Evidence That Human NKT Cells Enhance Haemopoiesis through Recognition of CD1d Expressed in Haemopoietic Stem Cells with Long Term Clonogenic Capacity.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4129-4129
Author(s):  
Ioannis Kotsianidis ◽  
Scott Patterson ◽  
Marianna Politou ◽  
Antonio Almeida ◽  
Despoina Pantelidou ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Cord Blood ◽  
Ex Vivo ◽  
Nkt Cells ◽  
Short Term ◽  
Gm Csf ◽  
Haemopoietic Stem Cells ◽  
Cd34 Cells

Abstract NKT cells, a novel class of regulatory T cells, secrete haemopoietic cytokines (GM-CSF, IL-3, IL-6) upon engagement of their TCR. Because of this property we hypothesized that NKT cells are involved in the regulation of haemopoiesis. NKT cells constitute <0.1% of T cells in blood, bone marrow and cord blood and are restricted by the glycolipid-presenting, non-polymorphic MHC-like molecule CD1d. CD1d is expressed in antigen presenting cells, thymocytes and in a variety of epithelial tissues including keratinocytes and enterocytes, but its expression in haematopoietic stem cells (HSC) has not been studied. Therefore we studied first the expression and function of CD1d in haemopoietic stem cells. Using multi-colour flow cytometry, we show that 1% of MACS-selected cord blood CD34+ cells are CD1d+ (n=6, range 0.4–1.67%). CD1d+CD34+ HSC express a variety of surface markers indicative of primitive HSC: CD7: 36.1% (35.6–36.4%), CD133: 68% (46.2–81.54%), CD117:74.2% (51–85.5%) and CD90 (Thy-1): 32.3% (26.7–39.1%); moreover, 6,2% (1.9–10.5%) and 12,8% (10.1–16%) of CD1d+CD34+ are CD1d+CD34+HLADR− and CD1d+CD34+CD38− respectively, consistent with an immature HSC phenotype. Expression of these markers by CD1d−CD34+ were identical to CD1d+CD34+ cells. Consistent with this, in long-term colony initiating cell (LTC-IC) assays (n=4), highly purified, flow-sorted, lineage-depleted (Stem Cell Technologies) Lin-CD1d+CD34+ HSC displayed a LTC-IC frequency of 1 in 35.7 cells (range 24.4–38) equivalent to those of CD1d−CD34+ HSC: LTC-IC frequency of 1 in 25.4 cells (range 20–30.3). Short term CFC activity of Lin−CD34+CD1d+ is slightly lower than their Lin−CD1d−CD34+ counterparts: 1 CFC per 15.3 cells (7.7–37.7) versus 1 CFC per 4.2 cells (3.8–5), respectively. To investigate the effect of cord blood NKT cells on the CFC activity of CD34+ cells, NKT were first activated ex vivo for 10 days in the presence of the CD1d-presented glycolipid a-galactosylceramide and subsequently were purified by flow-sorting using mAbs specific to their TCR a and b chains, i.e., anti-TCR Va24 and Vb11. Purified NKT were co-cultured in a ratio of 10:1 with CD34+ cells. In the absence of exogenous cytokines NKT enhanced the clonogenic capacity of CD34+ cells by 3-fold: 1 CFC per 14 cells (range 10.4–21) in the presence of NKT vs 1 per 43 cells (range 37–55.5) in the absence of NKT (n=4; p=0.024). By contrast, activated or resting autologous T cells co-cultured with CD34+ cells at the same ratio (10:1) had no effect on the CFC frequency, indicating that this enhancing effect on haemopoiesis is a unique property of NKT cells. The effect of NKT in the long-term clonogenic capacity is currently being evaluated. In summary, we have shown that a) CD1d is a novel marker expressed in HSC with long- and short -term clonogenic ability and b) CD1d-restricted NKT cells promote haemopoiesis These findings reveal a novel link between haemopoiesis and the CD1d-NKT axis of immune regulation and set the scene for the study of the role of NKT cells in the processes of engraftment and rejection in HSC transplantation.

