CD34-positive Hematopoietic Progenitor Cells Research Articles

Factors associated with the number of CD34-positive hematopoietic progenitor cells in the leukocyte concentrate in patients with lymphomas and acute T-lymphoblastic leukemia

Background. Autologous hematopoietic stem cell transplantation remains in demand for patients with hematological malignancies. The pool of hematopoietic stem cells is known to be heterogeneous. e studied various factors associated with previous treatment, patient and disease characteristics, as well as the composition of the C34+ pool in peripheral blood (), associated with the number of C34+ cells in the first leukocyte concentrate (LC).Aim. To determine the factors associated with C34+ , C34+ C143+ , C34+ C38– HLA-DR+/– and C34+ C38+/– HLA-DR– cells count in the first LC of patients with hematological malignancies.Materials and methods. Subpopulations of hematopoietic stem cells in the and first LC were studied in 80 patients with hematological malignancies (male to female ratio 1:1, median age 51 years). The control group included 24 healthy donors. Flow cytometry was used to determine the number of C34+, C34+ C38– HLA-DR+/–, C34+ C38+/– HLA-DR– and C34+ C143+ cells. Immunophenotyping was performed on samples of patients before mobilization and on the 1st day of leukapheresis, as well as on first LC samples on the 1st day of hematopoietic stem cells collection.Results. It was shown that the presence of early progenitor cells with C34+ C38– HLA-DR+/– immunophenotype in the before mobilization is associated with a higher first LC C34+ cells number (p = 0.003). In the presence of C34+ C38– HLA-DR+/– cells in the before mobilization, the median number of C34+ cells in the first LC was 2.6 %, and in their absence – 0.71 %. hen using granulocyte colonystimulating factor as monotherapy, the C34+ C143+ cells number in the first LC was significantly lower than when using chemotherapy and granulocyte colonystimulating factor (p = 0.03). However, the majority (9 of 16) of patients in the granulocyte colonystimulating factor monotherapy group had renal failure before mobilization. No factors associated with the number of C34+ C38– HLA-DR+/– cells in the first LC were found. In patients >60 years old, the number of C34+ C38+/– HLA-DR– cells in the first LC was lower than in patients ˂ 60 years old, the number of C34+ C38+/– HLA-DR– cells in the first LC was lower than in patients ˂ 60 years old (p = 0.043). In patients with infectious complications during hematopoietic stem cell mobilization, the number of C34+ C38+/– HLA-DR– cells in the first LC was lower than in patients without them (p = 0.019).Conclusion. The first LC number of C34+ cells is associated with number of C34+ C38– HLA-DR+/– cells, i. e. cells of the early differentiation stage with a high proliferation potential. A relationship was established between C34+ C143+ cells number before mobilization and first LC C34+ cells count: in the paired model – within the borderline values (p = 0.05), and in the multifactorial covariance model – with high significance (p = 0.0033). It has been proven that with increasing patient age and in the presence of infectious complications, the number of longterm repopulating C34+ C38+/– HLA-DR– cells decreases.

Specific Uptake in the Bone Marrow Causes High Absorbed Red Marrow Doses During [177Lu]Lu-DOTATATE Treatment.

Bone marrow suppression is a common side effect after [177Lu]Lu-DOTATATE treatment of neuroendocrine neoplasms. Neuroendocrine neoplasms share expression of somatostatin receptor type 2 with CD34-positive hematopoietic progenitor cells, potentially leading to active uptake in the radiosensitive red marrow region where these cells are located. This study aimed to identify and quantify specific red marrow uptake using SPECT/CT images collected after the first treatment cycle. Methods: Seventeen patients diagnosed with neuroendocrine neoplasms were treated with [177Lu]Lu-DOTATATE. Seven of them had confirmed bone metastases. After the first treatment cycle, each patient went through 4 SPECT/CT imaging sessions 4, 24, 48, and 168 h after administration. Monte Carlo-based reconstructions were used to quantify activity concentrations in tumors and multiple skeletal sites presumed to house red marrow: the T9-L5 vertebrae and the ilium portion of the hip bones. The activity concentration from the descending aorta was used as input in a compartment model intended to establish a pure red marrow biodistribution by separating the nonspecific blood-based contribution from the specific activity concentration in red marrow. The biodistributions from the compartment model were used to perform red marrow dosimetry at each skeletal site. Results: Increased uptake of [177Lu]Lu-DOTATATE was observed in the T9-L5 vertebrae and hip bones in all 17 patients compared with activity concentrations in the aorta. The mean specific red marrow uptake was 49% (range, 0%-93%) higher than the nonspecific uptake. The median (±SD) total absorbed dose to the red marrow was 0.056 ± 0.023 Gy/GBq and 0.043 ± 0.022 Gy/GBq for the mean of all vertebrae and hip bones, respectively. The patients with bone metastases had an absorbed dose of 0.085 ± 0.046 Gy/GBq and 0.069 ± 0.033 Gy/GBq for the vertebrae and hip bones, respectively. The red marrow elimination phase was statistically slower in patients with fast tumor elimination, which is in line with transferrin transport of 177Lu back to the red marrow. Conclusion: Our results suggest that specific red marrow uptake of [177Lu]Lu-DOTATATE is in line with observations of somatostatin receptor type 2-expressing hematopoietic progenitor cells within the bone marrow. Blood-based dosimetry methods fail to account for the prolonged elimination of specific uptake and underestimate the absorbed dose to red marrow.

