In vitro cytotoxic study of immunoliposomal doxorubicin targeted to human CD34(+) leukemic cells

The expression of CD34 antigen in acute myelogenous leukemias is considered an unfavourable prognosis marker for response to anticancer drugs and duration of remission. This study investigated the applicability of long-circulating immunoliposomes loaded with doxorubicin targeted to CD34 antigen present on MDR(+) human myelogenous leukemia KG-1a cell line. Immunoliposomal doxorubicin showed a higher cytotoxicity against KG-1a cells than non-targeted liposomal doxorubicin, but it did not improve over that of free drug. Although no reversal of doxorubicin resistance was found to occur through its liposomal encapsulation, a therapeutic benefit can be obtained by the selective cytotoxicity observed. Endocytosis studies demonstrated that, after binding to CD34 antigen, the immunoliposomes are not internalized by the KG-1a cells and so the cytotoxic effect might be due to drug released into the space near the cell membrane. Thus, immunotargeting of liposomal doxorubicin to CD34(+) leukemic cells may only provide an ex vivo strategy for site-selective CD34(+) leukemia cell killing.     

Regulation of cell shape and adhesion by CD34

We previously reported that expression of CD43/leukosialin induces cell rounding and microvillus formation via inhibition of cell adhesion. Here, we found that CD34, a cell surface sialomucin and marker for hematopoietic progenitor cells, also inhibited cell adhesion and induced cell rounding and microvillus formation. Forced expression of CD34-induced cell rounding, microvillus formation, and phosphorylation of ezrin/radixin/moesin (ERM) proteins in HEK293T cells, while inhibiting integrin-mediated cell re-attachment. Furthermore, CD34+ blood cells and KG-1 cells, which express endogenous CD34 on their surface, were spherical in shape, surrounded by microvilli, and non-adherent to substrata. In addition, cleavage of O-sialomucin augmented integrin-mediated cell adhesion of KG-1 cells. These results suggest the involvement of CD34 in the inhibition of integrin-mediated cell adhesion and formation of the cell surface structure. The inhibitory function of CD34 in cell adhesion may affect cell shape organization via phosphorylation of ERM proteins. Cellular structures such as the spherical shape and microvilli of CD34+ cells may also contribute to regulation of cell adhesion.     

In a human lymphomyeloid progenitor cell line (KG-1) HIV-1 gp120 binds to chemokine-receptors CXC-R4 and CCR5, only in the presence of CD4.

A CD34+ human hematopoietic progenitor cell lines, KG-1, became susceptible to HIV-1 infection in the presence of a concurrent infection by human herpesvirus-6 (HHV-6). We have now analyzed the possible mechanism(s) underlying this phenomenon, in the light of the recent demonstration that at least two members of the chemokine receptor family, CXCR4 (LESTR/fusin) and CCR5 molecules, are the HIV-1-specific co-receptors, necessary, together with the high affinity receptor CD4, for the entry into target cells of HIV-1. Cytofluorimetric analysis demonstrated that in KG-1 cells, after HHV-6 infection, more than 40% of cell population became CD4 positive and only in KG-1 cells expressing the CD4+ phenotype, the exposure to r-gp120 masks a significant amount, not only of CD4, but also of both CXCR4 and CCR5 chemokine receptors. In fact, only when pre-infected by HHV-6, KG-1 cells, after exposure to r-gp120, exhibit a significant reduction in the percentage of CXCR4 or CCR5-positive cells.     

JC virus infection of hematopoietic progenitor cells, primary B lymphocytes, and tonsillar stromal cells: implications for viral latency.

