scholarly journals The physical microenvironment of hematopoietic stem cells and its emerging roles in engineering applications

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
Pan Zhang ◽  
Chen Zhang ◽  
Jing Li ◽  
Jiyang Han ◽  
Xiru Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Fate ◽  
Hematopoietic Stem Cell ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Stem Cell Fate ◽  
Stem Cell Engineering

AbstractStem cells are considered the fundamental underpinnings of tissue biology. The stem cell microenvironment provides factors and elements that play significant roles in controlling the cell fate direction. The bone marrow is an important environment for functional hematopoietic stem cells in adults. Remarkable progress has been achieved in the area of hematopoietic stem cell fate modulation based on the recognition of biochemical factors provided by bone marrow niches. In this review, we focus on emerging evidence that hematopoietic stem cell fate is altered in response to a variety of microenvironmental physical cues, such as geometric properties, matrix stiffness, and mechanical forces. Based on knowledge of these biophysical cues, recent developments in harnessing hematopoietic stem cell niches ex vivo are also discussed. A comprehensive understanding of cell microenvironments helps provide mechanistic insights into pathophysiological mechanisms and underlies biomaterial-based hematopoietic stem cell engineering.

Hematopoietic Stem Cell Expansion by HOXB4 Is Greatly Enhanced in p21 Deficient Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1688-1688 ◽  
Author(s):  
Noriko Miyake ◽  
Ann C.M. Brun ◽  
Mattias Magnusson ◽  
David T. Scadden ◽  
Stefan Karlsson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Ex Vivo ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract Hox transcription factors have emerged as important regulators of hematopoiesis. In particular, enforced expression of HOXB4 is a potent stimulus for murine hematopoietic stem cell (HSC) self-renewal. Murine HSCs engineered to overexpress HoxB4 expand significantly more than control cells in vivo and ex vivo while maintaining a normal differentiation program. HSCs are regulated by the cell proliferation machinery that is intrinsically controlled by cyclin-dependent kinase inhibitors such as p21Cip1/Waf1(p21) and p27Kip1 (p27). The p21 protein restricts cell cycling of the hematopoietic stem cell pool and maintains hematopoietic stem cell quiescence. In order to ask whether enhanced proliferation due to HOXB4 overexpression is mediated through suppression of p21 we overexpressed HOXB4 in hematopoietic cells from p21−/− mice. First, we investigated whether human HOXB4 enhances in vitro expansion of BM cells from p21−/− mice compared to p21+/+ mice. 5FU treated BM cells from p21−/− or p21+/+ mice were pre-stimulated with SCF, IL-6, IL-3 for 2 days followed by transduction using retroviral vector expressing HOXB4 together with GFP (MIGB4) or the control GFP vector (MIG). The cells were maintained in suspension cultures for 13 days and analyzed for GFP positive cells by flow-cytometry. Compared to MIG transduced BM cells from p21+/+ mice (MIG/p21+), the numbers of GFP positive cells were increased 1.1-fold in MIG/p21−, 3.2-fold in MIGB4/p21+ and 10.0-fold in MIGB4/p21− respectively (n=5). CFU assays were performed after 13 days of culture. The numbers of CFU were increased 4.8-fold in MIG/p21−, 19.5-fold in MIG/p21+ and 33.9 -fold in MIGB4/p21− respectively. Next, we evaluated level of HSCs expansion by bone marrow repopulation assays. After 12-days of culture, 1.5 x 105 MIGB4 or MIG-transduced cells (Ly5.2) were transplanted into lethally irradiated mice in combination with 8 x 105 fresh Ly5.1 bone marrow cells. Sixteen weeks after transplantation, no Ly5.2 cells could be detected in MIG/p21+ or MIG/p21− transplanted mice (n=6). In contrast, Ly5.2 positive cells were detected in both MIGB4/p21+/+ and MIGB4/p21−/− cells. The % of Ly5.2 positive cells in MIGB4/p21− transplanted mice was 9.9-fold higher than MIGB4/p21+ transplanted mice. (38.4 % vs 3.9 %, P<0.02, n=5). These Ly5.2 positive cells differentiated into all lineages, as determined by proportions of Mac-1, B-220, CD3 and Ter119 positive populations. Currently, we are enumerating the expansion of HOXB4 transduced HSCs in p21 deficient BM cells using the CRU assay. Our findings suggest that HOXB4 increases the self-renewal of hematopoietic stem cells by a mechanism that is independent of p21. In addition, the findings demonstrate that deficiency of p21 in combination with enforced expression of HOXB4 can be used to rapidly and effectively expand hematopoietic stem cells.

