High Proliferative Potential Colony-forming Cells Research Articles

Targeted Gene Correction in Osteopetrotic-Induced Pluripotent Stem Cells for the Generation of Functional Osteoclasts

SummaryAutosomal recessive osteopetrosis is a human bone disease mainly caused by TCIRG1 gene mutations that prevent osteoclasts resorbing activity, recapitulated by the oc/oc mouse model. Bone marrow transplantation is the only available treatment, limited by the need for a matched donor. The use of induced pluripotent stem cells (iPSCs) as an unlimited source of autologous cells to generate gene corrected osteoclasts might represent a powerful alternative. We generated iPSCs from oc/oc mice, corrected the mutation using a BAC carrying the entire Tcirg1 gene locus as a template for homologous recombination, and induced hematopoietic differentiation. Similarly to physiologic fetal hematopoiesis, iPSC-derived CD41+ cells gradually gave rise to CD45+ cells, which comprised both mature myeloid cells and high proliferative potential colony-forming cells. Finally, we differentiated the gene corrected iPSC-derived myeloid cells into osteoclasts with rescued bone resorbing activity. These results are promising for a future translation into the human clinical setting.

EMP Emergence from Hemogenic Endothelium in the Mammalian Yolk Sac Is Independent of Flow and Arterial Identity, but Is Regulated By Canonical Wnt Signaling

Hematopoietic stem cells (HSCs) emerge from arterial vessels of the mouse embryo through a Runx1-dependent process of endothelial-to-hematopoietic transition beginning at embryonic day 10.5 (E10.5). This arterial endothelial-to-hematopoietic transition is known to require embryonic circulation as well as beta-catenin signaling within the endothelial precursor, known as hemogenic endothelium. However, embryonic survival is dependent on the earlier emergence of a robust wave of yolk sac-derived definitive erythro-myeloid progenitors (EMPs), which have unilineage as well as multilineage potential, including high-proliferative potential colony forming cell (HPP-CFC) potential (Palis et al., PNAS, 2001). Like HSCs, EMP specification is dependent on Runx1, suggesting that they also emerge from a hemogenic endothelial precursor. However, the spatial localization of EMPs in the yolk sac and the mechanisms governing their emergence are not well understood. To visualize emerging EMPs in the yolk sac, we performed whole-mount immunohistochemistry for Kit, which we have demonstrated to contain nearly all EMP potential at E9.5. Kit+ cells coexpress Runx1 and CD31, and a subset have a polygonal/endothelial morphology, appear integrated into the vascular network, and are associated with rounded Kit+ cells in clusters, features consistent with an endothelial-to-hematopoietic transition. However, unlike HSCs, which emerge from major embryonic arteries, clusters of EMPs are located in larger and smaller caliber vessels in branches of both the arterial and venous vasculature, which is spatially organized within the yolk sac. To determine if EMP emergence from the vasculature is dependent on embryonic blood flow, which is required for HSC emergence, we analyzed the yolk sacs of Ncx1-null embryos, which fail to initiate heart contractions and subsequently lack embryonic circulation. Despite the lack of vascular remodeling in these circulation-deficient yolk sacs, Ncx1-null EMPs displayed normal cluster morphology, including both polygonal and rounded kit+ cells, indicating the endothelial-to-hematopoietic transition can occur without the mechanical influence of blood flow.To address whether EMP formation is responsive to other developmental signals, we utilized a yolk sac explant culture to evaluate the propensity of hemogenic endothelial cells to commit to hematopoiesis ex vivo. Culture of intact E8.5 yolk sacs for 48 hours with the canonical Wnt ligand Wnt3a resulted in an increase in both day 6-7 colony forming cells and day 13-14 HPP-CFC when compared with control yolk sacs. Preliminary treatment with Dkk1 alone did not adversely affect colony-forming activity when compared with untreated yolk sacs, and potentiation of endogenous canonical Wnt signaling with HLY78 did not augment colony production, suggesting that low levels of endogenous Wnt ligands are produced ex vivo. Despite the positive effect of Wnt3a on whole yolk sacs, treatment of isolated E9.5 Kit+CD41+CD16/32+ EMPs with Wnt3a did not increase colony formation, suggesting that Wnt signaling augments progenitor production at, or prior to, the hemogenic endothelial stage. Preliminary results utilizing imaging flow cytometry demonstrated increased beta-catenin intensity within the nuclear region in E9.5 Kit+VE-Cadherin/AA4.1+ endothelium following Wnt3a treatment, suggesting that hemogenic endothelial cells in the yolk sac are Wnt responsive. Consistent with this finding, in vitro Wnt3a treatment on primary E8.5-9.5 VE-Cadherin/AA4.1+CD16/32- endothelial cells resulted in upregulation of the beta-catenin target gene Axin2. To address whether Wnt signaling is endogenously active in vivo, we analyzed E8.5-E9 yolk sacs of BAT-gal reporter mice (Maretto et al., PNAS, 2003), and visualized a subset of cells with endothelial morphology expressing LacZ. Taken together, these data support the concept that EMPs, like HSCs, emerge from hemogenic endothelium. Surprisingly, this earlier endothelial-to-hematopoietic transition in the yolk sac is not dependent on blood flow or an arterial identity. However, similar to HSC emergence, EMP emergence from hemogenic endothelium is positively regulated by canonical Wnt signaling. These data highlight the presence of spatially, temporally, and functionally heterogeneous populations of hemogenic endothelium in the mammalian conceptus. DisclosuresNo relevant conflicts of interest to declare.