The human polyomavirus JC virus (JCV) infects myelin-producing cells in the central nervous system, resulting in the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML). JCV-induced PML occurs most frequently in immunosuppressed individuals, with the highest incidence in human immunodeficiency type 1-infected patients, ranging between 4 and 6% of all AIDS cases. Although JCV targets a highly specialized cell in the central nervous system, infection is widespread, with more than 80% of the human population worldwide demonstrating serum antibodies. A number of clinical and laboratory studies have now linked the pathogenesis of PML with JCV infection in lymphoid cells. For example, JCV-infected lymphocytes have been suggested as possible carriers of virus to the brain following reactivation of a latent infection in lymphoid tissues. To further define the cellular tropism associated with JCV, we have attempted to infect immune system cells, including CD34+ hematopoietic progenitor cells derived from human fetal liver, primary human B lymphocytes, and human tonsillar stromal cells. Our results demonstrate that these cell types as well as a CD34+ human cell line, KG-1a, are susceptible to JCV infection. JCV cannot, however, infect KG-1, a CD34+ cell line which differentiates into a macrophage-like cell when treated with phorbol esters. In addition, peripheral blood B lymphocytes isolated by flow cytometry from a PML patient demonstrate JCV infection. These results provide direct evidence that JCV is not strictly neurotropic but can infect CD34+ hematopoietic progenitor cells and those cells which have differentiated into a lymphocytic, but not monocytic, lineage.     

Blood progenitor cell separation from clinical leukapheresis product by magnetic nanoparticle binding and magnetophoresis

Positive selection of CD34+ blood progenitor cells from circulation has been reported to improve patient recovery in applications of autologous transplantation. Current magnetic separation methods rely on cell capture and release on solid supports rather than sorting from flowing suspensions, which limits the range of therapeutic applications and the process scale up. We tested CD34+ cell immunomagnetic labeling and isolation from fresh leukocyte fraction of peripheral blood (leukapheresis) using the continuous quadrupole magnetic flow sorter (QMS), consisting of a flow channel (SHOT, Greenville, IN) and a quadrupole magnet with a maximum field intensity (B(o)) of 1.42 T and a mean force field strength (S(m)) of 1.45 x 10(8) TA/m(2). Both the sample magnetophoretic mobility (m) and the inlet and outlet flow patterns highly affect the QMS performance. Seven commercial progenitor cell labeling reagent combinations were quantitatively evaluated by measuring magnetophoretic mobility of a high CD34 expression cell line, KG-1a, using the cell tracking velocimeter (CTV). The CD34 Progenitor Cell Isolation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) showed the strongest labeling of KG-1a cells and was selected for progenitor cell enrichment from 11 fresh and 11 cryopreserved clinical leukapheresis samples derived from different donors. The CD34+ cells were isolated with a purity of 60-96%, a recovery of 18-60%, an enrichment rate of 12-169, and a throughput of (1.7-9.3) x 10(4) cells/s. The results also showed a highly regular dependence of the QMS performance on the flow conditions that agreed with the theoretical predictions based on the CD34+ cell magnetophoretic mobility.     

Replication of Colorado tick fever virus within human hematopoietic progenitor cells.

Significant neutropenia, as well as thrombocytopenia and a mild anemia, occurs in patients infected with Colorado tick fever virus. In this study, human bone marrow CD34+ cells and KG-1a cells, a human hematopoietic progenitor cell line, were infected in vitro with Colorado tick fever virus. The time course and morphological appearance of viral replication in human progenitor cells were similar to those seen in erythroblasts and in HEL cells and suggest one possible mechanism for the clinical hematologic findings.     

Myeloblastic Cell Lines Mimic Some but Not All Aspects of Human Cytomegalovirus Experimental Latency Defined in Primary CD34+ Cell Populations

