Normal Short-Term but Reduced Long-Term Engraftment Capacity of CML Hematopoietic Cells with Skewed Myeloid Lineage Differentiation Is Seen in an Improved Mouse Model of Human Hematopoiesis.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 3383-3383 ◽  
Author(s):  
Jeff Tauer ◽  
Leonard Shultz ◽  
Tessa Holyoake ◽  
Ravi Bhatia
Keyword(s):  
Stem Cells ◽  
Mouse Model ◽  
Human Cell ◽  
Hematopoietic Cells ◽  
Short Term ◽  
Myeloid Lineage ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Nog Mice

Abstract The NOD/SCID mouse model has been widely used to assay human hematopoietic cells capable of long-term multilineage engraftment (SCID-repopulating cells or SRC). Leukemia cells from CML patients can also engraft in NOD-SCID mice. However the utility of this model for studying CML stem cells is limited by inconsistent and low levels of engraftment, and lack of a leukemia-related phenotype of engrafted cells. Since NOD/SCID IL2Rγchain KO (NOG) mice support superior engraftment of human hematopoietic cells compared with NOD/SCID mice, they may allow for improved evaluation of CML stem cells. In the current study we investigated engraftment of CML CD34+ cells in the NOG mouse model. CD34+ cells were transplanted into irradiated (300 cGy) 8 wks old NOG mice via tail vein injection. Blood samples were obtained at 4 wks intervals to monitor human cell engraftment. At the end of the maintenance period of 16–18 wks, animals were euthanized and marrow content of femora, spleen cells and peripheral blood were obtained. Human cell engraftment was analyzed by flow cytometry after labeling cells with anti-human CD45 mAb and specific human cell subsets detected by staining with human CD34, CD33, CD14, CD11b, CD19 and CD3 mAbs. Establishment of human hematopoiesis was consistently seen 4 weeks after injection. Larger numbers of CML (1–2 x106) compared to normal bone marrow (1–8x105) CD34+ cells were required to establish engraftment, which is consistent with previous reports. Interestingly CML CD34+ cells established their highest levels of engraftment in blood 4 wks after transplantation (14.9±5.9% CD45+ cells, n=13) and engraftment levels were significantly lower at 8 wks (1.8±0.3%) and beyond. However engraftment was consistently detected in blood up to 16–20 wks. The engraftment kinetics of CML cells was strikingly different from that of normal CD34+ cells which while showing similar levels of engraftment at 4 wks (12.6±3.8%, n=16), showed considerably higher engraftment at 8 wks (19.2±3.8%) with a gradual decline subsequently (11.5±2.7% at 12 wks). Analysis after autopsy at 16–18 wks revealed higher levels of human cell engraftment in mouse BM and spleen compared with blood. CML CD34+ cells showed reduced BM engraftment compared with normal CD34+ cells (7.3±4.8% vs. 22.1±9.0% CD45+ cells, n=7, p<0.01). CML CD34+ cells engrafted with myeloid lineage cells and minimal lymphoid engraftment was seen (CD33: 5.2±3.7%, CD11b: 2.6±1.6% and CD19: 0.1±0.1%), whereas normal CD34+ cells engrafted with both myeloid and lymphoid cells (CD33: 7±3%, CD11b: 1.6±0.6% and CD19: 11±4.3%). We also observed reduced frequency of CD34+ cells in mice receiving CML compared with normal cells (2.6±2.5% vs. 6.2±3.0%, p<0.05). In conclusion we show, using an improved mouse model of human hematopoiesis, that CML SRC have similar short-term but reduced long-term engraftment capacity compared with normal SRC, possibly indicating a preponderance of short-term repopulating cells or increased HSC turnover. CML SRC demonstrated predominantly myeloid differentiation compared with balanced myeloid and lymphoid engraftment of normal SRC. These results support the utility of the NOG mouse model to investigate functional characteristics of CML stem cells and study the activity of specific therapeutic interventions against primitive CML cells.