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

2007 ◽  
Vol 16 (6) ◽  
pp. 579-585 ◽  
Author(s):  
Guo-Ping Huang ◽  
Zhi-Jun Pan ◽  
Bing-Bing Jia ◽  
Qiang Zheng ◽  
Chun-Gang Xie ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Mesenchymal Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord Blood ◽  
Ex Vivo ◽  
Hematopoietic Stem ◽  
Cd34 Cells

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.

Fibronectin Enhances Ex Vivo Expansion and Maturation of Megakaryocyte Progenitors from Umbilical Cord Blood.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 698-698 ◽  
Author(s):  
Varda Deutsch ◽  
Einav Hubel ◽  
Kay Sigi ◽  
Ariel Many ◽  
Elizabeth Naparstek ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Ex Vivo ◽  
Ex Vivo Expansion ◽  
Target Cells ◽  
Short Term ◽  
Hematopoietic Stem ◽  
Platelet Production ◽  
Cd34 Cells

Abstract Following cord blood (CB) transplant and bone marrow (BM) protracted thrombocytopenia remains a serious clinical problem. Platelet production following transplant depends on the availability of adequate numbers of cytokine responsive stem and megakaryocyte progenitor cells (MK-p). Thrombopoietin (TPO), had no clinical impact on thrombopoiesis when given to patients post BMT due to the paucity of MK-p in the grafts. If expanded, Mk-p would supply the appropriate target cells to maximize the effect of TPO and provide efficient earlier platelet engraftment. We propose a novel strategy to facilitate thrombopoiesis, by expanding MK-p from CB mononuclear cells (MNC) prior to transplantation in short term cultures. While CB CD34+ cells can be expanded by several reported methods, isolation of CD34+ cells from the fresh CB is not practical due to the limited number of stem and progenitor cells in the CB units. Additionally, MK expansion from purified stem cells requires long culture periods which are inappropriate for transplantation. We aimed to improved techniques for enrichment and ex-vivo expansion of MK-p and hematopoietic stem cells, from small aliquots of whole CB, using 7–10 days cultures and new growth conditions. CB progenitors were enriched by separation of MNC from RBC on gelatin followed by centrifugation on ficoll, as we previously reported (1). MNC were expanded on fibronectin (FN) coated dishes in the presence of autologous plasma with various new cytokine combinations. These included r-hu-TPO (10 ng/ml), b- FGF (10 ng/ml), r-hu-SCF (50 ng/ml) and ARP a peptide derived from the stress variant of acetylcholinesterase (AChE-R) recently discovered to have potent hematopoietic stem cell and MK growth factor activity (2). The cell populations, MK and MK-p were characterized by high resolution flow cytometry on day 0 and 10 of culture using SSC, CD41 and CD34. True MK expansion was assessed by appropriate gating out of granulocyte and monocytes, which acquire CD41+ adherent platelets in culture. FN alone, without any other growth supplement increased the viability of cells in culture and expansion of MK-p (CD41high, SSClow and FSClow) by 2.8±1.1 (P < 0.05) fold. The combination of FN with TPO enhanced MK-p number by 4.8±2.7 and the addition of either SCF or b-FGF or ARP further stimulated the expansion of MK-p all producing about a 6 fold increase (P < 0.05). Further analysis was performed on the maturing MKs which were characterized as CD41high, CD45low/negative, CD34negative. Increased Mk ploidy was found when either b-FGF or ARP were added to cultures containing TPO, grown on FN coated plates. Significant MK maturation, as measured by GPIIb/IIIa expression using real time quantitative PCR, was also found. The combination of FN and TPO increased the MK colony forming progenitors in culture by 9 fold and up to 35 fold when other supplements were added. We demonstrate that short term expansion of enriched MK-p from a small fraction of the CB unit is feasible and easy to perform and can comply with GTP regulations. This approach may lead to the development of more effective cell therapy modalities to facilitate platelet production and decrease the time of thrombocytopenia in severely myelosuppressed patients during the extended nadir before platelet engraftment.