Human cytomegalovirus (HCMV) is a significant human pathogen that achieves lifelong persistence by establishing latent infections in undifferentiated cells of the myeloid lineage, such as CD34⁺ hematopoietic progenitor cells. When latency is established, viral lytic gene expression is silenced in part by a cellular intrinsic defense consisting of Daxx and histone deacetylases (HDACs) because pp71, the tegument transactivator that travels to the nucleus and inactivates this defense at the start of a lytic infection in differentiated cells, remains in the cytoplasm. Because the current in vitro and ex vivo latency models have physiological and practical limitations, we evaluated two CD34⁺ myeloblastic cell lines, KG-1 and Kasumi-3, for their ability to establish, maintain, and reactivate HCMV experimental latent infections. Tegument protein pp71 was cytoplasmic, and immediate-early (IE) genes were silenced as in primary CD34⁺ cells. However, in contrast to what occurs in primary CD34⁺ cells ex vivo or in NT2 and THP-1 in vitro model systems, viral IE gene expression from the laboratory-adapted AD169 genome was not induced in the presence of HDAC inhibitors in either KG-1 or Kasumi-3 cells. Furthermore, while the clinical strain FIX was able to reactivate from Kasumi-3 cells, AD169 was not, and neither strain reactivated from KG-1 cells. Thus, KG-1 and Kasumi-3 experimental latent infections differ in important parameters from those in primary CD34⁺ cell populations. Aspects of latency illuminated through the use of these myeloblastoid cell lines should not be considered independently but integrated with results obtained in primary cell systems when paradigms for HCMV latency are proposed.     

Collection and analysis of hematopoietic progenitor cells from cynomolgus macaques (Macaca fascicularis): assessment of cross-reacting monoclonal antibodies

Previous studies have shown that hematopoietic progenitor cells can be isolated from human or nonhuman primate bone marrow (BM) cells. In the present study, we studied the cross-reactivity of 13 anti-human CD34, two anti-human c-Kit, and one anti-human CD133 monoclonal antibodies (mAbs) with cynomolgus macaque (Macaca fascicularis) BM cells, using flow cytometric analysis, cell enrichment, and clonogenic assay. Among the 13 anti-human CD34 mAbs assessed, six cross-reacted as previously reported by other groups. However, only three of these six mAbs (clones 561, 563, and 12.8) recognized cynomolgus CD34+ cells that formed progenitor colonies when grown in methylcellulose culture. Similarly, of the two anti-human c-Kit mAbs (clones NU-c-kit and 95C3) that were previously reported to cross-react with cynomolgus BM cells, only one (clone NU-c-kit) resulted in a similar outcome. The anti-human CD133 mAb (clone AC133) also cross-reacted with cynomolgus BM cells, although these cells did not give rise to colonies when grown in culture. These results suggest that antibodies that cross-react with nonhuman primate cells may not identify the hematopoietic cells of interest. In addition, while the CD34 mAb (clone 561) results in the selection of hematopoietic progenitor cells of all lineages when assessed in methylcellulose culture, the c-Kit(high) fraction (NU-c-kit) exclusively identifies erythroid-specific progenitor cells after growth in culture. It is important to consider these findings when selecting cross-reacting mAbs to identify cells of hematopoietic lineages in macaque species.     

Functional involvement of E-cadherin in TGF-beta 1-induced cell cluster formation of in vitro developing human Langerhans-type dendritic cells

Epithelial Langerhans cells (LC) represent immature dendritic cells that require TGF-beta 1 stimulation for their development. Little is known about the mechanisms regulating LC generation from their precursor cells. We demonstrate here that LC development from human CD34+ hemopoietic progenitor cells in response to TGF-beta 1 costimulation (basic cytokine combination GM-CSF plus TNF-alpha, stem cell factor, and Flt3 ligand) is associated with pronounced cell cluster formation of developing LC precursor cells. This cell-clustering phenomenon requires hemopoietic progenitor cell differentiation, since it is first seen on day 4 after culture initiation of CD34+ cells. Cell cluster formation morphologically indicates progenitor cell development along the LC pathway, because parallel cultures set up in the absence of exogenous TGF-beta 1 fail to form cell clusters and predominantly give rise to monocyte, but not LC, development (CD1a-, lysozyme+, CD14+). TGF-beta 1 costimulation of CD34+ cells induces neoexpression of the homophilic adhesion molecule E-cadherin in the absence of the E-cadherin heteroligand CD103. Addition of anti-E-cadherin mAb or mAbs to any of the constitutively expressed adhesion molecule (CD99, CD31, LFA-1, or CD18) to TGF-beta 1-supplemented progenitor cell cultures inhibits LC precursor cell cluster formation, and this effect is, with the exception of anti-E-cadherin mAb, associated with inhibition of LC generation. Addition of anti-E-cadherin mAb to the culture allows cell cluster-independent generation of LC from CD34+ cells. Thus, functional E-cadherin expression and homotypic cell cluster formation represent a regular response of LC precursor cells to TGF-beta 1 stimulation, and cytoadhesive interactions may modulate LC differentiation from hemopoietic progenitor cells.     