scholarly journals Osteoblast ablation reduces normal long-term hematopoietic stem cell self-renewal but accelerates leukemia development

Blood ◽  
2015 ◽  
Vol 125 (17) ◽  
pp. 2678-2688 ◽  
Author(s):  
Marisa Bowers ◽  
Bin Zhang ◽  
Yinwei Ho ◽  
Puneet Agarwal ◽  
Ching-Cheng Chen ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Key Points ◽  
Leukemia Development

Key Points Bone marrow OB ablation leads to reduced quiescence, long-term engraftment, and self-renewal capacity of hematopoietic stem cells. Significantly accelerated leukemia development and reduced survival are seen in transgenic BCR-ABL mice following OB ablation.

scholarly journals Phenotypic analysis of mouse hematopoietic stem cells shows a Thy-1- negative subset

Blood ◽  
1992 ◽  
Vol 80 (8) ◽  
pp. 1957-1964 ◽  
Author(s):  
GJ Spangrude ◽  
DM Brooks
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Bone Marrow Cells ◽  
Cell Activity ◽  
Mouse Strains ◽  
Hematopoietic Stem ◽  
Stem Cell Activity

Mouse hematopoietic stem cells can be identified and enriched from populations of normal bone marrow cells by immunofluorescent labeling of cell surface molecules followed by flow cytometric separation. We show here that the majority of hematopoietic stem cell activity, as defined by long-term competitive repopulation of irradiated animals and by a secondary transplant assay for spleen colony-forming units (CFU- S), could be localized in Ly-6b haplotype mice to a fraction of bone marrow cells that expresses the Ly-6A/E (Sca-1) molecule. Further, an analysis of hematopoietic stem cell activity in bone marrow of mouse strains expressing the Thy-1.1 allele indicated that the vast majority of activity was included in the Thy-1low population. In contrast, hematopoietic stem cell activity found in the bone marrow of Thy-1.2 genotype mouse strains was recovered in both the Thy-1neg and the Thy- 1low populations. However, similar to Thy-1.1 strains, most activity was localized to the Ly-6A/E+ population of cells. The difference in Thy-1 phenotype of hematopoietic stem cell activity apparent between Thy-1.1- and Thy-1.2-expressing mouse strains was not caused by differences in the staining intensity of monoclonal antibodies (MoAbs) specific for the Thy-1 alleles. Furthermore, an antiframework MoAb that stains both alleles of Thy-1 separated hematopoietic stem cell activity from mice expressing the two alleles in the same manner as did allele- specific MoAb. The results of this study show that Thy-1 expression is not an invariant characteristic of mouse hematopoietic stem cells, and that mice expressing the Thy-1.1 allele are unique in that hematopoietic stem cell activity is found exclusively in the Thy-1low population.

DNA Microarray Analysis of GPI-Negative Hematopoietic Stem Cells in Paroxysmal Nocturnal Hemoglobinuria Patients.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1048-1048
Author(s):  
Kazuhiko Ikeda ◽  
Tsutomu Shichishima ◽  
Yoshihiro Yamashita ◽  
Yukio Maruyama ◽  
Hiroyuki Mano
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Paroxysmal Nocturnal Hemoglobinuria ◽  
Bone Marrow Failure ◽  
Data Set ◽  
Hematopoietic Stem ◽  
Pnh Clone

Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired clonal hematological disorder which is manifested by complement-mediated hemolysis, venous thrombosis, and bone marrow failure. Deficiencies of glycosylphosphatidylinositol (GPI)-anchored proteins, due to mutations in the phosphatidylinositol glycan-class A (PIG-A) gene, contribute to complement-mediated hemolysis and affect all hematopoietic lineages in PNH. However, it is unclear how a PNH clone with a PIG-A gene mutation expands in bone marrow. Although some genes, including the Wilms’ tumor gene (Shichishima et al, Blood, 2002), the early growth response gene, anti-apoptosis genes, and the gene localized at breakpoints of chromosome 12, have been reported as candidate genes that may associate with proliferations of a GPI-negative PNH clone, previous studies were not intended for hematopoietic stem cell, indicating that the differences in gene expressions between GPI-negative PNH clones and GPI-positive cells from PNH patients remain unclear at the level of hematopoietic stem cell. To identify genes contributing to the expansion of a PNH clone, here we compared the gene expression profiles between GPI-negative and GPI-positive fractions among AC133-positive hematopoietic stem cells (HSCs). By using the FACSVantage (Becton Dickinson, San Jose, CA) cell sorting system, both of CD59+AC133+ and CD59− AC133+ cells were purified from bone marrow mononuclear cells obtained from 11 individuals with PNH. Total RNA was isolated from each specimen with the use of RNeasy Mini column (Qiagen, Valencia, CA). The mRNA fractions were amplified, and were used to generate biotin-labeled cDNAs by the Ovation Biotin system (NuGEN Technologies, San Carlos, CA). The resultant cDNAs were hybridized with a high-density oligonucleotide microarray (HGU133A; Affymetrix, Santa Clara, CA). A total of &gt;22,000 probe sets (corresponding to &gt;14,000 human genes) were assayed in each experiment, and thier expression intensities were analyzed by GeneSpring 7.0 software (Silicon Genetics, Redwood, CA). Comparison between CD59-negative and CD59-positive HSCs has identified a number of genes, expression level of which was statistically different (t-test, P &lt;0.001) between the two fractions. Interestingly, one of the CD59− -specific genes isolated in our data set turned out to encode a key component of the proteasome complex. On the other hand, a set of transcriptional factors were specifically silenced in the CD59− HSCs. These data indicate that affected CD59-negative stem cells have a specific molecular signature which is distinct from that for the differentiation level-matched normal HSCs. Our data should pave a way toward the molecular understanding of PNH.

Growth Factor Independence 1 b (Gfi1b) as a New Regulator of Hematopoietic Stem Cell Fate

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 837-837
Author(s):  
Cyrus Khandanpour ◽  
Lothar Vassen ◽  
Marie-Claude Gaudreau ◽  
Christian Kosan ◽  
Tarik Moroy
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Fold Increase ◽  
Surface Proteins ◽  
Expression Array ◽  
Hematopoietic Stem