The adult livers of immunodeficient mice support human hematopoiesis: evidence for a hepatic mast cell population that develops early in human ontogeny.

The liver plays a vital role in hematopoiesis during mammalian prenatal development but its hematopoietic output declines during the perinatal period. Nonetheless, hepatic hematopoiesis is believed to persist into adulthood. We sought to model human adult-liver hematopoiesis by transplantation of fetal and neonatal hematopoietic stem cells (HSCs) into adult immunodeficient mice. Livers were found to be engrafted with human cells consisting primarily of monocytes and B-cells with lesser contributions by erythrocytes, T-cells, NK-cells and mast-cells. A resident population of CD117++CD203c+ mast cells was also documented in human midgestation liver, indicating that these cells comprise part of the liver's resident immune cell repertoire throughout human ontogeny. The murine liver was shown to support human multilineage hematopoiesis up to 321 days after transplant. Evidence of murine hepatic hematopoiesis was also found in common mouse strains as old as 2 years. Human HSC engraftment of the murine liver was demonstrated by detection of high proliferative-potential colony-forming cells in clonal cultures, observation of CD38−CD34++ and CD133+CD34++ cells by flow cytometry, and hematopoietic reconstitution of secondary transplant recipients of chimeric liver cells. Additionally, chimeric mice with both hematopoietic and endothelial reconstitution were generated by intrasplenic injection of immunodeficient mice with liver specific expression of the urokinase-type plasminogen activator (uPA) transgene. In conclusion, the murine liver is shown to be a hematopoietic organ throughout adult life that can also support human hematopoiesis in severely immunodeficient strains. Further humanization of the murine liver can be achieved in mice harboring an uPA transgene, which support engraftment of non-hematopoietic cells types. Thus, offering a model system to study the interaction of diverse human liver cell types that regulate hematopoiesis and immune function in the liver.

High proliferative potential endothelial colony-forming cells contribute to hypoxia-induced pulmonary artery vasa vasorum neovascularization

Angiogenic expansion of the vasa vasorum (VV) is an important contributor to pulmonary vascular remodeling in the pathogenesis of pulmonary hypertension (PH). High proliferative potential endothelial progenitor-like cells have been described in vascular remodeling and angiogenesis in both systemic and pulmonary circulations. However, their role in hypoxia-induced pulmonary artery (PA) VV expansion in PH is not known. We hypothesized that profound PA VV neovascularization observed in a neonatal calf model of hypoxia-induced PH is due to increased numbers of subsets of high proliferative cells within the PA adventitial VV endothelial cells (VVEC). Using a single cell clonogenic assay, we found that high proliferative potential colony-forming cells (HPP-CFC) comprise a markedly higher percentage in VVEC populations isolated from the PA of hypoxic (VVEC-Hx) compared with control (VVEC-Co) calves. VVEC-Hx populations that comprised higher numbers of HPP-CFC also demonstrated markedly higher expression levels of CD31, CD105, and c-kit than VVEC-Co. In addition, significantly higher expression of CD31, CD105, and c-kit was observed in HPP-CFC vs. the VVEC of the control but not of hypoxic animals. HPP-CFC exhibited migratory and tube formation capabilities, two important attributes of angiogenic phenotype. Furthermore, HPP-CFC-Co and some HPP-CFC-Hx exhibited elevated telomerase activity, consistent with their high replicative potential, whereas a number of HPP-CFC-Hx exhibited impaired telomerase activity, suggestive of their senescence state. In conclusion, our data suggest that hypoxia-induced VV expansion involves an emergence of HPP-CFC populations of a distinct phenotype with increased angiogenic capabilities. These cells may serve as a potential target for regulating VVEC neovascularization.

A Chemical Approach for Ex Vivo Expansion of Human Hematopoietic Stem Cells

Abstract 481Hematopoietic stem cells (HSCs) have been applied to treatment of a wide variety of malignant and non-malignant blood disorders. Because of low risk of graft versus-host disease, use of human cord blood (hCB) as a source of HSCs continues to increase. However, wide application of hCB is limited by the relatively low number of HSCs obtained from a single CB unit. Numerous attempts have been made to expand hCB HSCs in cultures to acquire a sufficient number of transplantable HSCs. Chemical approaches have especially showed promise for ex vivo expansion of HSCs. As an example, Boitano et al. reported that SR1, a chemically synthesized purine derivative, induced hCB HSC expansion by antagonizing the aryl hydrocarbon receptor.In this study, we initially screened several tens of thousands of small-molecule compounds for the capability to induce STAT5 activation but obtained active compounds showed no positive effect on proliferation of hCB CD34+ hematopoietic stem and progenitor cells (HSPCs). However, a derivative of the STAT5 activating compounds proved to increase the population of CD34+CD38– cells when added to hCB CD34+ cell culture. Thus, we optimized the chemical structure of the compound and identified MISK303 as the most potent compound. We next evaluated the effects of MISK303 on the ex vivo expansion of hCB CD34+ cells in detail; hCB CD34+ cells were cultured in serum-free medium supplemented with MISK303 in addition to rhTPO and rhSCF for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assays. Although the total and CD34+ cell number when cultured with MISK303 was comparable to that cultured with DMSO (vehicle), the cultures with MISK303 exhibited a >2-fold increase in the number of CD34+CD38– cells and contained 1.7-fold more high proliferative potential colony-forming cells (HPP-CFCs; >1mm in diameter) compared to those with DMSO. Correspondingly, in the NOD/SCID repopulation assay, hCB CD34+ cells cultured with MISK303 for 7 days displayed up to 2-fold higher levels of engraftment compared to control cultures and uncultured HSPCs. These data suggest that MISK303 promotes the net expansion of hematopoietic stem and progenitor cells. Furthermore, combination of SR1 and MISK303 had additive effects; cell cultures with SR1 and MISK303 showed a >4-fold increase in the number of CD34+CD38– cells. In accordance with the data, MISK303 had no antagonizing activity against the aryl hydrocarbon receptor. We also observed that a chemically-synthesized TPO receptor (c-mpl) agonist could be substituted with rhTPO and provided better expansion of CD34+CD38– cells in combination with SR1 and MISK303.To investigate the molecular mechanism by which MISK303 increases HSPCs, we first conducted gene expression profiling of CD34+ cells cultured with MK303 by DNA microarray analysis. Treatment of CD34+ cells with MISK303 for 24 hours led to up-regulation of 343-genes (>2-fold) and down-regulation of 112 genes (<0.5-fold). We also profiled effect of MISK303 over 311 protein kinases and found no inhibitory activity. We are now validating the expression of differentially expressed genes identified by DNA microarray analysis.In conclusion, we have conducted high-throughput screening and identified MK303, a novel chemically synthesized small-molecule compound, which induces the expansion of human HSCs ex vivo. The approach using MISK303 will facilitate the development of novel and efficient technologies for hematopoietic stem cell and gene therapies. Disclosures:No relevant conflicts of interest to declare.