High Resolution Purification and Characterization of Human Cord Blood-Derived CD34–negative SCID-Repopulating Cells with a Very Immature Phenotype.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 815-815
Author(s):  
Mari Murakami ◽  
Yoshikazu Matsuoka ◽  
Ryusuke Nakatsuka ◽  
Masaya Takahashi ◽  
Tsuyoshi Nakamoto ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Human Cell ◽  
Scid Mice ◽  
Human Cord Blood ◽  
Cd34 Cells ◽  
Nog Mice ◽  
Cell Repopulation ◽  
Limiting Dilution

Abstract Abstract 815 We have successfully identified human cord blood (CB)-derived CD34-negative (CD34−) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) with an extensive lymphoid and myeloid repopulating ability using the intra-bone marrow injection (IBMI) method (Blood 101:2924,2003). In that study, 13 lineage specific antibodies (Abs), including anti-CD2, CD3, CD4, CD7, CD10, CD14, CD16, CD19, CD20, CD24, CD41, CD56, and CD235a, were used to purify CD34− SRCs. A limiting dilution analysis demonstrated the frequency of CD34− SRCs in the 13 lineage-negative (Lin−) CD34− cells to be approximately 1/25,000. In this study, we added 5 more lineage specific Abs, including anti-CD11b, CD33, CD45RA, CD66c, and CD127, in order to highly purify CD34− SRCs. The 18 Lin−CD34− cells showed a homogeneously blast-like morphology, and their incidence in the CB-derived nucleated cells ranged from 0.0002 to 0.001%. The colony-forming capacity of these highly purified 18 Lin−CD34− cells was quite unique, since 50% of the total colony-forming cells (CFCs) were mixed colony-forming cells (CFU-Mix). In contrast, the 18 Lin−CD34+ cells formed myeloid, erythroid, and mixed colonies, however, only <10% of the total CFCs were CFU-Mix. The phenotypic and functional characterizations of these 18 Lin−CD34− cells were then further investigated by cocultures with the HESS-5 murine stromal cell line in the presence of a cocktail of cytokines, such as SCF, flt3 ligand, TPO, IL-3, IL-6, and G-CSF. After 7 days of coculture, the total number of cells was observed to expand by 20 to 30 folds, 40 to 60% of which were consisted of CD34+ cells. Next, we investigated the SRC activity of these 18 Lin−CD34− cells using NOD/Shi-scid mice. When 4×104 18 Lin−CD34− cells were transplanted using IBMI, all 4 mice were highly repopulated with human CD45+ cells, including CD19+ B-lymphoid and CD33+ myeloid cells. In addition, the level of human cell engraftment in the bone marrow (BM) ranged from 93.3 to 97.5% (median, 96.8%), at 12 weeks after the transplantation. Interestingly, the BM cells obtained from the primary engrafted NOD/ Shi-scid mice that received transplants of 1,000 to 2,000 18 Lin−CD34− cells showed a secondary repopulating capacity. Furthermore, a limiting dilution analysis demonstrated the frequency of CD34− SRCs in these 18 Lin−CD34− cells to be approximately 1/1,000. The next approach to characterize the CD34− SRCs with respect to the self-renewal potential as well as the long-term repopulating potential, was to serially analyze the kinetics of engraftment for 24 weeks in the NOD/Shi-scid/IL-2Rgcnull (NOG) mice that received transplants of 2,000 18 Lin−CD34− cells containing only 2 CD34− SRCs (estimated number). All 6 mice showed signs of human cell repopulation (0.4 to 28.9%, median 2.5%) at 5 weeks after the transplantation at the contra-lateral sites of IBMI. The repopulation rates gradually increased, and reached a high level of repopulation (47.0 to 87.9%, median 72.0%) at 18 weeks after the transplantation. These results indicated that CD34− SRCs could thus sustain a long-term human cell repopulation in NOG mice, thereby suggesting that CD34− SRCs are a distinct class of primitive HSCs in comparison to the previously reported CD34+ SRCs. Disclosures: No relevant conflicts of interest to declare.

HOXA10 Affects the Fate of Hematopoietic Progenitors and Stem Cells in a Concentration Dependent Manner.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 3550-3550 ◽  
Author(s):  
Ann C.M. Brun ◽  
Mattias Magnusson ◽  
Noriko Miyake ◽  
Eva Nilsson ◽  
Jon Mar Björnsson3 ◽  
...  
Keyword(s):  
Stem Cells ◽  
Stem Cell ◽  
Hematopoietic Cells ◽  
Fold Increase ◽  
Dependent Manner ◽  
Hematopoietic Progenitors ◽  
Short Term ◽  
Cells Cultured