Abstract Abstract 837 Donor matched transplantation of bone marrow or hematopoietic stem cells (HSCs) are widely used to treat hematological malignancies, but are associated with high mortality. Methods for expansion of HSC numbers and their mobilization into the bloodstream of a donor could significantly improve therapy. We show here that the zinc finger transcriptional repressor Gfi1b is highly expressed in hematopoietic stem cells (defined as CD 150+, CD 48-, Lin-, Sca1+ and c-kit+) cells and is down-regulated more than 10 fold upon differentiation into multipotential progenitors (defined as CD 150+ or CD150-, CD 48+, Lin-, Sca1+ and c-kit+). Constitutive germline deletion of Gfi1b is lethal at midgestation due to impaired development of erythrocytes and megakaryocytes. We have therefore developed a conditional knock-out of Gfi1b to study its role specifically in the adult hematopoietic system. Deletion of Gfi1b leads to a 30-fold increase of HSC numbers in bone marrow and around a100 fold increase in spleen and peripheral blood. This was due to a higher rate of HSCs undergoing cell cycling. Concomitantly, the number of quiescent HSCs was reduced 5–6 times. We then performed an gene expression array of wt and Gfi1b deficient HSCs and observed that loss of Gfi1b leads to an altered RNA expression of integrins and adhesion molecules, for instance CXCR4, VCAM-1 and Tenascin C, which usually retain HSCs in a dormant state in the endosteal niche. These changes were also confirmed on protein level. Finally, we could observe a higher levels of Reactive Oxygen Species (ROS) in the Gfi1b deficient HSCs compared to wt HSCs. We verified whether elevated level of ROS are causative for the expansion of HSCs and noticed that application of N-Acetyl-Cystein, which counteracts the effects of ROS, limits significantly the expansion of HSCs, underscoring the important role of ROS in the expansion of Gfi1b deficient HSCs. Despite markedly increased proliferation, Gfi1b-/- HSCs can reconstitute lymphoid and myeloid lineages to the same extent as wt HSCs when transplanted in competition with wt HSCs. Furthermore, Gfi1b deficient HSCs also feature an expansion after transplantation and expand 5–10 fold more than wt HSC when transplanted initially in equal numbers with wt HSCs. It is possible that lower expression of CXCR4, VCAM-1 and other surface proteins leads to release and egression of Gfi1b deficient HSCs from the hypoxic endosteal stem cell niche and exposes the HSCs to more oxygen which in turn increases ROS levels. Elevated ROS could promote entry of Gfi1b-/- HSCs into cell cycle. In conclusion Gfi1b regulates HSC dormancy, pool size and potentially also the egress and mobilization of HSCs and might offer a new therapeutic approach to improve human HSC transplantation. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Human hematopoietic microenvironments

PLoS ONE ◽  
2021 ◽  
Vol 16 (4) ◽  
pp. e0250081
Author(s):  
Helene Bjoerg Kristensen ◽  
Thomas Levin Andersen ◽  
Andrea Patriarca ◽  
Klaus Kallenbach ◽  
Birgit MacDonald ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Treatment Modalities ◽  
Computer Assisted ◽  
Hematopoietic Stem ◽  
Multipotent Progenitors ◽  
Murine Hematopoietic Stem Cell

Dormancy of hematopoietic stem cells and formation of progenitors are directed by signals that come from the bone marrow microenvironment. Considerable knowledge has been gained on the murine hematopoietic stem cell microenvironment, while less so on the murine progenitor microenvironment and even less so on these microenvironments in humans. Characterization of these microenvironments is decisive for understanding hematopoiesis and finding new treatment modalities against bone marrow malignancies in the clinic. However, it is equally challenging, because hematopoietic stem cells are difficult to detect in the complex bone marrow landscape. In the present study we are characterizing the human hematopoietic stem cell and progenitor microenvironment. We obtained three adjacent bone marrow sections from ten healthy volunteers. One was used to identify a population of CD34+/CD38- “hematopoietic stem cells and multipotent progenitors” and a population of CD34+/CD38+ “progenitors” based on immunofluorescence pattern/intensity and cellular morphology. The other two were immunostained respectively for CD34/CD56 and for CD34/SMA. Using the combined information we performed a non-computer-assisted quantification of nine bone marrow components (adipocytes, megakaryocytes, bone surfaces, four different vessel types (arteries, capillaries, sinusoids and collecting sinuses), other “hematopoietic stem cells and multipotent progenitors” and other “progenitors”) within 30 μm of “hematopoietic stem cells and multipotent progenitors”, “progenitors”, and “random cell profiles”. We show that the microenvironment of the “hematopoietic stem cells and multipotent progenitors” is significantly enriched in sinusoids and megakaryocytes, while the microenvironment of the “progenitors” is significantly enriched in capillaries, other “progenitors”, bone surfaces and arteries.