Prostaglandin E2 Signaling Through the EP4 Receptor Regulates the Proliferation of Hematopoietic Stem/Progenitor Cells Under Stress Conditions

Abstract 2377Hematopoietic stem cells (HSCs) maintain the balance between self-renewal and differentiation in order to replenish all blood cell lineages throughout the lifespan of the individual. Signaling molecules produced by various niche cells regulate the function and fate of HSCs. Prostaglandin E2 (PGE2) is a known mediator of various pathological states and physiological systems. In the hematopoietic system, PGE2 signaling regulates HSC development and enhances repopulation activity through its interactions with the Wnt/β-catenin pathway. However, it is not been clear which receptor is responsible for PGE2 signaling in adult HSCs.Here we analyzed the function of PGE2 receptors, EP2 and EP4, in the regulation of adult bone marrow (BM) hematopoietic stem/progenitor cells (HSPCs). Quantitative real time PCR (Q-PCR) analysis revealed that EP4 was highly expressed in Lin−Sca-1+c-Kit+ (LSK) fractions compared to Lin− or Lin+ fractions. On the other hand, EP2 mRNA expression was detected at a higher level in LSKCD34+Flt3+ fractions than in other fractions. Digital PCR analysis showed that EP2 transcripts were present in much lower numbers than EP4 transcripts in HSCs.We next analyzed the effects of short-term treatment with an EP2 or EP4 agonist on the colony forming activity of LSK cells. Although the EP2 agonist did not affect the colony formation activity of LSK cells, the EP4 agonist increased the number of colony forming unit in culture (CFU-C) and high proliferative potential-colony forming cells (HPP-CFC) in a dose dependent manner, indicating that PGE2/EP4 signaling affects the proliferation of HSPCs. A knockdown of EP4 reduced the engraftment capacity of LSK cells, suggesting that PGE2/EP4 signaling contributes to the BM repopulation activity of HSPCs.Next, we examined the effect of PGE2/EP4 signaling on the recovery of HSCs after myelosuppression. 5-FU-injected mice were treated with PGE2 and the proportion of LSK cells in the mice at various days after 5-FU injection was examined. The frequency of LSK cells at the early phase of post 5-FU treatment was comparable in control and PGE2-treated mice. In the later phase of post 5-FU treatment, mice treated with PGE2 showed a higher frequency of LSK cells, and the LSK cells derived from the PGE2-treated mice had significantly higher expression of Cdk4, Cdk6, Ctnnb1, and Myc compared to the control mice. These data indicate that PGE2 promoted BM recovery by accelerating the proliferation of HSPCs. The EP4 agonist was as efficient as PGE2 in enhancing BM recovery after myelosuppression. In addition, we found that EP4 agonist treatment induced an increase in the nuclear localization of β-catenin, indicating that PGE2 signaling through EP4 impinges on Wnt/β-catenin signaling in HSCs. Altogether, these data suggest that EP4 plays a crucial role in the PGE2 signaling involved in the proliferation of HSCs under stress conditions. This study was conducted under the collaboration with Dr. Narumiya (Kyoto University Graduate School of Medicine) Disclosures:No relevant conflicts of interest to declare.

Homeostasis of hematopoietic stem cells regulated by the myeloproliferative disease associated-gene product Lnk/Sh2b3 via Bcl-xL

Hematopoietic stem cells (HSCs) are maintained at a very low frequency in adult bone marrow under steady-state conditions. However, it is not fully understood how homeostasis of bone marrow HSCs is maintained. We attempted to identify a key molecule involved in the regulation of HSC numbers, a factor that, in the absence of Lnk, leads to HSC expansion. Here, we demonstrate that upon stimulation with thrombopoietin, expression of Bcl-xL, an antiapoptotic protein, was highly enhanced in Lnk-deficient HSCs compared to normal HSCs. As a result, Lnk-deficient HSCs underwent reduced apoptosis following exposure to lethal radiation. Downregulation of Bcl-xL expression in Lnk-deficient HSCs by short-hairpin RNA resulted in a great reduction of their capacity for reconstitution. These findings suggest that Lnk/Sh2b3 constrains the expression of Bcl-xL and that the loss of Lnk/Sh2b3 function enhances survival of HSCs by inhibiting apoptosis. Furthermore, our observations indicate that HSCs in patients with an Lnk/Sh2b3 mutation might become resistant to apoptosis due to thrombopoietin-mediated enhanced expression of Bcl-xL. Consequently, reduced apoptosis could facilitate accumulation of HSCs with oncogenic mutations leading to development of myeloproliferative disorders.