Abstract Several studies have demonstrated that homeobox (Hox) genes are involved in the regulation of hematopoietic stem cells (HSC), and overexpression with retroviral vectors containing HOXB4 generate increased numbers of repopulating stem cells in vitro, but may also perturb differentiation of hematopoietic cells when the concentration of HOXB4 is very high. HOXA10 is expressed in primitive hematopoietic cells and myeloid progenitors. To study the effect of this gene we generated an inducible system based on a tetracycline transactivator, controlling the expression of HOXA10, aiming to study how different concentrations of HOXA10 affect the fate of hematopoietic progenitors and stem cells. We mated our tetO-HOXA10 mouse with the Rosa26rtTA strain, allowing activation of HOXA10 in all hematopoietic tissues after administration of doxycycline. Mice were born at normal ratios with no hematopoietic pathology. Inducible bone marrow was harvest and cultured for 12 days in 6 different concentrations of doxycyclin, revealing an increased proliferation at low concentrations, but a decline in proliferation capacity with higher concentrations. To verify that hematopoietic progenitors were affected, a CFU-GM colony assay was performed on cells cultured for 12 days, showing a two fold increase in the number of CFU-GM formed from the highly proliferating cells compared to wt and uninduced HOXA10 cells (p = 0.01). To study the effect of HOXA10 in more primitive cells, sorted inducible HOXA10 lin−, Sca1+, c-kit+ (LSK) cells were cultured for 13 days in different concentrations of doxycyclin. Lower concentrations of doxycyclin resulted in increased proliferation, while increasing concentrations resulted in decreased proliferation. Furthermore, using Q-RT-PCR, we found that the expression of HOXA10 was directly proportional to the concentration of doxycycline and no leakiness was detected in the uninduced LSK cells. The cultured cells were transplanted in a competitive setting into lethally irradiated mice to evaluate the repopulating ability of the expanded cells. Three weeks post BMT (short-term repopulation), intermediate levels of HOXA10 (0.08–0.2 mg/ml doxycyclin) resulted in a three-fold increase in repopulating capacity of the HOXA10 LSK cells whereas uninduced and higher levels of HOXA10 resulted in decreased reconstitution compared with fresh LSK cells (fresh LSK = 100%, intermediate: 313±182%, high: 45±35%, uninduced 35±33%, n=7 p< 0.01). However, sixteen weeks after transplantation we found that cells cultured for 13 days at intermediate levels of HOXA10 (0.08–0.2 mg/ml doxycyclin) preserved the stem cell reconstitution capacity compared to fresh LSK cells (fresh LSK = 100%, 0.2 mg/ml 153±82% n=7). Furthermore, uninduced LSK cells and higher levels of HOXA10 resulted in a 3 fold lower long-term reconstitution compared to Fresh LSK cells (0 mg/ml 34±32 %, high HOXA10 9±8% significant to both fresh cells and cells cultured in 0.2 mg/ml, p<0.003, n=7). These findings show that intermediate expression of HOXA10 can increase the short-term HSCs repopulating potential and can maintain the long-term repopulating stem cells for up to 13 days of in vitro culture. These results suggest that HOXA10 plays an important role in the regulation of HSCs and indicate that the effect of HOXA10 on stem cell fate decisions is dependent on the level of HOXA10 expression.

Use of the NOD/SCID/γcnull Mouse Model To Assess the Hepatocyte-Producing Ability of Human Hematopoietic Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1695-1695
Author(s):  
Hisanori Fujino ◽  
Hidefumi Hiramatsu ◽  
Atsunori Tsuchiya ◽  
Haruyoshi Noma ◽  
Mitsutaka Shiota ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Hematopoietic Stem Cells ◽  
Liver Damage ◽  
Hematopoietic Cells ◽  
Human Albumin ◽  
Hematopoietic Stem ◽  
Hepatic Cells ◽  
Cd34 Cells

Abstract Hematopoietic cells have been shown to generate nonhematopoietic cells, although the true plasticity of stem cells has been questioned. Here we used the NOD/SCID/γcnull mouse model, which permits efficient engraftment of human hematopoietic stem cells and their multi-lineage differentiation including T cells, to investigate whether human hematopoietic stem cells can differentiate into human hepatocytes. Freshly collected cord blood was depleted of phagocytes with Silica® followed by CD34 positive selection using auto MACS®. These cells were intravenously transplanted into irradiated mice, after which the liver was either undamaged or damaged by chemicals. The livers of these mice contained hepatocyte-specific (albumin, CYP family, TAT, alpha1AT, CPSI, prealbumin, transferrin and RBP4), cholangiocyte-specific (CK19) and vascular endothelial cell-specific (eNOS) human mRNAs. Immunohistochemistry detected the human hepatocyte specific antigens, albumin and alpha-1-antitrypsin-positive hepatocytes, cholangiocytes and CD68+ Kupffer cells. We also found human albumin in the murine bloodstream. Human albumin levels in the peripheral blood of transplanted mice correlate with the degree of PB chimerism and increase with time after transplantation. Furthermore, after obtaining liver cells by collagenase perfusion, flow cytometry revealed the presence of human albumin-positive cells that bear both human and murine MHC molecules, suggesting cell fusion occurs. All of the above phenomena were found in both liver-damaged and undamaged mice. In addition, we found human CD34+ cells are recruited from the murine bone marrow to the liver only in the case of acute liver injury but do not acquire hepatic stem/progenitor characteristics. Our observation suggests there are two pathways that yield hepatic cells from hematopoietic stem cells. The first requires liver damage that recruits CD34+ cells from the bone marrow via the circulation while the second pathway does not involve liver damage and appears to represent a constitutive default pathway of hematopoietic to nonhematopoietic transition. Our model is thus a versatile tool for investigating the development of functional human hepatic cells from hematopoietic cells and the feasibility of using hematopoietic cells in clinical situations.