Genetic Uncoupling of the Regulation of Hematopoietic Stem Cell Quiescence and Self-Renewal.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1347-1347
Author(s):  
Yan Liu ◽  
Yasuhiko Miyata ◽  
Goro Sashida ◽  
Anthony Debalsio ◽  
Yuhui Liu ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Hematopoietic Stem Cell ◽  
Quiescent State ◽  
Double Knockout ◽  
Ki 67 ◽  
Self Renewal ◽  
Hematopoietic Stem

Abstract It is usually stated that HSCs must choose to either self-renew or to differentiate and lose some of their multi potentiality. Recently, we demonstrated that MEF, an ETS family of transcription factor, played an important role in regulating HSC quiescence, illustrating a third choice for the HSC, namely to make an “active” choice and remain quiescent, without undergoing either self-renewal, or differentiation. MEF null HSCs are more quiescent than normal HSCs. In addition, MEF null mice exhibit greater numbers of hematopoietic stem cells and show resistance to chemotherapy and radiation. Little is known about the regulation of self-renewal vs. quiescence of HSCs, however the cdk inhibitor p21 has been implicated in regulating both HSC quiescence and proliferation. In the absence of p21, hematopoietic stem cell numbers are reported to be increased, but so is proliferation, leading to stem cell exhaustion. This implies that while p21 may maintain HSCs in their quiescent state, MEF functions to facilitate the entry of quiescent HSCs into the cycle, To investigate the potential opposing roles of MEF and p21 in HSC quiescence and self-renewal and to test whether the quiescent state of MEF null HSCs is dependent on the presence of p21, we have generated MEF / p21 double-knockout (DKO) mice. These mice are viable and born at normal mendelian frequency. MEF / p21 DKO mice have a higher than normal proportion of HSCs in the G0 phase, based on Pyronin Y/Hoechst staining and staining for the proliferation antigen Ki-67. Thus, the increased quiescence is not dependent on the presence of p21. However, by measuring LSK cells, we have observed a normal number of HSCs in the bone marrow of MEF / p21 DKO mice, in contrast to the increased number of HSCs in the bone marrow of MEF null mice. This suggests that the increased number of hematopoietic stem cells in MEF null mice is dependent on p21. Ongoing studies will further address the unique mechanisms that control HSC vs. stem cell expansion.

Foxo3 Modulation of ATM and Oxidative Stress Mediates Distinct Functions in the Regulation of Hematopoietic Stem and Progenitor Cell Fate.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 1272-1272 ◽  
Author(s):  
Safak Yalcin ◽  
Julia P. Luciano ◽  
Xin Zhang ◽  
Cecile Vercherat ◽  
Reshma Taneja ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Stem Cells ◽  
Cell Fate ◽  
Hematopoietic Stem Cell ◽  
Progenitor Cell ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Atm Gene

Abstract FOXO transcription factors are required for hematopoietic stem cell self renewal. In this study, we demonstrate that Foxo3 plays a specific and essential function in the regulation of both hematopoietic stem and progenitor cell fate. Foxo3 null mice display a myeloproliferative syndrome characterized by splenomegaly, a major expansion of the myeloid compartment in the blood, bone marrow and spleen, cytokine hypersensitivity of progenitors in hematopoietic organs and associated with the repression of the B lymphoid compartment. In addition, loss of Foxo3 leads to significant defects in hematopoietic stem cell numbers and activity. In particular, the numbers of long-term culture initiating cells (LTC-IC) was significantly reduced and the ability to repopulate lethally irradiated mice was severely compromised in Foxo3-defcient mice. This effect was mediated at least partially by enhanced accumulation of reactive oxygen species (ROS) in Foxo3-deficient hematopoietic stem cells as demonstrated by reduced QRT-PCR expression of several anti-oxidant enzymes leading to accumulation of ROS, (as measured by chloromethyl,dichlorodihydrofluorescein diacetate assay) in Foxo3 null hematopoietic stem cells, and in vitro and in vivo rescue of the phenotype using ROS scavengers. Furthermore, we demonstrate that while ROS accumulation results in suppression of Foxo3 null hematopoietic stem cell compartment, it enhances the activity of multipotential cells. By measuring RNA versus DNA content, and BrdU uptake, we determined that Foxo3-deficient hematopoietic stem cells exit quiescence (G0) and are impaired in their cycling at the G2/M phase. In particular, we identified ROS activation of p19ARF/p53 pathway and ROS-independent modulation of ataxia telangiectasia mutated (ATM) gene and p16INK4a, as major contributors to the interference with Foxo3-deficient hematopoietic stem cell self renewal and cycling. Loss of ATM has been shown to lead to hematopoietic stem cell deficiency. Importantly, we show that ATM gene expression is significantly suppressed in Foxo3-deficient hematopoietic stem cells suggesting that ATM lies downstream of Foxo3. Retroviral expression of a constitutively active form of Foxo3 in Foxo3-deficient bone marrow mononuclear cells enhances significantly the ATM expression suggesting that Foxo3 regulate expression of ATM gene. These combined findings suggest that Foxo3 functions in a tumor suppressor network to protect hematopoietic stem cells against deleterious effects of oxidative damage, to maintain hematopoietic lineage fate determination and to restrict the activity of long term repopulating hematopoietic stem cells. These findings provide insights into the mechanisms underlying hematopoietic stem cell fate.