Bmi1 Confers Stress Resistance to Self-Renewing Hematopoietic Stem Cells.

AbstractAbstract 1607The polycomb group (PcG) proteins form chromatin-modifying complexes that implement transcriptional silencing. There are two major PcG complexes, polycomb repressive complex (PRC) 1 and PRC2. PRC1 ubiquitylates histone H2A at lysine 119 and PRC2 trimethylates lysine 27 of histone H3. Among PcG proteins, Bmi1, a core component of PRC1, plays an essential role in the self-renewal and maintenance of various kinds of stem cells including hematopoietic stem cells (HSCs), neural stem cells, and leukemic stem cells. We previously reported that forced expression of Bmi1 using a Bmi1 retrovirus promotes symmetrical cell division of HSCs, resulting in a marked ex vivo expansion of multipotent progenitors and an enhancement of repopulating capacity of HSCs in vivo. However, the impact of overexpression of Bmi1 in HSCs remains to be precisely addressed. To this end, we generated a mouse line where Bmi1 can be conditionally overexpressed under the control of the Rosa promoter in a tissue-specific fashion by the Cre-LoxP system. We crossed the mice to Tie2-Cre mice (Tie2-Cre; Rosa-Bmi1fl/+) and induced overexpression of Bmi1 in hematopoietic cells in vivo. Real-time PCR analysis demonstrated that expression of Bmi1 inpurified bone marrow (BM) c-Kit+Sca-1+Lineage-marker- (KSL) cells was 6-fold higher in Tie2-Cre; Rosa-Bmi1fl/+ mice than control Tie2-Cre mice mice. Overexpression of Bmi1 did not significantly affect steady state hematopoiesis. The number of HSCs and progenitors (multipotent progenitors, CMPs, GMP, MEPs, and CLPs) and lineage composition (myeloid cells, B cells, and T cells) in BM of Tie2-Cre; Rosa-Bmi1fl/+ mice was not significantly changed compared to those in control mice. We then performed serial transplantation assay. The repopulating capacity of Tie2-Cre; Rosa-Bmi1fl/+ BM cells was comparable to those of the control cells in primary recipients. However, Tie2-Cre; Rosa-Bmi1fl/+ cells retained higher repopulating capacity during serial transplantation compared to those of the control cells. Moreover, ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs for 10 days contained approximately 2-fold more high proliferative potential colony-forming cells (HPP-CFCs; colony diameter>1mm) and colony-forming unit-neutrophil/macrophage/erythroblast/megakaryocyte (CFU-nmEM) which retain multi-lineage differentiation potential along myeloid lineage than the control. Of note, cells in ex vivo culture of Tie2-Cre; Rosa-Bmi1fl/+ HSCs exhibited significantly augmented repopulating capacity in recipient mice and we are now engaged in competitive repopulation unit (CRU) assays to determine the net HSC expansion by overexpression of Bmi1 during ex vivo culture. These results indicate that overexpression of Bmi1 confers stress resistance to HSCs during ex vivo culture and serial transplantation. Our findings also provide Bmi1 as a potential target for efficient manipulation of HSCs ex vivo. Disclosures:No relevant conflicts of interest to declare.

Role of Protection of Telomeres 1A (Pot1a) In the Regulation of Hematopoietic Stem Cells.

AbstractAbstract 2617Hematopoietic stem cells (HSCs) undergo self-renewing cell divisions and maintain blood cell production throughout the lifetime. Appropriate control of HSC self-renewal is critical for the maintenance of hematopoietic homeostasis. Telomeres are nucleoprotein structures that cap the ends of eukaryotic chromosomes, and shelterin is required for the stability of telomeres. It is known that HSCs have telomerase activity and maintains telomere lengths longer than those of differentiated cells. The accelerated telomere erosion reduces the long-term repopulating capacity of HSCs in mutant mice, suggesting that keeping the telomerase activity and telomere structures is critical for the maintenance of HSCs.On the other hand, it has been shown that the maintenance of cell cycle quiescence and self-renewal activity of HSCs largely depend on the interaction with the bone marrow niches. We previously reported that the interaction of Tie2 in HSCs with its ligand angiopietin-1 (Ang-1) in niche cells in bone marrow (BM) endosteum is critical for the maintenance of HSC quiescence (Arai 2004).In this study, we found that Ang-1 upregulated the expression of protection of telomeres 1A (Pot1a) in side-population (SP) cells within Lin–Sca-1+c-Kit+ (LSK) fraction, and further investigated the role of Pot1a in the regulation of HSCs. Pot1 has been proposed to form a part of the six-protein shelterin complex at telomeres. In mice, there are two genes encoding Pot1-related proteins, Pot1a and Pot1b. Knockout of Pot1a results in early embryonic lethality, whereas mice lacking Pot1b are alive and fertile, suggesting that Pot1a is essential for mouse development. We found that long-term HSC population, LSK-CD34– cells, expressed higher levels of Pot1a than short-term HSCs population, LSK-CD34+ cells, both in transcriptional and protein level. To analyze the function of Pot1a in the maintenance of HSCs, we transduced Pot1a in LSK cells and examined the colony formation and long-term BM reconstitution capacities. Overexpression of Pot1a increased the size of colonies compared to control. In addition, the number of high proliferative potential colony-forming cells (HPP-CFC) was increased by the overexpression of Pot1a after long-term culture. There was no significant difference in long-tern reconstitution capacity after the primary bone marrow transplantation (BMT) between Pot1a-transduced LSK cells and control. After the secondary BMT, however, Pot1a-transduced LSK cells showed higher reconstitution activity than control. Moreover, Pot1a-transduced cells increased the frequency of Ki67-negative cells after the primary and the secondary BMT compared with control. Next, we transduced Pot1a shRNA into LSK cells and examined the effect of Pot1a-knockdown on the regulation of HSCs. The number of colonies derived from Pot1a-knockdown LSK cells was significantly decreased compared to control. In addition, knockdown of Pot1a significantly reduced long-term reconstitution activity of LSK cells after BMT.These data suggest that Pot1a plays a critical role in the maintenance of self-renewal activity and cell cycle quiescence of HSCs. We will also discuss about the dependence of the Pot1a function in HSCs on the telomerase activity. Disclosures:No relevant conflicts of interest to declare.