Tracking Differential Repopulation Kinetics of Human Hematopoietic Progenitor Cells Using MRI Detection of Nanoparticles.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 1274-1274
Author(s):  
Todd E. Meyerrose ◽  
Andrew J. Hope ◽  
Shao P. Lin ◽  
Phillip E. Herrbrich ◽  
Michael H. Creer ◽  
...  
Keyword(s):  
Stem Cell ◽  
Human Cell ◽  
Hematopoietic Progenitor Cells ◽  
Hematopoietic Progenitor ◽  
Short Term ◽  
Post Transplant ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Kinetics Of

Abstract Even within cell populations enriched for hematopoietic stem cells, there exists phenotypic and functional heterogeneity, with subsets of progenitors differentially contributing to the total character of the transplant. Here, we have used a dynamic in vivo imaging technology to track the differential homing kinetics of such subsets, within a well defined stem cell pool, and identified patterns of short term homing unique to CD31 versus CD34 cells. Human umbilical cord blood derived (hUCB) hematopoietic progenitor cells (HPC) were isolated on high expression of aldehyde dehydrogenase (ALDH) to yield a population of cells we have previously reported to be enriched for stem cell activity by phenotypic and functional parameters. Cells were plated 12 hours on fibronectin in X-Vivo15 supplemented with SCF, flt-3, and TPO. During incubation, cells were labeled with Bang’s Laboratories 0.9um SPIO nanoparticles containing the Dragon Green fluorescent dye at a ratio of approximately 10:1 beads to cells. Positively loaded cells were immunomagnetically selected and transplanted into sublethally irradiated NOD/SCID/MPS7 mice. The MPS7 mouse model is highly permissive of human cell engraftment, and allows easy identification of donor cells by virtue of a genetic defect in beta-glucuronidase production. Post-transplant, animals underwent MRI every 30 minutes for 12 hours to develop a dynamic, real-time survey of donor migration between organs, and then sacrificed at optimal times for analysis by flow cytometry. The utility of this approach was validated by quantitative PCR for human beta-globin versus murine rapsyn to correlate loss of MR signal intensity to human cell engraftment. At 12 hours, MR signal intensity had decreased by 29% relative to time 0, with internal standardization against a water control. In contrast, repetitive imaging of a non-transplanted animal resulted in deviation of only +/− 4% from the mean. This loss of MR signal correlated to a spleen engraftment of as few as 10,000 cells (homing efficiency of ~2.5%) in some cohorts. This demonstrates the efficacy and resolution of this paradigm for real time analysis and manipulation of homing in vivo. We have noted a preferential affinity of CD31+ ALDH high HPC that highly co-express markers for CD117 and CD133 to the splenic vasculature as early as 1 hour post-transplant and peaking at 12 hours post-transplant. Alternatively, we observed initial marrow seeding to contain almost exclusively CD34+ cells with little co-expression of CD31 or even CD133. Additionally, we found that the spleen-homed fractions of cells also express classical markers of activation such as CD71 and CD38, whereas the marrow homed fraction does not. From a functional standpoint, we believe these two subsets comprise two facets of contribution to the total graft, providing short-term radioprotection and cytokine delivery from the spleen-homed compartment, and long term durable engraftment and self-renewal from the marrow-homed compartment.

Bone Marrow Osteoblast Ablation Enhances Short-Term Hematopoietic Stem Cells Without Altering Long-Term Hematopoietic Stem Cell Populations and Accelerates Leukemia Development

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 586-586
Author(s):  
Marisa Bowers ◽  
YinWei Ho ◽  
Ravi Bhatia
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mouse Model ◽  
Lower Extremity ◽  
Hematopoietic Stem Cells ◽  
Myelogenous Leukemia ◽  
Short Term ◽  
Hematopoietic Stem