Application of flow cytometry in transfusion medicine: The Sanjay Gandhi Post Graduate Institute of Medical Sciences, India experience.

The application of flow cytometry (FC) is diverse and this powerful tool in used in multiple disciplines such as molecular biology, immunology, cancer biology, virology, and infectious disease screening. FC analyzes a single cell or a particle very rapidly as they flow past single or multiple lasers while suspended in buffered solution. FC has a great impact in the field of transfusion medicine (TM) due to its ability to analyze individual cell population and cell epitopes by sensitive, reproducible, and objective methodologies. The main uses of FC in TM are detection of fetomaternal hemorrhage, diagnosis of paroxysmal nocturnal hemoglobinuria, quantification of D antigen, detection of platelet antibody, quality control of blood components, for example, residual leukocyte counts and evaluation of CD34-positive hematopoietic progenitor cells in stem cell grafts. In recent years, FC has been implemented as an alternative method for the detection and characterization of red cell autoantibodies in autoimmune hemolytic anemia. Many workers considered FC as a very good complement when aberrant expression of various erythrocyte antigens needs to be elucidated. It has been extensively used in the resolution of ABO discrepancies and chimerism study. FC has also been used successfully in various platelet immunological studies. In the recent past, FC has been used in several studies to assess the platelet storage lesions and elucidate granulocyte/monocyte integrity and immunology. FC analysis of CD34+ stem cells is now the method of choice to determine the dosage of the collected progenitor cells. The technique is vastly used to evaluate residual leukocytes in leukodepleted blood components. We conclude that flow cytometers are becoming smaller, cheaper, and more user-friendly and are available in many routine laboratories. FC represents a highly innovative technique for many common diagnostic and scientific fields in TM. Finally, it is the tool of choice to develop and optimize new cellular and immunotherapeutic trials.

An experimental study of menopause induced by bilateral ovariectomy and mechanistic effects of mesenchymal stromal cell therapy on the parotid gland of a rat model

The current study was conducted on a menopause rat model induced by ovariectomy to assess the histological and immunohistochemical alterations in the parotid glands and to verify the efficiency of human umbilical cord derived-mesenchymal stromal cell (hUCB-MSCs) in treating this condition. Eighteen adult female rats were equally divided into three groups: sham-operated (SHAM), ovariectomized (OVX) and OVX injected with hUCB-MSCs (OVX+hUCB-MSCs). At 3months post-ovariectomy, the salivary flow rate and size of the parotid glands were measured. The parotid glands were histologically investigated via H&E stained sections. Furthermore, immunohistochemical analysis for human CD105, human CD34, proliferating cell nuclear antigen (PCNA), single strand DNA (ssDNA), caspase 3, aquaporin (AQP)1, α-smooth muscle actin (α-SMA) and mouse CD34 were performed. The OVX group showed interstitial hemorrhage, dispersed acini and intracytoplasmic vacuoles in the acinar cells. Furthermore, immunohistochemical staining revealed a significant decrement in the number of ssDNA positive apoptotic cells, but a significant increment of PCNA positive proliferating cells, AQP1 positive blood capillaries, α-SMA positive myoepithelial cells and endogenous CD34 positive hematopoietic progenitor cells in the OVX+hUCB-MSCs group as compared with the OVX group. These findings suggest a potential regenerative therapy of MSCs to injured parotid gland structures. However, further investigations are required to illustrate the mechanism of hUCB-MSCs mediated parotid gland regeneration.

Efficient Site-Specific Multi-Gene Engineering of Renewable Pluripotent Cells for Generation of Off-the-Shelf Hematopoietic Immunotherapeutics