Critical Roles of Lysosomal Acid Lipase in Myelopoiesis

Lysosomal acid lipase (LAL) is a key enzyme that cleaves cholesteryl esters and triglycerides to generate free fatty acids and cholesterol in lysosomes. Genetic ablation of the lal gene (lal(-/-)) in mice has resulted in a systemic increase of macrophages and neutrophils, causing severe inflammation and pathogenesis in multiple organs. We hypothesized that aberrant growth and differentiation of myeloid cells in lal(-/-) mice arises from dysregulated production of progenitor cells in the bone marrow. Indeed, lal(-/-) mice displayed increased numbers of primitive lin(-)Sca-1(+)c-Kit(+) (LSK) cells and granulocyte-macrophage precursors (GMP). Increased high proliferative potential colony-forming cells (HPP-CFC) were enumerated from cultured lal(-/-) bone marrow cells, as were significantly more CFU-GM, CFU-G, and CFU-M colonies. As a consequence, lal(-/-) mice developed significant myeloid infiltration, particularly with CD11b+/Gr-1+ myeloid-derived suppressive cells in multiple organs. Both decreased apoptosis and increased proliferation contribute to the systemic increase of myeloid cells in lal(-/-) myeloid cells. These lal(-/-) CD11b(+)/Gr-1(+) cells displayed suppressive activity on T cell proliferation and function in vitro. Bone marrow chimeras confirmed that the myeloproliferative disorder in lal(-/-) mice was primarily attributable to autonomous defects in myeloid progenitor cells, although the hematopoietic microenvironment in the lal(-/-) mice did not support hematopoiesis normally. These results provide evidence that LAL is an important regulator of myelopoiesis during hematopoietic development, differentiation, and homeostasis.

Decreased colony formation of high proliferative potential colony-forming cells and granulocyte-macrophage colony-forming units and increased Hes-1 expression in bone marrow mononuclear cells from patients with psoriasis

Psoriasis is a chronic inflammatory disease of the skin. The dysfunctional immunity experienced by patients with psoriasis is believed to influence the bone marrow haematopoietic cells and their surrounding microenvironment. Phagocytes derived from the bone marrow of patients with active psoriasis exhibit enhanced monocytopoietic activity and hyperplasia in vitro. However, direct evidence supporting the hypothesis that bone marrow is involved in the pathogenesis of psoriasis has yet to be established. To investigate the involvement of bone marrow in the pathogenesis of psoriasis. Bone marrow mononuclear cells (BMMNCs) were isolated from patients with psoriasis and healthy individuals. The high proliferative potential colony-forming cells (HPP-CFCs), granulocyte-macrophage colony-forming units (CFU-GM) and erythroid colony-forming units (CFU-E) were cultured in the presence of defined cytokines, and the effects of secreted factors from psoriatic peripheral blood mononuclear cells (PBMCs) on colony formation of normal haematopoietic cells were analysed. Furthermore, the telomere activity of psoriatic and normal BMMNCs was determined using the polymerase chain reaction (PCR)-based telomeric repeat amplification protocol, while the expression of human telomerase reverse transcriptase (hTERT) and HES1 mRNA was detected by reverse transcription-PCR assay. The numbers of HPP-CFCs and CFU-GM, but not CFU-E, were significantly reduced in cultured haematopoietic cells from patients with psoriasis. The culture supernatant of PBMCs from patients with psoriasis was found to inhibit the colony formation capacity of HPP-CFCs, CFU-GM and CFU-E of normal haematopoietic cells. We also detected low levels of telomerase activity and hTERT gene expression in psoriatic and control BMMNCs that was statistically similar between the two groups. In contrast, the HES1 gene expression appeared to be significantly elevated in psoriatic BMMNCs (P < 0.05). Together, our results indicate the involvement of bone marrow in the immunopathogenesis of psoriasis, and suggest a mechanism mediated by certain inflammatory or haematopoietic cytokines present in the bone marrow microenvironment. Elevated expression levels of HES1 mRNA suggest a potential role for the Notch signalling pathway in this process.