Abstract Hematopoietic stem cells (HSCs) within the bone marrow (BM) microenvironment reside in close proximity to endosteal osteoblasts (OBs). Although OBs have been considered to provide a HSC niche, other studies suggest that perivascular mesenchymal cells or endothelial cells may be the primary HSC niches, and the specific role of OBs in regulation of HSCs requires further clarification. Moreover, the role of OBs in regulating leukemic stem cells (LSC) is even less well studied. To address these questions, we used a conditional OB ablation mouse model (Col2.3Δtk) in which a truncated version of the herpes simplex virus thymidine kinase (Δtk) is expressed under an OB-specific promoter. In these mice, daily intraperitoneal (IP) administration of ganciclovir (GCV) leads to production of a toxic DNA base analogue in OBs, resulting in their death. We crossed Col2.3Δtk mice with Col2.3GFP mice that specifically express GFP in OBs to facilitate assessment of OB ablation. We confirmed that 4 weeks of GCV administration resulted in ablation of endosteal OBs in this model using both immunofluorescence microscopy and flow cytometry analysis. OB ablation was associated with reduced BM cellularity (Δtk+ 3.7e7±3.0e6, Δtk- 4.8e7±3.8e6 per 4 lower extremity bones, p=0.04), but did not alter spleen (SP) cellularity (Δtk+ 5.1e7±5.3e6, Δtk- 6.3e7±7.4e6 cells per SP, p=0.19). OB ablation was also associated with significantly increased numbers of cells with long-term HSC (LTHSC) phenotype (Lin-Kit+Sca-1+Flt3-CD150+CD48-) in both the BM (Δtk+ 6490±1315, Δtk- 4236±922 per 4 lower extremity bones; p=0.03) and SP (Δtk+ 980±473, Δtk- 96±40 per SP; p=0.04). Significant increases in common myeloid progenitor (CMP) (Δtk+ 145114±43608, Δtk- 82200±26754; p=0.002) and granulocyte/monocyte progenitor (GMP) (Δtk+ 51411±17349, Δtk- 20206±9279, p=0.003, p=0.02) numbers were seen in SP of OB-ablated mice, whereas significant alterations in other hematopoietic populations in BM, SP or PB were not seen. We performed limiting-dilution competitive repopulation assays to determine the functional LTHSC potential of BM cells from OB-ablated and control mice. OB-ablated mice demonstrated a higher frequency of short-term repopulating cells compared to LTHSCs from non-ablated mice (5 weeks: Δtk+ 1 in 4,941; Δtk- 1 in 17,351 BM cells) but similar long-term engraftment (15 weeks: Δtk+ 1 in 22,853; Δtk- 1 in 23,137 BM cells). Transplantation of BM cells from primary transplant recipients into secondary recipients demonstrated similar long-term engraftment potential after second transplant. These results suggest that despite increased numbers of phenotypic LTHSCs in OB-ablated mice, the long-term repopulating and self-renewing capacity of BM cells remains unchanged in OB-ablated mice, but on the other hand there is an increase in functional short-term repopulating capacity. Next, to examine the role of OBs in regulation of Chronic Myelogenous Leukemia (CML) stem cells, we crossed the Col2.3GFPΔtk mice with an inducible transgenic BCR-ABL mouse model of CML (ScltTA-BCR/ABL). In these mice withdrawal of tetracycline results in induction of BCR-ABL expression in HSCs and development of a CML-like myeloproliferative disorder. GCV administration to achieve OB ablation was initiated one week prior to BCR-ABL induction by tetracycline withdrawal, and was continued for the duration of the experiment. CML development was monitored by checking blood counts every 2 weeks after induction and mice were followed for survival. We observed significantly accelerated development of CML in OB-ablated versus non-ablated mice, with 50% of the OB-ablated mice dying within 47 days of CML induction, whereas >50% of the non-ablated mice survived to day 73 (p=0.017). Collectively, these studies suggest that BM OBs are not essential for maintenance of long-term repopulating and self-renewing HSCs, but regulate the expansion of short-term HSCs in the BM. Our studies also indicate an important and previously unrecognized role for OBs in regulating the leukemogenicity of CML LSCs. Disclosures: No relevant conflicts of interest to declare.

scholarly journals The Bluegreen Algae (AFA) Consumption over 48 h Increases the Total Number of Peripheral CD34+ Cells in Healthy Patients: Effect of Short-Term and Long-Term Nutritional Supplementation (Curcumin/AFA) on CD34+ Levels (Blood)

2020 ◽  
Vol 10 (2) ◽  
pp. 49
Author(s):  
José Joaquín Merino ◽  
María Eugenia Cabaña-Muñoz ◽  
María Jesús Pelaz
Keyword(s):  
Stem Cells ◽  
Side Effects ◽  
Healthy Subjects ◽  
Nutritional Supplementation ◽  
Double Blind Study ◽  
Short Term ◽  
Cd34 Cells ◽  
Term Administration ◽  
Bluegreen Algae