Human induced pluripotent stem cells (hiPSCs) are a unique population of cells that can serve as an unlimited source for "off-the-shelf" cellular immunotherapeutics. Similar to master cell lines used in the manufacture of monoclonal antibodies, engineered hiPSC lines have the potential to serve as a renewable cell source for the consistent manufacture of homogeneous populations of effector cells for the treatment of thousands of patients. However, the creation of an effective master line is largely dependent on the ability to genetically edit hiPSCs in a precise, efficient and clonal manner. Furthermore, the genetically edited hiPSCs must maintain their inherent ability to continuously self-renew while retaining ability to express engineered modalities upon directed differentiation to the cell type of choice.We have previously reported the use of stage-specific media compositions to enable the footprint-free generation and long-term maintenance of single cell naïve hiPSCs with enhanced clonogenicity, an attribute critical for the derivation of engineered single cell-derived lines. Here we demonstrate the use of our naïve hiPSC platform to precisely introduce, in a site-specific manner, multiple genes into multiple safe harbor loci. By combining our single-cell naïve hiPSC platform with different nuclease-independent and -dependent strategies, we are able to generate large numbers of precisely engineered iPSC clones. The single cell-derived hiPSC clones were subsequently screened in a multiplexed fashion for successful multi-parameter engineering, maintained pluripotency and propensity for differentiation with lack of undesired phenotypes and genomic alterations. Using this approach, we derived individual clones containing a uniform population (>99%) of multi-engineered modalities consisting of tumor targeting, a controllable safety switch and a tracking marker. Moreover, we show that engineered modalities are expressed in undifferentiated and differentiated hiPSCs, including being expressed in >95% of both CD34 positive hematopoietic progenitor cells and CD56 positive natural killer cells. Furthermore, we have generated hiPSC clones with dual suicide genes (inducible Caspase 9 (iCasp9) and Herpes simplex virus thymidine kinase (HSV-TK)) targeted into two independent safe harbor loci, in both mono- and bi-allelic manner. The dual-targeted hiPSC clones were confirmed to have specific insertions into the predicted sites and were screened to exclude random insertions. Concurrent activation of both suicide genes led to complete elimination of engineered hiPSCs and no treatment-refractory cells were observed unlike the case when only one suicide gene was activated. In addition to robust targeted insertions, we were able to generate small insertions and deletions in up to 70% of naïve hiPSCs without selection and homozygous knockout of gene of interest in 100% of cells after selection. Finally, we will discuss efforts to temporally synchronize ectopic gene expression through endogenously regulated promoters by simultaneous endogenous gene disruption and transgene insertion. Overall, we show our naïve hiPSC platform is an ideal renewable source to efficiently generate, genetically engineer, isolate and bank clonally-derived homogenous population of pluripotent cells. These highly-stable pluripotent clonal banks can be repeatedly tapped to facilitate the consistent production of homogenous populations of potent, persistent, scalable and safer off-the-shelf cellular immunotherapeutics. DisclosuresAbujarour:Fate Therapeutics: Employment. Lan:Fate Therapeutics: Employment. Lee:Fate Therapeutics: Employment. Bonello:Fate Therapeutics: Employment. Meza:Fate Therapeutics: Employment, Equity Ownership. Robinson:Fate Therapeutics: Employment. Clarke:Fate Therapeutics: Employment. Truong:Fate Therapeutics: Employment, Equity Ownership. Robbins:Fate Therapeutics: Employment, Equity Ownership. Rezner:Fate Therapeutics, Inc: Employment, Equity Ownership. Abbot:Fate Therapeutics: Employment. Shoemaker:Fate Therapeutics: Employment, Equity Ownership. Valamehr:Fate Therapeutics, Inc: Employment.

Late Adherent Subpopulation in Umbilical Cord Blood Has the Same Characteristics and Hematopoiesis-Supporting Capacity As Mesenchymal Stromal/Stem Cells

Mesenchymal stromal/stem cells (MSCs) are a major source of cell for cell therapy. MSCs derived from bone marrow (BMMSCs) have been mostly used in clinical applications. BMMSCs can be easily isolated as a cell population that adheres to plastic culture dishes within 1 week of culture. A recent report has demonstrated that cells that remain in suspension and fail to form adherent colonies contain a fraction of late adherent cells that resembles BMMSCs (Biomed Res Int, 2013; 2013: 790842). Umbilical cord blood (UCB) is as accessible as bone marrow for the isolation of MSCs. In this study, we identified a late adherent subpopulation in UCB and determined its hematopoiesis-supporting activity.Forty-five UCB units, which were not matched to the eligibility criterion defined in the Japan UCB donation program, were collected after delivery of placenta. Written informed consent was obtained before delivery from all pregnant women who participated in the study. The study protocol was approved by the ethics committee of the Kyoto University Graduate School of Medicine.Mononuclear cells were isolated from UCB by the density gradient centrifugation method with (n = 19) and without (n = 18) subsequent separation of CD34 negative cells using anti-CD34 immunomagnetic microbeads (Miltenyi Biotec, Bergisch Gladbach, Germany). Nucleated cells were separated by the hydroxyethyl starch sedimentation method from the other eight UCB units. The cells were then seeded into a culture flask and cultured in alpha minimal essential medium supplemented with 15% FBS (Culture 1; C1). After 1 week of culture, non-adherent cells in C1 supernatant were collected and re-seeded into a new flask (C2). The attached cells in C1 were cultured until adherent colonies emerged, after which they were detached using trypsin/EDTA and twice passaged to obtain a sufficient number of cells (C1 cells). In the same way, after 1 week of culture, non-adherent cells in C2 supernatant were collected and re-seeded into a new flask (C3). The attached cells in C2 were cultured to obtain C2 cells. Afterwards, re-seeding and culture (C4, C5c) were repeated until no new colonies were formed. Collected cells were cryopreserved and thawed when required in experiments. BMMSCs were isolated from human bone marrow cells purchased from AllCells (Emeryville, CA).C1 cells, the so-called UCBMSCs, were successfully isolated from 18 units (40 %). Adherent cells isolated from C2 and later were defined as elate adherent cellsf and, were obtained from 9 units: these cells were referred to as C2 cells (from 9 units), C3 cells (from 9 units), C4 cells (from 6 units) and C5 cells (from 2 units). The interval from seeding to the first colony formation in C1 was shorter in these 9 units than that in the other 9 units that contained only C1 cells: 10.8 } 1.4 vs 15.9 } 4.5 days, p < 0.01. The volume of the former 9 units tended to be large compared to the latter 9 units: 49.6 } 10.5 vs 33.7 } 21.0 mL, p = 0.07. These findings indicated that UCB containing late adherent cells was suitable for a cell source of MSCs. Next, we examined whether these late adherent cells (C2 and C3 cells) had properties consistent with those of MSCs. Both C2 and C3 cells showed spindle-shaped fibroblast-like morphology and the same immunophenotype as C1 cells: positive for CD73, CD90 and CD105, and negative for CD34, CD45 and HLA-DR. They had osteogenic, adipogenic and chondrogenic differentiation potentials in vitro. These findings are the minimal criteria for MSCs (Cytotherapy, 2006; 8:315). Finally, we evaluated the hematopoiesis-supporting activity of these cells in vitro and in vivo. CD45-positive hematopoietic cells were expanded when co-cultured of CD34-positive hematopoietic progenitor cells (6 ~ 102 cells) with C2 or C3 cells (2 ~ 104 cells) in vitro as much as when co-cultured with C1 cells (Figure A). In vivo analysis was conducted by using subcutaneous transplantation of MSCs on NOD/SCID mice (Int J Hematol, 2015; 102: 218). C2 cells induced trabecular bone formation and bone marrow hematopoiesis as well as C1 cells, however, C3 cells did not induce hematopoiesis (Figure B).In conclusion, we demonstrated that UCB contains a late adherent cell subpopulation with the same characteristics and hematopoiesis-supporting activity as those of UCBMSCs isolated using the conventional method. The continuance of cell culture without discarding suspension cells could improve the efficiency of isolation of MSCs from UCB. [Display omitted] DisclosuresHirai:Kyowa Hakko Kirin: Research Funding; Novartis Pharma: Research Funding. Maekawa:Bristol-Myers K.K.: Research Funding.