Promoter methylation status of p15 and p21 genes in HPP-CFCs of bone marrow of patients with psoriasis

Psoriasis is an inflammatory disease related to dysfunctional immunity. The dysfunctional immunity may influence the haematopoietic microenvironment or haematopoiesis in psoriasis. However, direct evidence is lacking. Our objective was to investigate the proliferation of hematopoietic cells from psoriatic patients and any link between the promoter methylation status of p15 and p21 genes and the colony formation ability of high proliferative potential colony-forming cells (HPP-CFCs). Marrow mononuclear cells were isolated from the bone marrow of psoriatic patients and normal controls by density gradient centrifugalization. Colony forming assays of HPP-CFCs were performed in vitro in methylcellulose semi-solid culture medium. mRNA expression and the promoter methylation status of p15 and p21 genes in HPP-CFCs were studied by semi-quantitative RT-PCR and methylation-specific PCR respectively. In methycellulose semi-solid culture system, the colony count of HPP-CFCs in bone marrow of psoriatic patients was significantly less than that of normal controls. Moreover, significantly lower positive frequencies of promoter methylation and higher transcription levels for p15 and p21 genes were observed in psoriasis in comparison to normal volunteers. The lower promoter methylation of p15 and p21 genes may be an important mechanism for the dysfunctional growth regulation pathways in HPP-CFCs in psoriasis.

Influence of hemoglobin vesicles, cellular‐type artificial oxygen carriers, on human umbilical cord blood hematopoietic progenitor cells in vitro

Hemoglobin vesicles (HbVs), liposomal oxygen carriers containing human hemoglobin, are candidates for development as clinically useful blood substitutes. Although HbVs are shown to distribute transiently into the bone marrow in animal models, the influence of HbVs on human hematopoietic stem/progenitor cells has not yet been studied. Therefore, we investigated the influence of HbVs at a concentration of up to 3 vol/vol % on the clonogenic activity (in semisolid culture) and proliferative activity (in liquid culture) of human hematopoietic progenitor cells derived from umbilical cord blood (CB) in vitro. Continuous exposure of CB mononuclear cells to HbVs tended to decrease the number and size of mature-committed colonies and most notably reduced the number of colonies of high-proliferative potential colony-forming cells (HPP-CFC). In contrast, exposure to HbVs for 20 h or 3 days, which is more relevant to the clinical setting, had no effect on the number of mature-committed colonies and only modestly decreased the number of HPP-CFC. Continuous exposure (10 days) to HbVs significantly suppressed the cellular proliferation and differentiation of both the erythroid and myeloid lineages in liquid culture. Again, short exposure (20 h or 3 days) did not affect these parameters. Thus, our results show that HbVs, under conditions relevant to the clinical setting, have no adverse effect on human CB hematopoietic progenitor activity in vitro.

Ex Vivo Expansion and Transplantation of Hematopoietic Stem/Progenitor Cells Supported by Mesenchymal Stem Cells from Human Umbilical Cord Blood

Human mesenchymal stem cells (MSCs) are multipotential and are detected in bone marrow (BM), adipose tissue, placenta, and umbilical cord blood (UCB). In this study, we examined the ability of UCB-derived MSCs (UCB-MSCs) to support ex vivo expansion of hematopoietic stem/progenitor cells (HSPCs) from UCB and the engraftment of expanded HSPCs in NOD/SCID mice. The result showed that UCB-MSCs supported the proliferation and differentiation of CD34+ cells in vitro. The number of expanded total nucleated cells (TNCs) in MSC-based culture was twofold higher than cultures without MSC (control cultures). UCB-MSCs increased the expansion capabilities of CD34+ cells, long-term culture-initiating cells (LTC-ICs), granulocyte-macrophage colony-forming cells (GM-CFCs), and high proliferative potential colony-forming cells (HPP-CFCs) compared to control cultures. The expanded HSPCs were transplanted into lethally irradiated NOD/SCID mice to assess the effects of expanded cells on hematopoietic recovery. The number of white blood cells (WBCs) in the peripheral blood of mice transplanted with expanded cells from both the MSC-based and control cultures returned to pretreatment levels at day 25 posttransplant and then decreased. The WBC levels returned to pretreatment levels again at days 45-55 posttransplant. The level of human CD45+ cell engraftment in primary recipients transplanted with expanded cells from the MSC-based cultures was significantly higher than recipients transplanted with cells from the control cultures. Serial transplantation demonstrated that the expanded cells could establish long-term engraftment of hematopoietic cells. UCB-MSCs similar to those derived from adult bone marrow may provide novel targets for cellular and gene therapy.

The mRNA expression and promoter methylation status of the p16 gene in colony-forming cells with high proliferative potential in patients with psoriasis

Psoriasis is a chronic and relapsing inflammatory disease of the skin associated with various immune abnormalities. It is tempting to speculate that the dysfunctional immunity may influence the haematopoietic microenvironment or haematopoiesis in psoriasis. However, direct evidence of involvement of bone-marrow haematopoietic cells in the pathogenesis of psoriasis is lacking. To investigate the proliferative activity of haematopoietic cells of patients with psoriasis and a link between the promoter methylation status and transcriptional activity of the p16 gene and the colony-forming ability of high proliferative potential colony-forming cells (HPP-CFCs). Marrow mononuclear cells were isolated from the bone marrow of patients with psoriasis and normal controls by density gradient centrifugation. A comparison of HPP-CFC colony formation counts between patients with psoriasis and normal controls was carried out by colony-forming assays of HPP-CFCs in methylcellulose semisolid culture medium in vitro. Subsequently, genomic DNA and RNA in HPP-CFCs were isolated, and mRNA expression and p16 promoter methylation status were studied by reverse transcriptase PCR and methylation-specific PCR (MSP), respectively. In comparison with normal controls, patients with psoriasis showed less HPP-CFC colony formation in the methycellulose semisolid culture system, and showed upregulated mRNA transcriptional level and downregulated promoter methylation of p16. Our data demonstrate for the first time the abnormal proliferative activity of haematopoietic cells in patients with psoriasis and a profound link between the promoter methylation status and transcriptional activity of p16 and the colony-forming ability of HPP-CFCs, suggesting that haematopoietic cells are involved in psoriasis.