Cytokine Production and Hematopoiesis Supporting Activity of Cord Blood Derived Unrestricted Somatic Stem Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 1279-1279
Author(s):  
Gesine Koegler ◽  
Teja Radke ◽  
Aurelie Lefort ◽  
Sandra Sensken ◽  
Johannes Fischer ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Cytokine Production ◽  
Ex Vivo ◽  
Somatic Stem Cells ◽  
Expression Array ◽  
Gm Csf ◽  
Cd34 Cells ◽  
Marrow Mesenchymal Stem Cells ◽  
Unrestricted Somatic Stem Cells

Abstract Particularly within the last several years cord blood (CB) has become a well established hematopoietic source for unrelated transplantation in children. Assuming, however, a threshold of 2x107 nucleated cells (NC)/kg body weight required for transplantation in adults over 70kg, an average of only 25% of the CB units presently stored in the major CB banks contain sufficient NC to safely engraft patients. Thus far the majority of studies have assessed selective cytokine driven expansion of progenitors for transplantation, which however may be associated with exhaustion of stem cells and an increase of GvHD in adult patients. Therefore, we assessed cytokine production and hematopoiesis supporting stromal activity of CB derived unrestricted somatic stem cells (USSC, Koegler et al. JEM2004: 200: 2: 1–13) in comparison to bone marrow mesenchymal stem cells (BMMSC) and hematopoietic progenitor expansion solely driven by recombinant cytokines. Initiation of USSC generation was initiated from fresh (n=325) and cryopreserved CB (n=66). In the presence of H5100 medium/10−7M dexamethasone, USSC cultures from fresh CB were initiated from 57.1% of CB samples (n= 52 out of 91 CB samples), whereas in DMEM-medium/10−7M dexamethasone the frequency was 31.6% (n=74 out of 234 CB samples). The generation frequency from frozen HES separated CB was only 19.5%. Cytokine production by USSC and BMMSC was determined qualitatively by cytokine mRNA expression array analyses or quantitatively by Luminex or ELISA analyses. To analyze hematopoiesis supporting activity, CB CD34+ cells were expanded in co-cultures with USSC, BMMSC or in the presence of Flt3-L, SCF, TPO. Expansion of CD34+ cells, total cells and colony-forming cells (CFC) were determined after 1, 2, 3 and 4 weeks of culture. USSC constitutively produced SCF, LIF, TGF-1ß, M-CSF, GM-CSF, VEGF, IL-1ß, IL-6, IL-8, IL-11, IL-12, IL-15, SDF-1a and HGF. When USSC were stimulated with IL-1ß, G-CSF was released. Production of SCF (p= 0.0104) and LIF (p= 0.046) was significantly higher in USSC compared to BMMSC. At 1, 2, 3 and 4 weeks, respectively co-cultivation of CD34+ cells on the USSC layer resulted in a 14.62±1.1-fold, 110.1±17.9-fold, 151.8±39.7-fold and 183.6±40.4-fold amplification of total cells and in a 30.56±4.4-fold, 101.4±27.5-fold, 64.67±15.8-fold and 29.4±3.1-fold amplification of CFC. Expansion achieved was significantly higher for USSC compared to BMMSC feeder layers. USSC produce functionally significant amounts of hematopoiesis supporting cytokines and are able to expand CD34+ cells from CB in significantly higher amounts compared to BMMSC. Therefore the USSC is a suitable candidate to direct ex-vivo expansion of hematopoietic CB cells for short term reconstitution.

scholarly journals Engraftment and Retroviral Marking of CD34+ and CD34+CD38− Human Hematopoietic Progenitors Assessed in Immune-Deficient Mice

Blood ◽  
1998 ◽  
Vol 91 (4) ◽  
pp. 1243-1255 ◽  
Author(s):  
Mo A. Dao ◽  
Ami J. Shah ◽  
Gay M. Crooks ◽  
Jan A. Nolta
Keyword(s):  
Stem Cells ◽  
Ex Vivo ◽  
Human Stem Cells ◽  
Hematopoietic Stem ◽  
Human Cd34 ◽  
Cd34 Cells ◽  
Low Levels ◽  
Ex Vivo Culture ◽  
Daunting Challenge