Identification of novel target genes of nerve growth factor (NGF) in human mastocytoma cell line (HMC-1 (V560G c-Kit)) by transcriptome analysis

BackgroundNerve growth factor (NGF) is a potent growth factor that plays a key role in neuronal cell differentiation and may also play a role in hematopoietic differentiation. It has been shown that NGF induced synergistic action for the colony formation of CD34 positive hematopoietic progenitor cells treated with macrophage-colony stimulating factor (M-CSF or CSF-1), or stem cell factor (SCF). However, the exact role of NGF in hematopoietic system is unclear. It is also not clear whether NGF mediated signals in hematopoietic cells are identical to those in neuronal cells.ResultsTo study the signal transduction pathways induced by NGF treatment in hematopoietic cells, we utilized the mastocytoma cell line HMC-1(V560G c-Kit) which expresses the NGF receptor, tropomyosin-receptor-kinase (Trk)A, as well as the constitutively activated SCF receptor, V560G c-Kit, which can be inhibited completely by treatment with the potent tyrosine kinase inhibitor imatinib mesylate (imatinib). NGF rescues HMC-1(V560G c-Kit) cells from imatinib mediated cell death and promotes proliferation. To examine the NGF mediated proliferation and survival in these cells, we compared the NGF mediated upregulated genes (30 and 120 min after stimulation) to the downregulated genes by imatinib treatment (downregulation of c-Kit activity for 4 h) by transcriptome analysis. The following conclusions can be drawn from the microarray data: Firstly, gene expression profiling reveals 50% overlap of genes induced by NGF-TrkA with genes expressed downstream of V560G c-Kit. Secondly, NGF treatment does not enhance expression of genes involved in immune related functions that were down regulated by imatinib treatment. Thirdly, more than 55% of common upregulated genes are involved in cell proliferation and survival. Fourthly, we found Kruppel-like factor (KLF) 2 and Smad family member 7 (SMAD7) as the NGF mediated novel downstream genes in hematopoietic cells. Finally, the downregulation of KLF2 gene enhanced imatinib induced apoptosis.ConclusionNGF does not induce genes which are involved in immune related functions, but induces proliferation and survival signals in HMC-1(V560G c-Kit) cells. Furthermore, the current data provide novel candidate genes, KLF2 and SMAD7 which are induced by NGF/TrkA activation in hematopoietic cells. Since the depletion of KLF2 causes enhanced apoptosis of HMC-1(V560G c-Kit), KLF2 may play a role in the NGF mediated survival signal.