Several active principles from plants could trigger the release of stem cells from the bone marrow. Stem cell mobilizers have shown side effects in patients. Thus, the purpose of this paper is to find the natural products from plants (curcuminoids, glycosinolate of sulforaphane, AFA bluegreen algae), which could be potential stem mobilizes without adverse side effects. The antioxidant curcumin [1,7-bis(4-hydroxy-3-methoxyphenyl)-1,6-heptadiene-2,5-dione], glycosinolate of sulforaphane (broccoli) or AFA (Aphanizomenon flos) extract promote beneficial effects in patients. The number of circulating stem cells were monitored by HSC marker-CD34 by flow cytometry in peripheral blood from healthy subjects. CD34 is a hematological stem cells (HSC) marker. A double-blind study was conducted in 22 healthy subjects. We have evaluated whether short-term AFA—Aphanizomenon flos aquae—algae or curcuminoids consumption (powder or liquid formulation) over 48 consecutive hours could increase the total number of peripheral CD34+ blood cells (n = 22, n = 5 subjects/group). The total number of circulating CD34+ cells were quantified after short-term and long-term nutritional supplementation; their levels were compared with their own basal levels (n = 5/group, controls: before taking any supplement) or placebo-treated patients (n = 7); their average age was 54 years old. We also evaluated whether long-term nutritional supplementation with several nutraceuticals could enhance HSC mobilization by increasing the total number of peripheral CD-34+ cell after seven or 38 consecutive days of administration (n = 5, with seven placebo-treated patients). The long-term administration take place with these doses/day [curcuminoids: 2000 mg/day, equivalent to 120 mg of curcuminoids/day), glycosinolate of sulforaphane (66 mg/day), plus AFA Algae bluegreen extract (400 mg/day)]. On the last day (10 a.m.) of treatment, blood samples were collected six hours after taking these supplements; the average age was 54 years old. Notably, the blue green AFA algae extract consumption over 48 h enhances HSC mobilization by increasing the total number of peripheral CD34+ cells. The long-term administration with curcuminoids, glycosinolate of sulforaphane, and AFA bluegreen algae extract also increased the total number of CD34-HSC cells after seven or 38 days of consecutive of administration in healthy subjects.

scholarly journals Purified hematopoietic stem cell engraftment of rare niches corrects severe lymphoid deficiencies without host conditioning

2005 ◽  
Vol 203 (1) ◽  
pp. 73-85 ◽  
Author(s):  
Deepta Bhattacharya ◽  
Derrick J. Rossi ◽  
David Bryder ◽  
Irving L. Weissman
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
Mouse Model ◽  
Cd4 T Cells ◽  
General Mechanism ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Cell Engraftment

In the absence of irradiation or other cytoreductive conditioning, endogenous hematopoietic stem cells (HSCs) are thought to fill the unique niches within the bone marrow that allow maintenance of full hematopoietic potential and thus prevent productive engraftment of transplanted donor HSCs. By transplantation of purified exogenous HSCs into unconditioned congenic histocompatible strains of mice, we show that ∼0.1–1.0% of these HSC niches are available for engraftment at any given point and find no evidence that endogenous HSCs can be displaced from the niches they occupy. We demonstrate that productive engraftment of HSCs within these empty niches is inhibited by host CD4+ T cells that recognize very subtle minor histocompatibility differences. Strikingly, transplantation of purified HSCs into a panel of severe combined immunodeficient (SCID) mice leads to a rapid and complete rescue of lymphoid deficiencies through engraftment of these very rare niches and expansion of donor lymphoid progenitors. We further demonstrate that transient antibody-mediated depletion of CD4+ T cells allows short-term HSC engraftment and regeneration of B cells in a mouse model of B(-) non-SCID. These experiments provide a general mechanism by which transplanted HSCs can correct hematopoietic deficiencies without any host conditioning or with only highly specific and transient lymphoablation.