Abstract Retroviral-mediated transduction of human hematopoietic stem cells to provide a lifelong supply of corrected progeny remains the most daunting challenge to the success of human gene therapy. The paucity of assays to examine transduction of pluripotent human stem cells hampers progress toward this goal. By using the beige/nude/xid (bnx)/hu immune-deficient mouse xenograft system, we compared the transduction and engraftment of human CD34+progenitors with that of a more primitive and quiescent subpopulation, the CD34+CD38− cells. Comparable extents of human engraftment and lineage development were obtained from 5 × 105 CD34+ cells and 2,000 CD34+CD38− cells. Retroviral marking of long-lived progenitors from the CD34+ populations was readily accomplished, but CD34+CD38− cells capable of reconstituting bnx mice were resistant to transduction. Extending the duration of transduction from 3 to 7 days resulted in low levels of transduction of CD34+CD38− cells. Flt3 ligand was required during the 7-day ex vivo culture to maintain the ability of the cells to sustain long-term engraftment and hematopoiesis in the mice.

scholarly journals Cord-Blood-Stem-Cell-Derived Conventional Dendritic Cells Specifically Originate from CD115-Expressing Precursors

Cancers ◽  
2019 ◽  
Vol 11 (2) ◽  
pp. 181 ◽  
Author(s):  
Maud Plantinga ◽  
Colin G. de Haar ◽  
Ester Dünnebach ◽  
Denise A.M.H. van den Beemt ◽  
Kitty W.M. Bloemenkamp ◽  
...  
Keyword(s):  
Stem Cells ◽  
T Cells ◽  
Dendritic Cells ◽  
Stem Cell ◽  
Cord Blood ◽  
Ex Vivo ◽  
Treatment Success ◽  
Gene Set Enrichment Analysis ◽  
Long Term Memory ◽  
Vaccination Strategies

Dendritic cells (DCs) are professional antigen-presenting cells which instruct both the innate and adaptive immune systems. Once mature, they have the capacity to activate and prime naïve T cells for recognition and eradication of pathogens and tumor cells. These characteristics make them excellent candidates for vaccination strategies. Most DC vaccines have been generated from ex vivo culture of monocytes (mo). The use of mo-DCs as vaccines to induce adaptive immunity against cancer has resulted in clinical responses but, overall, treatment success is limited. The application of primary DCs or DCs generated from CD34+ stem cells have been suggested to improve clinical efficacy. Cord blood (CB) is a particularly rich source of CD34+ stem cells for the generation of DCs, but the dynamics and plasticity of the specific DC lineage development are poorly understood. Using flow sorting of DC progenitors from CB cultures and subsequent RNA sequencing, we found that CB-derived DCs (CB-DCs) exclusively originate from CD115+-expressing progenitors. Gene set enrichment analysis displayed an enriched conventional DC profile within the CD115-derived DCs compared with CB mo-DCs. Functional assays demonstrated that these DCs matured and migrated upon good manufacturing practice (GMP)-grade stimulation and possessed a high capacity to activate tumor-antigen-specific T cells. In this study, we developed a culture protocol to generate conventional DCs from CB-derived stem cells in sufficient numbers for vaccination strategies. The discovery of a committed DC precursor in CB-derived stem cell cultures further enables utilization of conventional DC-based vaccines to provide powerful antitumor activity and long-term memory immunity.