Factors Associated with Peripheral Blood Stem Cell Yield in Volunteer Donors Mobilized with Granulocyte Colony-Stimulating Factors: The Impact of Donor Characteristics and Procedural Settings

Peripheral blood stem cells (PBSCs) are increasingly used as the source of hematopoietic stem cells, but there are large variations in harvest outcome between individuals mobilized by granulocyte colony-stimulating factor (G-CSF). We examined the effects of donor characteristics and procedure factors on the day 1 CD34+ cell yield in 373 unrelated healthy donors. G-CSF was administered subcutaneously at a planned dose of 8.3 to 11 microg/kg daily for 5 days, followed by harvest started on day 5 of G-CSF treatment. Of the 373 donors, 159 (42.6%) had the radial artery as the inlet access for harvest. Poor day 1 cell yield was defined as <10x10(6) CD34+ cells/L of processed blood for the first apheresis; 62 donors (16.6%) did not attain this threshold. The male donors had significantly higher yields at harvest compared with the female donors. The female donors had higher CD34+ cell yields if the circulation access was through an artery than if is was through a vein. In a multiple regression analysis, donor age, sex, body mass index (BMI), preharvest white blood cell and circulating immature cell counts, access type, and flow rate correlated with day 1 yield. Female sex, older age, venous access, and a higher flow rate were significantly associated with greater risk for a day 1 poor yield of CD34+ cells (odds ratio=3.0074, 1.045, 4.3362, and 1.1131, respectively). A higher BMI may decrease the risk (odds ratio=0.8472). In donors at higher risk for poor CD34+ cell yield, strategies for increasing CD34+ cells must be considered.

Vav promoter- tTA conditional transgene expression system for hematopoietic cells drives high level expression in developing B and T cells

We previously showed that Vav promoter-tetracycline transactivator (Vav-tTA)-driven tetracycline-regulated element (TRE)-NRAS(V12) expression resulted in mastocytosis development in mice. To investigate which hematopoietic cells express TRE-driven transgenes when combined with Vav-tTA, we assayed hematopoietic cells, including bone marrow-derived mast cells (BMMC) and CD34-positive hematopoietic progenitor cells (HPC) as well as myeloid and lymphoid lineages. To determine if suppression of NRAS(V12) expression early in life would delay mastocytosis we treated developing and juvenile mice with doxycycline (Dox). Vav-tTA-driven luciferase expression was assayed by live mouse imaging and relative light unit measurement before or after treating Vav-tTA and TRE-luciferase (TRE-Luc) cotransgenic mice with Dox. Magnetic cell sorting and fluorescence-activating cell sorting methods were used to sort hematopoietic cells. To suppress TRE-mediated luciferase or NRAS(V12) expression in Vav-tTA cotransgenic mice, we added Dox to the drinking water. B cells in the bone marrow and T cells in the thymus expressed Vav-tTA-driven luciferase at much higher levels than in myeloid cells, BMMC, and CD34-positive HPC, which showed relatively low levels. Dox treatment completely eliminated the luciferase expression from all hematopoietic cells. Repression of TRE-NRAS(V12) expression early in life was sufficient to increase the latency of mastocytosis development. The Vav-tTA transgenic line will be very useful for conditional transgene expression in developing B and T cells. Vav-tTA-driven NRAS(V12) expression is sufficient for mastocytosis development, but not for myeloid leukemia. Lymphoid cells are resistant to NRAS(V12) transformation despite high level of expression.

Role of TPO in the Differentiation of Embryonic Stem Cells into Hematopoetic Cells.

Thrombopoetin (TPO) is a primary regulator of megakaryocyte and platelet production. However, studies in c-mpl-deficient mice and in congenital amegakaryocytic thrombocytopenia-patients with non-sense c-mpl mutation who develop pancytopenia during the first years of life suggest that TPO also play an important role on early hematopoesis. We demonstrated that TPO enhances FLK-1 (VEGF-receptor) expression on hemangioblasts during murine embryonic stem cell differentiation in embryoid body-liquid cultures (up to 73%). To extend our studies, we investigated the TPO signaling in FLK-1 positive cells. ES cells at different time point of differentiation showed that TPO enhances c-mpl-, BMP4-, Notch-, HOXB4-, HOXB9-, HOXA10-, Runx1-and CD133- mRNA expression. To investigate mesoderm formation, we also analyzed GATA-4 and T-brachyury mRNA level expression. Interestingly, we found that TPO alone did not increase GATA-4- and T-brachyury- mRNA expression, suggesting that TPO requires other cytokines to form the mesoderm. We also found that TPO could maintain VEGF-A mRNA expression level during differentiation of ES-cells. We hypothesize that VEGF expression together with c-mpl expression is required in hematopoetic differentiation of ES cell. This activity of Tpo was also observed during Rhesus monkey embryonic stem cell differentiation into hematopoetic cell. Only combinations of TPO and VEGF were capable of increasing CD34 positive hematopoietic progenitor cells (up to 8%), but TPO alone failed to induce high levels of CD34+ cell. In addition, analysis of gene expression during hemangioblast development demonstrated that TPO was capable of increasing the expression of VEGF receptors (FLK-1) and TPO receptors (c-mpl) in mice and primates. The in-vitro differentiation of mouse and rhesus monkey ES cells provides an opportunity to better understand the role of TPO in the early stage of hematopoietic development from ES cells to mature hematopoietic cells.

Defects of the Tpo-Receptor, Mpl, Caused by Mutations Found in CAMT Patients.