Effect of Umbilical Cord Blood Hematopoietic Stem Cells on Formation of Angiogenic Structures.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 4054-4054
Author(s):  
Aaron Victor ◽  
Mary J. Laughlin ◽  
Marcie R. Finney ◽  
Nicholas J. Greco
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Coronary Artery ◽  
Hematopoietic Stem Cells ◽  
Cord Blood ◽  
Hematopoietic Stem Cell ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Stem

Abstract There is a significant unmet need for novel therapeutic treatments for patients presenting with chronic ischemic conditions such as coronary artery disease and diabetes. Revascularization measures, such as infusions with endothelial progenitor cells (EPC) characterized by the expression of early hematopoietic stem cell markers, hold significant potential in treating these patients. Pre-clinical and clinical studies using transplanted EPC to restore blood flow and improve cardiac function in animal models of ischemia have proven effective. Recent studies have used bone marrow mononuclear cells while some more recent studies have focused on enriched stem cell treatments, such as purified bone marrow hematopoietic stem cell (HSC) CD34+/133+ cell populations, in patients with coronary artery ischemia. In this study, the hypothesis to be tested was that umbilical cord blood-derived hematopoietic stem cells (CD34+/CD133+) cells may augment the formation and stability of angiogenic networks of cord-like structures derived from umbilical vein endothelial cells (HUVEC) cultured in growth factor-reduced Matrigel (GFR MG) assays. Umbilical cord blood MNC were isolated with ficoll and separated into HSC CD34+/133+ and CD34−/133− fractions. Positive fractions were flow cytometry, sorted for HSC, and stained with the lipophilic fluorescent red dye CM-DiI and the HUVEC were stained with the lipophilic fluorescent green dye Oregon Green. HUVEC alone or HSC and HUVEC were then co-cultured under hypoxic conditions (1% O2) on the GFR MG in 96 well plates. Cells were photographed with a fluorescent microscope at 16, 48, and 72 hours. Transwell experiments (0.4μm pores) were also performed with HSC CD34+/133+ and CD34−/133− fractions prepared and suspended in transwells above HUVEC plated on GFR MG on bottom wells. The presence of both HSC CD34+/133+ and CD34−/133− fractions increased the numbers of nodes (branch points of structures) and allowed the structures to persist when observed over three days (a representative experiment of N =3) (Table): Day 1 Day 1 Day 2 Day 2 Day 3 Day 3 Node # % Total Node # % Total Node # % Total HUVEC 11.6 ± 4.9 100 1.3 ± 1.2 9.2 0.33 ± 0.58 2.2 HUVEC + HSC CD34+/133+ 17.3 ± 9.2 100 6.3 ± 4.5 35.3 4.7 ± 5.5 21.4 HUVEC + HSC CD34−/133− 34 ± 13.2 100 19.7 ± 2.5 61.6 10 ± 3.6 29.8 The HSC CD34−/133− fraction resulted in a greater increase in node formation than the HSC CD34+/133+ and both fractions stimulated significant persistence in formed structures. In addition, CM-Dil labeled cells were localized at nodes points. Results with the transwell assay demonstrated that when either HSC CD34+/133+ or CD34−/133− fractions were suspended above HUVEC, augmentation of the formation of cord-like structures was not observed. In summary, both umbilical cord blood-derived HSC CD34+/133+ and CD34−/133− fractions possess properties that augment the formation of angiogenic structures. We observed that the number of nodes are greater in the presence of both HSC CD34+/133+ and CD34−/133− fractions than with HUVEC alone. The transwell experiment suggested that cell-to-cell interactions are necessary for augmentation of the cord structures. In future studies, we will address the mechanism of intercellular interactions that result in the augmentation of cord-like structures and which particular subpopulations within cord blood, both from HSC CD34+/133+ and CD34−/133− fractions are required for augmentation of structure formation.