Ex-Vivo Generation and Expansion of Both Lymphoid and Myeloid Lineages from Human Cord Blood (CB) HSC Using a Serum-Free Human Mesenchymal Stem Cell Based Culture System.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 2888-2888
Author(s):  
Ana Frias ◽  
Christopher D. Porada ◽  
Kirsten B. Crapnell ◽  
Joaquim M.S. Cabral ◽  
Esmail D. Zanjani ◽  
...  
Keyword(s):  
Stem Cell ◽  
Cord Blood ◽  
Culture System ◽  
Ex Vivo ◽  
Cell Number ◽  
Human Cord Blood ◽  
Serum Free ◽  
Gm Csf ◽  
Cd34 Cells

Abstract The in vitro culture of a hematopoietic stem cell (HSC) graft with either media containing animal-derived components or a feeder layer with ill-defined pathogenic potential such as xenogeneic cell lines or cells modified by viral transformation poses risks that concern scientists and regulatory agencies. In the present studies, we avoided these risks by evaluating the ability of a human stromal-based serum free culture system (hu-ST) to support the ex-vivo expansion/maintenance of human CB HSC. CB CD34+ enriched cells were cultured in serum free medium in the presence of hu-ST with SCF, bFGF, LIF and Flt-3, and the cultures were analyzed for expansion, phenotype and clonogenic ability. We have previously reported the ability of this culture system to allow the successful expansion/maintenance of HSC along the myeloid pathway. In the present study, we investigated whether we could further develop this culture system to simultaneously expand myelopoiesis and lymphopoiesis in vitro. To this end, cord blood CD34+ cells were cultured for a total of 28 days and analyzed every 3 days for expansion and phenotype. There was a progressive increase in CD34 cell number with time in culture. The differentiative profile was primarily shifted towards the myeloid lineage with the presence of CD33, CD15, and CD14. However, a significant number of CD7+ cells were also generated. At week 2 of culture, we observed that 30% of the cells in the culture were CD7 positive. These CD7+CD2-CD3-CD5-CD56-CD16-CD34- cells were then sorted and either plated on top of new irradiated hu-ST layers in the presence of SCF, FLT-3, IL-7, IL-2, and IL-15, or cultured with IL-4, GM-CSF, and FLT-3 in the absence of stroma. Both of these cultures were maintained for an additional 2 weeks. In both sets of cultures, further expansion in the total cell number occurred with the time in culture, and by the end of the week 2, we observed that 25.3±4.18% of the cells had become CD56+ CD3-, a phenotype consistent with that of NK cells. Furthermore, cytotoxicity assays were performed and showed cytotoxic activity that increased in an E:T ratio-dependent fashion. 38.6% of the CD7+ cells grown in the presence of IL-4, GM-CSF, and FLT-3 became CD123+CD11c-, a phenotype characteristic of nonactivated dendritic cells, while 7.3–12.1% adopted an activitated dendritic cell phenotype CD83+CD1a+. In summary, we developed an in vitro culture system that reproducibly allows the effective ex vivo expansion of human cord blood HSCs while maintaining the capability of generating both myeloid and lymphoid hematopoiesis in vitro.

Deliniation of the Ex Vivo Culture Conditions Supporting the Long-Term Engraftment of Cord Blood CD34+ Cells.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4954-4954
Author(s):  
Ronald L. Brown ◽  
J. Zhang ◽  
L. Qiu ◽  
A. Nett ◽  
G. Almeida-Porada ◽  
...  
Keyword(s):  
Cord Blood ◽  
Ex Vivo ◽  
Culture Conditions ◽  
Cell Populations ◽  
Multilineage Differentiation ◽  
Cd34 Cells ◽  
Ex Vivo Culture ◽  
Cytokine Combination ◽  
Cells Cultured