Congenital amegakaryocytic thrombocytopenia (CAMT) is a rare disorder that presents with severe thrombocytopenia and absence of megakaryocytes in the bone marrow. The disease may develop into bone marrow aplasia. In vitro, CD34-positive hematopoietic progenitor cells from CAMT patients did not show any megakaryocyte formation in a Tpo-driven expansion culture. We and others found genetic defects in the gene encoding the Tpo receptor, c-mpl (Van den Oudenrijn et al., Br J Haematol. 2002, 117: 390–398 and Ballmaier et al., Ann N Y Acad Sci. 2003, 996: 17–25). In our patients, we found four mutations that predicted amino-acid substitutions, of which three in the extracellular domain; Arg102Pro, Pro136His and Arg257Cys, and one in the intracellular signaling domain (Pro635Leu), which may result in either defective Tpo-binding and/or signaling. To investigate this, we transfected full-length Mpl (wt and mutants) into the erythroleukemic cell line K562 and truncated Mpl (encompassing the extracellular domain; wt and mutants) into Baby Hamster Kidney (BHK) cells. In the K562 cells, the mRNA level (RQ-PCR) of the Pro136His mutant was severely decreased compared to the wt transfectant, while the mRNA level of the other mutants was comparable to that of wt. On Western blot, wt Mpl migrated as two, presumably differently glycosylated, bands of 75 kD and 72 kD. The mutants showed an altered migration pattern, which might result from differences in glycosylation. With the Pro635Leu mutant lower signals were obtained when equal amounts of total protein were loaded. Since the Mpl mRNA level was comparable to that of wt, this suggests a higher level of protein degradation. Upon transfection of the Arg102Pro and the Arg257Cys mutants in BHK cells, we observed that these mutants did not gain endo-H resistency, which suggests an aberrant processing of these mutant Mpls through the Golgi apparatus and retention in the ER. However, in cell fractionation experiments with surface-biotinylated K562 cells, biotinylated wt Mpl and mutant Mpl (except Pro136His) could be detected. Apparently, in K562 cells, the amino-acid substitutions do not impair membrane expression completely. To examine whether the mutant receptors were still able to signal after Tpo incubation, K562 cells were serum-starved and subsequently stimulated with 50 ng/ml rhTpo for 5 to 30 minutes. All mutants, including Pro136His, showed increased ERK phosphorylation after 5 minutes. To summarize, the Pro136His mutant is hardly expressed in the K562 expression model, presumably because of instability of the mRNA, but is still able to induce signaling. In contrast to the results obtained in the BHK model, the Arg102Pro and Arg257Cys mutants, showed cell-surface expression in the K562 cell line. The obtained cell-surface expression in the K562 model may have been significantly increased compared to the in vivo situation on hematopoietic stem cells, because of artificially induced efficient expression. Finally, with a super-physiological concentration of rhTpo, we obtained evidence that all Mpl mutants were able to signal upon Tpo binding. Whether impaired signaling by the Mpl mutants in the presence of physiological levels of Tpo may contribute to the development of CAMT, will be investigated.

The effect of steroid on myeloid leukemic cells: The potential of short-course high-dose methylprednisolone treatment in inducing differentiation, apoptosis and in stimulating myelopoiesis

Several in vitro studies have shown that dexamethasone (Dex) and prednisolone can induce differentiation of some mouse and human myeloid leukemic cells to macrophages and granulocytes. Based on in vitro experiments, we have shown that short-course (3–7 days) high-dose methylprednisolone (HDMP) (20–30 mg/kg/day) treatment can induce differentiation of myeloid leukemic cells in vivo in children with different subtypes of acute myeloblastic leukemia (AML) (AML-M1, -M2, -M3, -M4, -M7). We have also shown that induction of apoptosis of myeloid leukemic cells with or without differentiation is possible by short-course HDMP treatment. In addition, short-course HDMP treatment has been shown to be effective in accelerating leukocyte recovery, possibly stimulating normal CD34-positive hematopoietic progenitor cells. Addition of HDMP to mild cytotoxic chemotherapy (low-dose cytosine arabinoside (LD-Ara-c), weekly mitoxantrone and Ara-c or 6-thioguanine) increased the remission rate (87–89%) and improved the outcome of AML children. We believe that the results of our 17-year clinical experience will provide important benefits to AML patients.

RNA-based gene transfer for adult stem cells and T cells.

Electroporation of mRNA has become an established method for gene transfer into dendritic cells for immunotherapeutic purposes. However, many more cell types and applications might benefit from an efficient mRNA-based gene transfer method. In this study, we investigated the potential of mRNA-based gene transfer to induce short-term transgene expression in adult stem cells and activated T cells, based on electroporation with mRNA encoding the enhanced green fluorescent protein. The results show efficient transgene expression in CD34-positive hematopoietic progenitor cells (35%), in in vitro cultured mesenchymal cells (90%) and in PHA-stimulated T cells (50%). Next to presentation of gene transfer results, potential applications of mRNA-based gene transfer in stem cells and T cells are discussed.

Hypermethylation of the imprinted NNAT locus occurs frequently in pediatric acute leukemia.