Murine allogeneic in vivo stem cell homing,

Stem cell homing has been studied in syngeneic models and appears to be rapid (<1 h) and dependent on cellular adhesion and migration factors. We utilized a full H2-mismatched transplantation model to determine the basics of allogeneic homing. C57BL/6J Lin-Sca-1+ cells were labeled with CFSE and injected in non-myeloablated BALB/c mice. Fluorescent cell detection was via high-speed FACS analysis. Alternatively, B6.SJL whole bone marrow cells were injected in lethally irradiated BALB/c mice (10 Gy). One, 3, 6, and 24 h after transplant, marrow was harvested and cells were either plated for high proliferative potential colony-forming cell (HPP-CFC) assay or secondarily injected into myeloablated (8 Gy) C57BL/6J mice using 10% competing C57BL/6J marrow. Chimerism was evaluated at 8 weeks. CFSE+ cells were detected in the bone marrow 1, 3, and 6 h after injection. The numbers were moderately lower when compared to syngeneic homing possibly due to strain effect. Conversely, utilizing a surrogate or secondary assay, we observed a decline of secondary engraftment of harvested cells over time, but not of HPP-CFC. Combining experiments and normalizing the 1-h time point to 100% (to allow comparison), we observed a mean relative engraftment of 87 +/- 29%, 72 +/- 21%, 84 +/- 35% of the 1 h level at 3, 6, and 24 h respectively. HPP-CFC assay showed no significant variation as a homing surrogate over 1-6 h. These data indicate a rapid homing into allogeneic recipients with a plateau at 1 h. The decline of secondary engraftability over time may indicate a phenotype alteration of homed cells.

Live Attenuated Salmonella Carrying Platelet Factor 4 cDNAs as Radioprotectors

To determine whether live attenuated Salmonella carrying platelet factor 4 cDNAs can protect mice from radiation damage, the attenuated Salmonella SL3261 was used as oral vector for targeted gene delivery. The recovery of mice receiving sublethal total-body irradiation (TBI) was investigated after the oral administration of attenuated Salmonella carrying cDNA for platelet factor 4 (PF4) or truncated PF4. This oral gene therapy protected mice from radiation damage after TBI. The number of bone marrow cells and high proliferative potential colony-forming cells (HPP-CFCs) increased significantly at day 7. Similarly, the administration of PF4 or PF4(17-70) protein also improved the survival of mice after TBI. Both PF4 gene therapy and protein administration accelerated hematopoietic recovery in vivo in mice after irradiation. In vitro, PF4 also promoted survival and proliferation of 5-fluorouracil-resistant hematopoietic stem/progenitor cells after irradiation. These data demonstrate a novel biological function of PF4 as a protector against radiation injury and suggest that attenuated Salmonella could be used in vivo as a PF4 DNA delivery vector in the management of radiation injury.

A Stem Cell Population Capable of Producing Both Lympho- and Hematopoietic Cells Can Be Used To Detect Increased Sensitivity and Residual Stem Cell Damage after Initial Drug Administration.

Detection of different in vitro stem cell populations has been possible using a number of different parameters, including membrane antigen expression, growth factor combinations, various culture conditions and the ability to produce colonies of specific cell types using the colony-forming differentiation assay. The colony-forming unit-blast (CFU-blast) which is considered to be at the junction point between the lympho- and hematopoietic systems, and the high proliferative potential colony-forming cell (HPP-CFC), regarded as the most primitive in vitro stem cell that feeds into hematopoiesis, are two such populations. Here we describe a primitive stem cell population, that can enter both lympho- and hematopoietic systems, based solely on its proliferative potential. Using a non-subjective, highly sensitive human bone marrow colony-forming proliferation assay that detects and measures changes in intracellular ATP concentration that are proportional to proliferation, a population designated the high proliferative potential stem and progenitor (HPP-SP) cell has been identified. Detection of the HPP-SP, and other in vitro proliferating cell populations (CFC-GEMM, BFU-E, GM-CFC, Mk-CFC, T-CFC, B-CFC), is performed in a 96-well plate format and release of intracellular ATP after lysing the cells drives a luciferin/luciferase reaction to produce luminescence that is detected in a plate luminometer. A variety of drugs, including Imatinib mesylate, 5-fluorouracil, doxorubicin, daunorubicin, hydroxyurea and busulphan were analyzed over a dose response range from 1pM to 10uM. The HPP-SP population was treated with each drug dose and “primed” out of quiescence using IL-3, IL-6, SCF and FL. After 7 days of incubation, 8 replicate 100μl cultures were used to measure proliferation in the primary (1°) stimulated cultures. The cells in a second set of 16 replicates for each dose, were removed from the primary cultures, pooled and re-plated without drug, into secondary (2°), “fully stimulated” cultures containing EPO, GM-CSF, G-CSF, IL-3, IL-6, SCF, TPO, FL, IL-2, IL-7 and IL-15 and incubated for a further 7 days followed by luminescence measurement. All dose response curves were fitted using a 4-parameter logistic statistic. The difference in drug dose response between the 1° and 2° cultures produced a “residual” dose response to the drug and therefore a measurement of how much of the population remained after initial drug treatment. Comparison of the 50% inhibitory concentrations (IC50 values) between the 1° and 2° dose responses indicated whether the remaining HPP-SP population exhibited the same (busulfan) or increased sensitivity (doxorubicin, daunorubicin) to the drug, thereby allowing a prediction to be made as to whether the cells would be further damaged by repeated rounds of drug administration. The cells from the final set of 8 replicate culture were again removed, pooled and analyzed by flow cytometry for the presence or absence of populations resulting from the 2° expansion conditions. These procedures provide a rapid and highly predictive method of ascertaining how primitive stem cells respond to drugs and therefore has a direct implication in chemotherapy and preparation of patients for stem cell transplantation prior to patient treatment.