Abstract Ex-vivo expansion regimens for cord blood (CB) CD34+ cells that maintain their long term engrafting ability hold great promise for adult transplantation but have been met with relatively little success. Data presented delineate the development of a cell cu1ture system composed of clinical grade serum-free medium (QBSF 60) and a cytokine combination that not only yields large numbers of CD34+ cell populations but also supports the long term engraftment of these cells. CBCD34+ cells were cultured for over 14 days in QBSF 60 medium supplemented with the following cytokine combination a.) SCF, Flt-3 and TPO, b.) SCF, Flt-3 and IL-6, c.) SCF, Flt-3 TPO and IL-3, d.) SCF Flt-3, TPO and IL-6, e.) SCF, Flt-3, TPO and IL-11, f.) SCF, Flt-3, TPO, IL-3, IL-6 and IL-11, g.) SCF, Flt-3, TPO, IL-3, IL-6, IL-11, G-CSF, and EPO. The following cytokine concentrations was used for each of the above combinations: SCF (50 ng/ml), Flt-3 (100 ng/ml), TPO (100 ng/ml), IL-3 (20 ng/ml), IL-6 (50 ng/ml), IL-11 (50 ng/ml), G-CSF (50 ng/ml) and EPO (10U), or 10 times lower concentrations of each cytokine. The ex vivo cultured were evaluated for the following cell populations: total nucleated cells, CD34+ cells, CD34+ CD38− cells, CFU-C, HPP-CFU, and LTC-IC. In all cases those combinations of cytokines containing either IL-3 and/or IL-6 yielded higher quantities of all the cellular populations studied. Those culture conditions having the fewest cytokines that yielded large quantities of total cells, CD34+ cells and/or CD34+ CD38− cells were subsequently examined after 14 days of culture for their long-term engrafting ability in the fetal sheep model for human hematopoiesis. Typically, after 14 days of ex vivo culture CD34+ cells fail to engraft long-term, therefore, all our cultures were maintained for at least this time frame. Based on these criteria, CD34+ cells cultured in the presence of the higher concentration of cytokines a, b d and f were examined. The cultured CD 34+ cells from all four cytokine combinations engraft and undergo multilineage differentiation in primary recipients (short-term engraftment) examined 63 days post-transplant. By contrast the secondary recipients (long-term engraftment) after 61 days post-transplant showed no engraftment from cells cultured in cytokine combinations a and f, very few human cells were found in secondary recipients engrafted with cells from cytokine concentration b, but cells cultured in cytokine combination d (SCF, Flt-3, TPO and IL-6) maintained their long-term engrafting ability and undergo multilineage differentiation. In conclusion, cytokine combinations of TPO and IL-6 with SCF and Flt-3 yielded successful long-term engraftment. The presence of IL-3 in any of there combinations supported excellent cellular proliferation and the increase in the various cell populations but failed to support engraftment. These studies suggest that it is possible to maintain/expand long-term engrafting CB stem cells after 14 days under clinically relevant culture conditions.

Role of Osteoclasts in Regulation of Human Hematopoietic Stem Cell Niche and Fate.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4253-4253
Author(s):  
Shmuel Yaccoby ◽  
Kenichiro Yata ◽  
Yun Ge ◽  
Bart Barlogie ◽  
Joshua Epstein ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Scid Mice ◽  
Common Mechanism ◽  
Hematopoietic Stem ◽  
Variable Expression ◽  
Cd34 Cells ◽  
Rabbit Bone