Recent studies have demonstrated imprinting of the human neuronatin (NNAT) gene. NNAT maps to 20q11.2-q12, a region exhibiting loss of heterozygosity in acute myeloid leukemia and myelodysplastic/myeloproliferative disease. To investigate possible epigenetic dysregulation of genes in this region relevant to leukemogenesis, we analyzed methylation of the NNAT gene in normal tissues and in leukemias. We found a differential methylation pattern, typical of imprinted genes, at sites in the CpG island containing NNAT exon 1 in normal pituitary, peripheral blood cells and bone marrow-derived CD34-positive hematopoietic progenitor cells. Substantial or complete loss of the unmethylated NNAT allele was observed in leukemia cell lines and in 20 of 29 (69%) acute myeloid or lymphoid leukemia samples. While most highly expressed in brain, NNAT mRNA was also detected in normal hematopoietic progenitor cells and in leukemia cells exhibiting the normal methylation pattern, although not in hypermethylated leukemia cells. Demethylation by treatment of hypermethylated leukemia cells with 5-aza-2'-deoxycytidine resulted in reactivation of NNAT expression, concomitant with a reversion to the normal methylation pattern. The data demonstrate that hypermethylation of the NNAT locus is a frequent event in both myeloid and lymphoid acute leukemias of childhood. Aberrant hypermethylation of the NNAT locus suggests that the dysregulation of genes at 20q11.2-q12 in leukemia may be the result of epigenetic as well as genetic events.

Flow cytometric analysis of hemetopoietic progenitor cells in peripheral blood stem cell harvest from patients with CD34 positive acute leukemia

We analyzed CD34 positive cells in peripheral blood stem cell harvest (PBSCH) using flow cytometry. PBSCH from CD34 positive acute myelogeous leukemia (AML-M2) patient contained 1.87% CD34 positive cells, of which 1.21% was represented by MRD.PBSCH from CD34 positive acute lymphoblast leukemia (ALL) patient contained 3.14% CD34 positive cells, of which 0.11% was accounted for by minimal residual disease (MRD). If PBSCH from CD34 positive acute leukemia patient is analyzed for CD34 monoclonal antibody alone, the presence of CD34 positive MRD may escape attention so that CD34 positive hematopoietic progenitor cells may be overestimated. To avoid this risk, it is necessary to analyze PBSCH using both CD34 monoclonal antibody and characteristic markers of leukemia cells that were found pre-treatment.

Principles of counting low numbers of leukocytes in leukoreduced blood components.

N THE LAST decade leukoreduced blood components have become widely used for the prevention of a variety of transfusion complications. A fundamental tool for the advance and evaluation of this technology has been new methods to count residual leukocytes in leukoreduced blood. Because the concentration of leukocytes is below the level of detection of most standard automated hematology analyzers, 1 methods have been specifically designed and developed to count low numbers of white blood cells (WBC) in red cell and platelet concentrates. This review summarizes the principles of rare event analysis as applied to counting leukocytes in leukoreduced blood components and describes some of the methods currently used for cell counting. Readers will note that many of the principles of rare event analysis are applicable to other areas of transfusion medicine. For example, accurate and precise enumeration of CD34-positive hematopoietic progenitor cells in the blood of patients undergoing growth-factor stimulation presents challenges similar to those encountered in the enumeration of residual donor leukocytes.

Immunocytochemical detection of breast cancer cells in marrow and peripheral blood of patients undergoing high dose chemotherapy with autologous stem cell support.

Detection of small numbers of breast cancer cells is important in staging the disease and can be helpful in assessing the efficacy of purging regimens prior to autologous stem cell infusion. Immunohistochemical methods are potentially useful and broadly applicable for this purpose since they are simple to perform, sensitive, and may be quite specific. We have used a combination of four monoclonal antibodies [260F9, 520C9, 317G5 (Baxter Corp); BrE-3 (Dr. R. Ceriani)] against tumor cell surface glycoproteins in a sensitive immunocytochemical assay to identify breast tumor cells in bone marrow and peripheral blood. Immunostained cytospin preparations were fixed prior to staining to preserve cytological details of immunopositive cells. After immunostaining, slides were counterstained with hematoxylin to confirm the identify of labeled cells. In cytocentrifuge experiments in which small numbers of CAMA human breast tumor cells were added to bone marrow mononuclear cells, a linear relationship between the number of tumor cells added and the number of tumor cells detected was obtained over a broad range of tumor cell concentrations. The probability of detecting tumor cells was dependent on the number of cytocentrifuge slides examined. When ten slides (5 million cells) were examined, the probability of detecting tumor at a concentration of 4 tumor cells per million bone marrow mononuclear cells was 98%. In clinical specimens, tumor cells were detected in marrow aspirates from 73 of 240 (30%) patients undergoing autologous transplantation, including 70 (37%) of 190 patients with clinical stage IV disease, 0 of 7 patients with clinical stage III disease, and 3 of 43 (7%) patients with clinical stage II disease. Seventy-three of 657 peripheral blood specimens from 26 of 155 patients (17%) contained breast cancer cells with counts ranging from 1 to 97 tumor cells per million leukocytes. Tumor cells were most frequently found in the blood of patients with stage IV disease [21 of 107 (20%)] but were also found in a substantial number [5 of 44 (11%)] of patients with stage II disease. Positive selection of CD34-positive hematopoietic progenitor cells as well as negative purging methods such as incubation with 4-hydroxyperoxy-cyclophosphamide (4-HC) were evaluated with respect to tumor cell depletion. Selection of CD34-positive progenitor cells from bone marrow or peripheral blood resulted in log reduction of 1 to > 4 tumor cells reinfused at autologous transplantation. A lesser log reduction (up to 1) was demonstrated following 4-HC purging. We conclude that properly performed and controlled immunocytochemical staining of bone marrow and peripheral blood cytospins is a sensitive and simple way to detect and quantitate breast cancer cells in hematopoietic specimens harvested for autotransplantation and that CD34-positive progenitor cell selection results in significant reduction in the number of breast cancer cells reinfused with marrow or peripheral blood stem cells.