An in-Vivo Model of T Cell-Mediated Rejection of Human Hematopoietic CD34+ Stem Cells Using NOD/SCID γnull (NOG) Mice.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4474-4474
Author(s):  
Benedetta Nicolini ◽  
Dolores Mahmud ◽  
Nadim Mahmud ◽  
Giuseppina Nucifora ◽  
Damiano Rondelli
Keyword(s):  
Bone Marrow ◽  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Mouse Model ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cell Engraftment ◽  
Cd34 Cells ◽  
Nog Mice

Abstract Abstract 4474 We have previously demonstrated that human CD34+ cells include subsets of antigen presenting cells capable of stimulating anti-stem cell T cell alloreactivity in-vitro. In this study we transplanted human CD34+ cells and allogeneic T cells in a NOD/SCID γnull (NOG) mouse model and evaluated the occurrence of stem cell rejection as well as xenogeneic graft-versus-host disease (GVHD) following the infusion of different doses of T cells. After sublethal irradiation NOG mice were cotransplanted with 2×105 CD34+ cells and HLA mismatched CD4+CD25- T cells at 1:0 (control), 1:2 or 1:10 CD34+ cell: T cell ratio (n=5-10 mice per group). Hematopoietic stem cell and T cell engraftment was assessed in the bone marrow and in the spleen 6 weeks following transplantation or earlier in case the animals died. Control mice transplanted with CD34+ cells alone showed a high level of stem cell engraftment (huCD45+ cells: 60±10%) in the bone marrow, encompassing CD19+ B cells (64±4%), CD34+ cells (18±1%), CD33+ myeloid cells (7±1%), CD14+ monocytes (3±1%), and no T cells within huCD45+ cells. In contrast, mice that were transplanted with CD34+ cells and 4×105 (1:2 ratio) or 2×106 (1:10 ratio) T cells had only 9±2% and 3±1% huCD45+ cells, respectively, in the bone marrow (p=0.01). Moreover, marrow samples of mice cotransplanted with CD34+ cells and T cells at 1:2 or 1:10 ratio included >98% huCD3+ T cells and no CD34+ cells. Spleen engraftment of huCD45+ cells was lower (25±8%) in control mice (1:0 ratio) as compared to 66±10% and 36±11% in 1:2 and 1:10 groups, respectively (p=0.05). As observed in the marrow, also the spleen of animals receiving CD34+ and T cells included >98% CD3+ T cells. Among the T cells, both in the marrow and in the spleen of mice in the 1:2 and 1:10 ratio groups, 60-70% were CD4+CD8- cells, 22-25% CD8+CD4- cells, 1-3% CD56+ cells, and 2-5% CD4+CD25+ cells. In mice receiving 4 ×105 T cells (1:2 ratio), on average 12±6% of the T cells in the bone marrow and spleen were CD4+CD8+. Only mice receiving 2×106 T cells (1:10 ratio) showed GVHD. This was demonstrated by fur changes, reduced survival (p=0.02) and weight loss (p=0.0001) compared to control mice or mice receiving a lower dose of T cells (1:2 ratio). The marrow engraftment of CD3+ cells with disappearance of CD34+ cells in mice receiving low doses of allogeneic T cells, in the absence of evident xenogeneic GVHD, suggests that NOG mouse model represents a useful tool to study human stem cell rejection. This model will be also utilized to investigate new strategies of immunosuppressive cell therapy applied to stem cell transplantation in an HLA mismatched setting. Disclosures: No relevant conflicts of interest to declare.

GPI-80 Defines Primitive Human Cord Blood-Derived CD34-Positive and Negative Hematopoietic Stem Cells

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 2365-2365
Author(s):  
Yoshikazu Matsuoka ◽  
Keisuke Sumide ◽  
Hiroshi Kawamura ◽  
Ryusuke Nakatsuka ◽  
Tatsuya Fujioka ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Human Cord Blood ◽  
Differentiation Potential ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Nog Mice ◽  
High Level