The changing cellular environments of hematopoiesis in human development in utero

The hematopoietic system is indispensable from the earliest stages of development and adapts to the rapidly changing anatomy of the embryo and fetus; this takes place in such different anatomic locations as the yolk sac blood island, hepatic parenchyme, aorta-gonads-mesonephros paravascular mesenchyme, and bone marrow primary logette. We herein summarize our investigation of these serial blood-forming events in the human embryo and fetus. The access to early stages of human development, availability of a large panoply of molecular markers for human blood cell lineages, and recent development of robust assays for the earliest human hematopoietic stem cells have allowed us to gain relatively clear insight into the developmental sequence that underlies the ontogeny of human blood cells. Conversely, the control exerted by these diverse cellular environments on the emergence of human hematopoietic cells remains elusive, as is the case in animal models. We nonetheless present preliminary attempts to decipher the structure and molecular characteristics of the distinct cellular "niches" in which blood cells are produced during human gestation.

Enhanced Self-Renewal of Hematopoietic Stem Cells Mediated by the Polycomb Gene Product, Bmi-1.

The Polycomb group (PcG) proteins form multiprotein complexes that play an important role in the maintenance of transcriptional repression of target genes. Loss-of-function analyses show abnormal hematopoiesis in mice deficient for PcG genes including Bmi-1, Mph-1/Rae28, M33, Mel-18, and Eed, suggesting involvement of PcG complexes in the regulation of hematopoiesis. Among them, Bmi-1 has been implicated in the maintenance of hematopoietic and leukemic stem cells. In this study, detailed RT-PCR analysis of mouse hematopoietic cells revealed that all PcG genes encoding components of the Bmi-1-containing complex, such as Bmi-1, Mph1/Rae28, M33, and Mel-18 were highly expressed in CD34−c-Kit+Sca-1+Lin− (CD34−KSL) hematopoietic stem cells (HSCs) and down-regulated during differentiation in the bone marrow. These expression profiles support the idea of positive regulation of HSC self-renewal by the Bmi-1-containing complex. To better understand the role of each component of the PcG complex in HSC and the impact of forced expression of PcG genes on HSC self-renewal, we performed retroviral transduction of Bmi1, Mph1/Rae28, or M33 in HSCs followed by ex vivo culture. After 14-day culture, Bmi-1-transduced but not Mph1/Rae28-transduced cells contained numerous high proliferative potential-colony forming cells (HPP-CFCs), and presented an 80-fold expansion of colony-forming unit-neutrophil/macrophage/Erythroblast/Megakaryocyte (CFU-nmEM) compared to freshly isolated CD34−KSL cells. This effect of Bmi-1 was comparable to that of HoxB4, a well-known HSC activator. In contrast, forced expression of M33 reduced proliferative activity and caused accelerated differentiation into macrophages, leaving no HPP-CFCs after 14 days of ex vivo culture. To determine the mechanism that leads to the drastic expansion of CFU-nmEM, we employed a paired daughter cell assay to see if overexpression of Bmi-1 promotes symmetric HSC division in vitro. Forced expression of Bmi-1 significantly promoted symmetrical cell division of daughter cells, suggesting that Bmi-1 contributes to CFU-nmEM expansion by promoting self-renewal of HSCs. Furthermore, we performed competitive repopulation assays using transduced HSCs cultured ex vivo for 10 days. After 3 months, Bmi-1-transduced HSCs manifested a 35-fold higher repopulation unit (RU) compared with GFP controls and retained full differentiation capacity along myeloid and lymphoid lineages. As expected from in vitro data, HSCs transduced with M33 did not contribute to repopulation at all. In ex vivo culture, expression of both p16INK4a and p19ARF were up-regulated. p16INK4aand p19ARF are known target genes negatively regulated by Bmi-1, and were completely repressed by transducing HSCs with Bmi-1. Therefore, we next examined the involvement of p19ARF in HSC regulation by Bmi-1 using p19ARF-deficient and Bmi-1 and p19ARF-doubly deficient mice. Although bone marrow repopulating activity of p19ARF-deficient HSCs was comparable to that of wild type HSCs, loss of p19ARF expression partially rescued the defective hematopoietic phenotypes of Bmi-1-deficient mice. In addition, transduction of Bmi-1 into p19ARF-deficient HSCs again enhanced repopulating capacity compared with p19ARF-deficient GFP control cells, indicating the existence of additional targets for Bmi-1 in HSCs. Our findings suggest that the level of Bmi-1 is a critical determinant for self-renewal of HSC and demonstrate that Bmi-1 is a novel target for therapeutic manipulation of HSCs.