IL-2 receptor gamma chain expression on CD34 positive hematopoietic progenitor cells from bone marrow and cord blood.

The IL-2 receptor (IL-2R) gamma chain is shared among receptors for IL-4, IL-7, IL-9 and IL-15 as well as IL-2. In order to clarify the functional role of these cytokines interacting with the common gamma chain in human early hematopoiesis, we studied expression of the IL-2R gamma chain on purified CD34 positive cells from bone marrow and cord blood. Broad populations of bone marrow mononuclear cells were all found to express the IL-2R gamma chain. CD34 positive cells were purified by CD34 monoclonal antibodies and immunomagnetic beads as representative hematopoietic progenitor cells. It was established that only 38 +/- 10% of CD34 positive bone marrow cells (n = 5) and 35 +/- 12% of CD34 positive cord blood cells (n = 11) expressed the IL-2R gamma chain. CD34(+) IL-2R gamma chain(+) and CD34(+) IL-2R gamma chain(-) cells fractionated by cell sorting were subjected to clonogenic assays that showed granulocyte-macrophage colony-forming cells (CFU-GM) were present evenly in both fractions, whereas erythroid burst-forming cells (BFU-E) were enriched in the CD34(+) IL-2R gamma chain(-) fraction approximately two- to six-fold as compared with CD34(+) IL-2R gamma chain(+) fraction. Such clonogenic features did not differ between the bone marrow and cord blood cases. These results indicate that CD34(+) IL-2R gamma chain(-) cells contain immature cells already committed to the erythroid lineage.

Large volume ex vivo expansion of CD34-positive hematopoietic progenitor cells for transplantation.

A large volume culture system was developed for the ex vivo expansion of CD34 positive (+) hematopoietic progenitors, using cell donated by 15 patients receiving high-dose chemotherapy with autologous hematopoietic progenitor cell support (AHPCS). Substantial expansion of myeloid (181-fold) and megakaryocyte (41-fold) progenitors cells was demonstrated, using the conditions that we determined to be optimal: CD34+ progenitors cultured unperturbed for 7 (marrow) or 10 (blood) days in Teflon-coated bags with X-Vivo-10 medium containing 10% autologous plasma, 100 ng/ml, respectively, of recombinant stem cell factor (SCF), interleukin 3 (IL-3), interleukin 6 (IL-6), and granulocyte colony-stimulating factor (G-CSF). The studies demonstrated that (a) CD34 selection was necessary to obtain large, clinically relevant numbers of hematopoietic progenitors, (b) the addition of G-CSF to the baseline regimen of SCF/IL-3/IL-6 significantly enhanced the expansion of myeloid progenitors, (c) the addition of IL-1 to SCF/IL-3/IL-6 did not significantly enhance myeloid progenitor cell expansion, (d) CD34+ G-CSF-mobilized peripheral blood progenitor cells (PBPC) produced higher numbers of myeloid progenitors in culture than CD34+ marrow cells, and (e) long-term tissue culture (LTC) assays demonstrate the preservation of long-term initiating cells in ex vivo culture. The short-term and long-term reconstituting capability of CD34+ PBPC cultured in this system remains to be determined and will be evaluated in a clinical trial where they will be used as the sole source of AHPCS following high-dose therapy.

Modified antisense oligodeoxynucleotides against the splice acceptor site of tat do not inhibit in vitro hematopoietic colony growth in HIV-positive patients

The hematopoietic failure in the majority of patients with progressive HIV infection is further aggravated by virustatic agents like azidothymidine. As an alternative therapeutic attempt, three derivatives of an antisense oligodeoxynucleotide (ODN) against the splice acceptor site of the tat gene have been shown to inhibit HIV replication in vitro. This study was aimed at examining whether these agents are toxic to the hematopoietic progenitor cells. To this end, bone marrow cells from HIV-positive and healthy persons were depleted from adherent cells to eliminate fibroblasts. In further experiments, the cells were additionally enriched for CD34-positive hematopoietic progenitor cells or were depleted from delta TCS-1-positive T lymphocytes. At concentrations of 1.25-10 microM, the three antisense ODN did not inhibit any erythrocyte or granulocyte-monocyte colony growth from CD34-positive cells, either from the HIV-positive or from the HIV-negative cohort. In contrast to azidothymidine, which served as inhibitory control, a significant increase of colony growth was seen after depletion of fibroblasts, of delta TCS-1-positive cells, or without cell separation. In conclusion, the three oligodeoxynucleotides do not exert any hematotoxic effect but do increase colony formation from low-density bone marrow cells in vitro and could therefore be useful in future clinical studies.