Down-Regulation of Homeobox Gene Ventx Promotes Expansion of Human Bone Marrow Hematopoietic Stem Cells (HSC)

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 1272-1272
Author(s):  
Hong (Jenny) Gao ◽  
Xiaoming Wu ◽  
Yan Sun ◽  
Jiayun Lu ◽  
Leslie E Silberstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Hematopoietic Stem Cells ◽  
Cell Fate ◽  
Conflicts Of Interest ◽  
Ex Vivo ◽  
Great Promise ◽  
Down Regulation ◽  
Hematopoietic Stem ◽  
Proliferation And Differentiation

Abstract Abstract 1272 Hematopoietic stem cells (HSC) give rise to mature cells of all lineages of blood and immune systems. HSC transplantation has shown great promise in the treatment of malignancies, reconstitution of hematopoietic systems and HSC-based gene therapy. Cell intrinsic factors/pathways have been the targets of intensive investigation for its potential application in HSC expansion. Over the past decades, several critical cell fate determination pathways, such as the Wnt signaling pathways and senescence pathways have been implicated in the proliferation and differentiation of HSC. Moreover, overexpression of HoxB4 and BMI1 was found to be able to expand human HSC 2∼3 folds. Nevertheless, the regulatory mechanisms of HSC proliferation and differentiation remain incompletely understood and safe and efficacious expansion of human HSC remains as a fundamental challenge that limits the clinical application of HSC-based therapy. VentX is a human homologue of the Xenopus homeobox protein Xom of the BMP4 signaling pathway. Using Xenopus model and methods of reverse genetics, our recent work showed that VentX is a LEF/TCF associated Wnt repressor and an activator of senescence pathways. VentX expression is highly regulated and restricted in hematopoietic cells and serves a major regulator of hematopoietic cell differentiation. To explore the potential role of VentX in proliferation and differentiation of HSC during hematopoiesis, we quantified VentX expression during hematopoiesis, using qRT-PCR methods and examined the effects of altered VentX expression on HSC properties in vitro and in vivo. Our data showed that VentX expression is significantly up-regulated during oncogenesis of hematopioetic cells. We demonstrated that lentiviral knockdown of VentX allowed for more than 5 fold ex vivo expansion of human HSC with balanced lineage development. Importantly, transient knockdown of VentX by siRNA also led to expansion of HSC. The effect of VentX down-regulation on the expansion of human HSC was also demonstrated by enhanced engraftment in the SCID/NODγ2null mouse model. Consistent with its role as a novel regulator of HSC, overexpression of VentX significantly inhibited clonal genesis of HSC. Mechanistically, we demonstrated that VentX controls the expression of cell cycle regulators downstream of the Wnt and senescence pathways, such as the C-myc, CyclinD1 and p21. In summary, using methods of reverse genetic and developmental modeling, we identified VentX as a novel regulator for expansion of human BM HSC. The results of our investigations provide novel insight in regulating HSC proliferation and differentiation. In addition, the findings that transient down-regulation of VentX by SiRNA lead to efficient expansion of bone marrow HSC suggests that VentX may serve as a novel target for safe expansion of HSC for its potential clinical applications. Disclosures: No relevant conflicts of interest to declare.

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