Abstract Recent studies indicate that osteoblasts play an important role in maintaining hematopoietic stem cells (HSCs) niche in the bone marrow microenvironment. The aim of study was to test the effect of osteoclasts on the fate of HSCs in a long term co-culture assay. To generate osteoclasts, peripheral blood mononuclear cells from mobilized donors were cultured for 6–10 days in αMEM media supplemented with 10% FCS, M-CSF and RANKL. After removal of non-adherent cells, the cultures contained 95% multinucleated osteoclasts and their precursors. These osteoclasts expressed TRAP and formed resorption pits on bone slices (Yaccoby et al., Cancer Res., 2004). CD34+ cells were purified from donor PBSCs and cord blood using immunomagnetic beads separation (>95% purity). Adult and cord blood HSCs were co-cultured with osteoclasts for up to 3 and 10 months, respectively, in media lacking any cytokines. Because osteoclasts do not survive long without M-CSF and RANKL, the HSCs were transferred to fresh osteoclast cultures every 6–10 days. Unlike their tight adherence to stromal cells, HSCs did not adhere to the osteoclasts and were easily recovered from co-cultures by gentle pipetting. Following 1 to 3 weeks of co-culture, committed HSCs rapidly differentiated into various hematopoietic cell lineage, followed by phagocytosis of terminal differentiated hematopoietic cells by the osteoclasts. The remaining HSCs were highly viable (>90% by trypan blue exclusion) and gradually lost their CD34 expression, so that the cultures contained subpopulations of HSCs expressing CD34−/lowCD38+ and CD34−/lowCD38−. Quantitive real time RT-PCR (qRT-PCR) revealed loss of expression of CD34 and reduced expression of CD45 by HSCs co-cultured with osteoclasts longer than 6 weeks. Variable expression of CD34 on HSCs was previously reported in murine but not human HSCs (Tajima et al., Blood, 2001). The co-cultured HSCs showed reduced capacity of generating in vitro hematopoietic colonies, and did not differentiate into osteoclasts upon stimulation with M-CSF and RANKL. We next tested the long term engraftment of these co-cultured HSCs in 2 animal models. In the first model, cord blood and adult HSCs from 2 donors recovered after >6 weeks in co-culture were injected I.V. into irradiated NOD/SCID mice. In the second novel model, co-cultured cord blood and adult HSCs from 2 donors were injected directly into rabbit bones implanted subcutaneously in SCID mice (SCID-rab model), 6–8 weeks after rabbit bone implantation. After 2–4 months, 10%±3% human CD45-expressing cells were identified in the NOD/SCID mice femora and 8%±4% in the SCID-rab mice rabbit bone. Our study suggests that osteoclasts promote rapid differentiation of committed HSCs and induce conversion of CD34+ cells to CD34− stem cells with self renewal potential. Intriguingly, long term co-culture of primary CD138-selected myeloma plasma cells (n=16) with osteoclasts resulted in dedifferentiation of tumor cells from a mature CD45− phenotype to an immature, CD45-expressing cells, suggesting a common mechanism of osteoclast-induced HSC and myeloma cell plasticity. This indicates that osteoclasts are important bone marrow component regulating human HSC niche, plasticity and fate.

Impaired Alloantigen Presenting Activity of Cord Blood Nucleated Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 2203-2203
Author(s):  
Sandeep Chunduri ◽  
Dolores Mahmud ◽  
Javaneh Abbasian ◽  
Damiano Rondelli
Keyword(s):  
T Cells ◽  
Stem Cell ◽  
T Cell ◽  
Cord Blood ◽  
Mononuclear Cells ◽  
Ex Vivo ◽  
Solid Organ Transplantation ◽  
T Cell Response ◽  
Solid Organ ◽  
Cd34 Cells

Abstract Transplantation of HLA-mismatched cord blood (CB) nucleated cells has limited risk of severe acute graft-versus-host disease and graft rejection. This may depend on naïve T cells not yet exposed to many antigens and on immature antigen-presenting cells (APC) not delivering appropriate signals to allogeneic T cells. In order to test the APC activity of human circulating CB cells in-vitro, we initially used irradiated CB mononuclear cells (MNC) or immunomagnetically selected CD34+ cells, CD133+ cells, or CD14+ monocytes to stimulate the proliferative response of incompatible blood T cells in mixed leukocyte culture (MLC). CB MNC failed to induce allogeneic T cell proliferation, while CD34+ and CD133+ progenitors or CD14+ monocytes induced potent T cell alloresponses. Nevertheless, since allogeneic T cell response was not restored after depletion of CD3+ cells in the CB, nor the add-back of irradiated CB MNC to CD34+ or CD14+ stimulators inhibited allo-T cells, a direct suppressive effect of CB MNC was excluded. Allogeneic peripheral blood cytotoxic T-lymphocyte (CTL) responses were not induced after 7 days of stimulation with irradiated CB MNC, although after 4 weekly rechallenges with CB MNC, on average a 23% lysis of antigen-specific CB PHA-blasts was observed at the highest effector:target ratio (50:1). To test the tolerogenic potential of CB MNC, T cells initially exposed to CB MNC were rechallenged in secondary MLC with CB MNC, or CD34+ cells, or monocyte-derived dendritic cells (Mo-DC) generated in liquid culture with GM-CSF and IL-4. Allogeneic T cells were still unresponsive upon rechallenge with CB MNC, but proliferated upon 3 days of restimulation with CD34+ cells or Mo-DC from the same CB. Surprisingly, the supernatant of these latter MLCs did inhibit completely a 3rd party MLC. Instead, the supernatant of blood T cells that had been activated by CB CD34+ cells or Mo-DC both in primary and secondary MLC did not. These results show an impaired allo-APC activity of CB MNC but not CB CD34+ cells, and suggest that T cells releasing immunosuppressive cytokines may be activated by CB MNC and then expanded by a second more potent stimulation with professional APC. This hypothesis could explain the sustained engraftment of HLA-mismatched CB stem cell transplants in humans. Based on these results, the in-vivo or ex-vivo downregulation of T cell alloreactivity induced by CB MNC will be tested in experimental models of stem cell, as well as solid organ transplantation.