Abstract Background. We identified very primitive CD34-negative (CD34-) severe combined immunodeficiency (SCID)-repopulating cells (SRCs) in human cord blood (CB) using the intra-bone marrow injection (IBMI) method (Blood 2003:101;2924). These CD34- SRCs possess myeloid-biased differentiation potential, which suggests that they are a distinct class of primitive hematopoietic stem cell (HSC) and that they reside at the apex of the human HSC hierarchy (Blood Cancer J 2015:5;e290). We recently developed high-resolution purification methods for CD34- SRCs using 18 Lineage (18Lin)-specific antibodies, which can enrich CD34- SRC at the 1/1,000 level in 18Lin- CD34- fractions (Exp Hematol 2011:39:203). In addition, we previously identified CD133 as a positive marker for CD34- SRCs as well as for CD34+ SRCs (Leukemia 2014:28;1308). The results showed that CD34+/- SRCs were enriched to approximately 1/100 and 1/140 in 18Lin- CD34+/- CD133+ fractions, respectively. Aim. In order to further elucidate the details of the characteristics of human CD34+/- HSCs, we aimed to identify additional positive markers for the high-level purification of CB-derived CD34+/- SRCs. Materials and Methods. First, weextensively analyzed the candidate positive markers, including cell adhesion molecules and homing receptors that are expressed on 18Lin- CD34+ CD38- and 18Lin- CD34- cells by multicolor FACS. Finally, we discovered that glycosylphosphatidylinositol-anchored protein GPI-80, which has recently been reported as a marker for human fetal liver hematopoietic stem/progenitor cells (HSPCs) (Cell Stem Cell 2015:16;80), was also expressed on human full-term CB-derived 18Lin- CD34+ CD38- and 18Lin- CD34- cells. Next, we sorted 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells from human CB. The HSPC characteristics of the 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells were assessed as follows: (1) the in vitro maintenance/production capacities of CD34+ and CD34+ CD38- cells were examined in co-cultures with mesenchymal stroma cells (MSCs) established from human bone marrow-derived CD45- Lin- CD271+ SSEA-4+ cells (Stem Cells 2015:33;1554); (2) an SRC assay was performed using NOD/Shi-scid/IL-2Rγcnull (NOG) mice; (3) limiting dilution analyses (LDAs) were performed to determine the SRC frequencies in these four fractions of cells. Results. In the CB-derived 18Lin- CD34+ CD38- and 18Lin- CD34- fractions, 10.1% and 14.4% of cells expressed GPI-80, respectively. The 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- cells were then co-cultured with human MSCs for 7 days in the presence of SCF and TPO. As a result, the 18Lin- CD34+ CD38- GPI-80+ cells maintained significantly higher percentages of CD34+ (86.4%) and CD34+ CD38- cells (24.8%) in comparison to 18Lin- CD34+ CD38- GPI-80- cells (78.7% and 14.3%, respectively). However, 18Lin- CD34- GPI-80+/- cells produced comparable levels of CD34+ (50.3% and 50.8%) and CD34+ CD38- cells (4.4% and 5.3%). These four fractions of cells were then transplanted into NOG mice using the IBMI method. All of these four fractions of cells showed long-term repopulating SRC activities with multi-lineage differentiation potential in mouse bone marrow. However, LDAs demonstrated that the frequencies of SRC in the 18Lin- CD34+ CD38- GPI-80+/- and 18Lin- CD34- GPI-80+/- fractions were 1/21, 1/35 and 1/28, 1/874, respectively. These data clearly demonstrate that both CD34+/- SRCs are enriched in GPI-80+ fractions. Surprisingly, a number of mice received a limited number of 18Lin- CD34+ CD38- GPI-80+ (2 cells) and 18Lin- CD34- GPI-80+ cells (10 cells), and thus also showed multi-lineage long-term human hematopoietic cell repopulation. Conclusion. These observations clearly demonstrated that GPI-80 defines CB-derived human primitive HSCs. Furthermore, these results indicate that GPI-80 is a useful marker for the high-level purification of human CB-derived CD34+/- SRCs (HSCs). Disclosures No relevant conflicts of interest to declare.

Local irradiation enhances congenic donor pluripotent hematopoietic stem cell engraftment similarly in irradiated and nonirradiated sites

Blood ◽  
2004 ◽  
Vol 103 (5) ◽  
pp. 1949-1954 ◽  
Author(s):  
Hiroshi Ito ◽  
Yasuo Takeuchi ◽  
Juanita Shaffer ◽  
Megan Sykes
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Local Irradiation ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Cell Engraftment ◽  
High Bone ◽  
Bone Marrow Doses

AbstractLong-term multilineage chimerism is achieved in CD45 congenic mice receiving high bone marrow doses with or without mediastinal irradiation (MI). Increased donor chimerism results in MI-treated compared with nonirradiated animals, suggesting that MI makes “space” for engraftment of donor pluripotent hematopoietic stem cells (PHSCs). We have now examined whether space is systemic or whether increased engraftment of donor marrow in locally irradiated mice is confined to the irradiated bones. While increased donor chimerism was observed in irradiated bones compared with nonirradiated bones of MI-treated animals 4 weeks following bone marrow transplantation (BMT), these differences were minimal by 40 weeks. MI-treated chimeras contained more adoptively transferable donor PHSCs in the marrow of both irradiated and distant bones compared with non-MI–treated chimeras. Similar proportions of donor PHSCs were present in irradiated and nonirradiated bones of locally irradiated mice at both 4 and 40 weeks. Irradiated bones contained more donor short-term repopulating cells than distant bones at 4 weeks, but not 40 weeks, after BMT. Our study suggests that local proliferation of donor PHSCs in mice receiving local irradiation rapidly leads to a systemic increase in donor PHSC engraftment.

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