypermethylation were largely distinct from CpG island hypermethylation, and showed a significant enrichment for putative enhancers. Analysis of three-dimensional genome interactions from primary hematopoietic cells showed that differentially methylated enhancers formed direct interactions with highly expressed genes, including MYC and ETV6. Taken together, these results suggest that focal hypermethylation in IDH-mutant AML cells occurs by disrupting the balance of DNA methylation and demethylation, which is highly active in genomic regions involved in gene regulation.

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Maintenance and Expansion of Hematopoietic Stem/Progenitor Cells in a Biomimetic Hematopoietic Cells Niche.

Blood ◽

10.1182/blood.v110.11.4115.4115 ◽

2007 ◽

Vol 110 (11) ◽

pp. 4115-4115

Author(s):

Ting Liu ◽

Jing Tan ◽

Li Hou ◽

Wentong Meng ◽

Yuchun Wang

Keyword(s):

Progenitor Cells ◽

Culture System ◽

Three Dimensional ◽

3D Culture ◽

Hematopoietic Progenitor Cells ◽

Hematopoietic Progenitor ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

3D Culture System ◽

2D System

Abstract As main hematopoietic organ, bone marrow have three dimensional microenvironment for hematopoietic stem/progenitor cells grow in, so call “hematopoietic cells niche”, which are composed by stromal cells and extracelluar matrix. The interactions of cell to cell and cell to matrix between stem/progenitor cells with hematopoietic niche are facilitated by its three dimensional conformation. The biology behaviors of hematopoietic stem cells are mediate by many signal transductions between stem/progenitor cells with their corresponding microenvironment. Now there are strong evidence from animal model study suggests that osteoblasts might play an essential role in creation of a hematopoietic stem cell niche and thereby regulation of stem cell maintenance, proliferation, and maturation. In light of the structure-function relationship of bone marrow topography, we conceived a biomimetic culture system (3D culture system) with bio-derived bone as framework, composited with human marrow mesenchymal stem cells, and induced the cells into osteoblasts to simulate the effects of hematopoietic osteobalst niche. CD 34+ cells or mononuclear cells separated from umbilical cord blood were cultured for 2∼5 weeks in the 3D culture system and also in a conventional 2D culture system as control without additional cytokine supplement. Based on our results, higher expression of extracelluar matrix and N-cadherin were observed in 3D culture system compared to 2D system. At 2 weeks culture, 3D culture system showed higher number of CD34+ cells and CD34+/CD38- cells when compared with the input (P<0.05), the increased cells were predominant CD34+/CD38-cells. Although CD34+ cells were decreased at 5 weeks culture; nevertheless, CD34+/CD38- cells were still maintained at high level. We also observed that imbedding MNCs with a higher percentage of CD34+/CD38-cells cultured in 3D system (P<0.05), which may represent a down regulation of CD38 phenotype during culture. The function of cultured cells was evaluated in colony forming unit (CFU) assay and long term culture initial cell (LTC-IC) assay. 3D system produced higher expansion of CFU progenitors than 2D system (7.13–8.89 times vs. 1.22–1.31times) after 2 weeks culture. Of note, the colony distribution of 3D system manifested higher percentage of BFU-E and CFU-GEMM, while 2D system showed mainly CFU-GM. LTC-IC represents the primitive progenitor, 3D system showed a 6.2 times increment over input after 2 weeks culture. Furthermore, it was competent to maintain the immaturation of hematopoietic progenitor cells (HPCs) over 5 weeks. This study demonstrated that our 3D culture system constructed with the bio-derived bone composited with induced osteoblast is capable to allow maintenance and expansion of primitive hematopoietic progenitor cells in vitro. It may open a new avenue to study HSCs/HPCs behaviors and to achieve sustained primitive progenitor cell expansion.

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Hypoxic Hematopoietic Stem and Progenitor Cells Reside in Structurally Diverse Perivascular Niches in the Bone Marrow,

Blood ◽

10.1182/blood.v118.21.3417.3417 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3417-3417

Author(s):

Cesar Nombela-Arrieta ◽

Gregory Pivarnik ◽

Beatrice Winkel ◽

Brendan Harley ◽

John E Mahoney ◽

...

Keyword(s):

Bone Marrow ◽

Progenitor Cells ◽

Cell Biology ◽

Laser Scanning ◽

Conflicts Of Interest ◽

Quantitative Imaging ◽

Three Dimensional ◽

Global Distribution ◽

Hematopoietic Stem ◽

Stem And Progenitor Cells

Abstract Abstract 3417 The identification of specific microenvironments, in which Hematopoietic Stem and Progenitor Cells (HSPCs), reside within the BM is a major challenge in stem cell biology. Yet the extreme rarity of HSCs, their dynamic nature, and the lack of unique specific markers to identify them, have precluded an accurate definition of HSC niches to date. Using Laser Scanning Cytometry, a powerful emerging quantitative imaging technology that enables analysis of whole femoral sections at the single cell level, we have mapped the global distribution of hematopoietic stem and progenitor cells within femoral bone marrow cavities, and analyzed their inmediate surrounding microenvironment. Systematic mapping of the global distribution of endogenous HSPC-enriched populations in the BM, revealed an accumulation of these cells inside endosteal regions (ER <100μm from inner bone surface), but not necessarily in contact with endosteal surfaces. Interestingly, the vast majority of HSPCs were found in direct association with BM micrrovessels, further supporting previous work, which suggested bone marrow endothelium as a major component of HSPC niches. By employing a novel imaging approach, we provide a three-dimensional (3D) microscopic overview of the unique BM vascular network found in endosteal zones, which contain the transition of bone-lining arterioles and capillaries to the sinusoidal network. Of note, HSPC association to vascular structures is not restricted to sinusoids. A significant fraction of HSPCs lied adjacent to non-sinusoidal endothelium. Using five-color imaging cytometry and pimonidazole incorporation, we have assessed the hypoxic state of HSPCs in different BM microenvironments. Our in situ analysis reveals that intracellular hypoxia is a hallmark of HSPCs, independent of their distance to bone surfaces, and more importantly, regardless of their perivascular localization. These studies provide unequivocal anatomical evidence for the intrinsic rather than environmental regulation of intracellular hypoxia in HSPCs and challenge the hypothesis of a “super hypoxic” HSPC niche. Disclosures: No relevant conflicts of interest to declare.

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