Use of Chromatin Modifying Agents for Ex Vivo Expansion of Human Umbilical Cord Blood Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4208-4208
Author(s):  
Hiroto Araki ◽  
Nadim Mahmud ◽  
Mohammed Milhem ◽  
Mingjiang Xu ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Functional Properties ◽  
Ex Vivo ◽  
Fold Increase ◽  
Scid Mice ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The fixed number of hematopoietic stem cells (HSCs) within a single cord blood (CB) unit has limited the use of CB grafts for allogeneic transplantation in adults. Efforts to promote self-renewal and expansion of HSCs have been met with limited success. Using presently available ex-vivo culture techniques HSCs lose their functional properties in proportion to the number of cellular divisions they have undergone. We hypothesized that chromatin modifying agents, 5-aza-2′-deoxycytidine (5azaD) and histone deacetylase inhibitor, trichostatin A (TSA) could reactivate pivotal genes required for retaining the functional properties of dividing HSC. We have demonstrated previously that the fate of human bone marrow CD34+ cells could be altered by the addition of 5azaD/TSA (Milhem et al. Blood.2004;103:4102). In our current studies we hypothesized that in vitro exposure of CB CD34+ cells to chromatin modifying agents might lead to optimal HSC expansion to permit transplantation of adults. A 12.5-fold expansion was observed in the 5azaD/TSA treated CD34+CD90+ cell cultures containing SCF, thrombopoietin and FLT3 ligand (cytokines) in comparison to the input cell number. Despite 9 days of culture, 35.4% ± 5.8% (n = 10) of the total cells in the cultures exposed to chromatin modifying agents were CD34+CD90+ as compared to 1.40 % ± 0.32% in the culture containing cytokines alone. The 12.5-fold expansion of CD34+CD90+ cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold expansion of cobblestone area-forming cells (CAFC). The frequency of SCID repopulating cells (SRC) was 1 in 26,537 in primary CB CD34+CD90+ cells but was increased to 1 in 2,745 CD34+CD90+ cells following 9 days of culture in the presence of 5azaD/TSA resulting in a 9.6-fold expansion of the absolute number of SRC. In contrast, the cultures lacking 5azaD/TSA had a net loss of both CFC/CAFC as well as SRC. The expansion of cells maintaining CD34+CD90+ phenotype was not due to the retention of a quiescent population of cells since all of the CD34+CD90+ cells in the culture had undergone cellular division as demonstrated by labeling with a cytoplasmic dye. CD34+CD90+ cells that had undergone 5–10 cellular divisions in the presence of 5azaD/TSA but not in the absence still retained the ability to repopulate NOD/SCID mice. 5azaD/TSA treated CD34+CD90+ cells, but not CD34+CD90- cells were responsible for in vivo hematopoietic repopulation of NOD/SCID assay, suggesting a strong association between CD34+CD90+ phenotype and their ability to repopulate NOD/SCID mice. We next assessed the effect of 5azaD/TSA treatment on the expression of HOXB4, a transcription factor which has been implicated in HSC self-renewal. A significantly higher level of HOXB4 protein was detected by western blot analysis after 9 days of culture in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. The almost 10-fold increase in SRC achieved using the chromatin modifying agents should be sufficient to increase the numbers of engraftable HSC within a single human CB unit so as to permit these expanded grafts to be routinely used for transplanting adult recipients.

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