Radiolabeling rhesus monkey CD34+ hematopoietic and mesenchymal stem cells with 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) for microPET imaging

Noninvasive positron emission tomography (PET) provides a potential method for in vivo tracking of radiolabeled cells. The goal of this study was to assess the potential toxicity of 64Cu-pyruvaldehyde-bis(N4-methylthiosemicarbazone) (PTSM) on rhesus monkey CD34+ hematopoietic and mesenchymal stem cells in vitro in preparation for developing imaging protocols posttransplantation. CD34+ hematopoietic cells were radiolabeled with 0 to 40 microCi/mL 64Cu-PTSM and viability and colony formation were assessed. Rhesus monkey mesenchymal stem cells (rhMSCs) were placed in culture postradiolabeling for assessments of growth and differentiation toward adipogenic, osteogenic, and chondrogenic lineages. The results indicated that CD34+ cells radiolabeled with 20 microCi/mL and rhMSCs radiolabeled with 10 microCi/mL 64Cu-PTSM did not result in adverse effects on growth or differentiation. Nonradioactive copper was also evaluated and showed that the presence of copper was not harmful to the cells. CD34+ cells and rhMSCs radiolabeled with the optimized concentrations of 20 and 10 microCi/mL, respectively, were also assessed using the microPET scanner. Studies showed that a minimum of 2.50x10(4) CD34+ cells (1.1 pCi/cell) and 6.25x10(3) rhMSCs (4.4 pCi/cell) could be detected. These studies indicate that CD34+ hematopoietic cells and rhMSCs can be safely radiolabeled with 64Cu-PTSM without adverse cellular effects.     

MERP1: a mammalian ependymin-related protein gene differentially expressed in hematopoietic cells

We have utilized differential display polymerase chain reaction to investigate the gene expression of hematopoietic progenitor cells from adult bone marrow and umbilical cord blood. A differentially expressed gene was identified in CD34+ hematopoietic progenitor cells, with low expression in CD34- cells. We have obtained the full coding sequence of this gene which we designated human mammalian ependymin-related protein 1 (MERP1). Expression of MERP1 was found in a variety of normal human tissues, and is 4- and 10-fold higher in adult bone marrow and umbilical cord blood CD34+ cells, respectively, compared to CD34- cells. Additionally, MERP1 expression in a hematopoietic stem cell enriched population was down-regulated with proliferation and differentiation. Conceptual translation of the MERP1 open reading frame reveals significant homology to two families of glycoprotein calcium-dependant cell adhesion molecules: ependymins and protocadherins.     

Autologous cell therapy as a new approach to treatment of radiation-induced bone marrow aplasia: preliminary study in a baboon model

The sparing of viable hematopoietic stem and progenitor cells located in underexposed bone marrow territories associated with the relative radioresistance of certain stem cell populations is the rationale for autologous cell therapy consisting of ex vivo expansion of residual cells after collection postirradiation. The feasibility of this treatment mainly depends on time constraints and hematopoietic cell threshold. We showed in this study that in the absence of early-acting mobilizing agent administration, subliminar amounts of CD34+ cells can be collected (1 x 10(6) CD34+ cells/100 mL bone marrow or for 1 L apheresis) from 6-Gy gamma globally irradiated baboons. Residual CD34+ cells were successfully expanded in serum-free medium in the presence of antiapoptotic cytokine combination (stem cell factor + FLT-3 ligand + thrombopoietin + interleukin 3, 50 ng/mL each, i.e., 4F): KCD34+ = x2.8 and x13.7 (n = 2). Moreover, we demonstrated the short-term neutrophil engraftment potential of a low-size mixed expanded graft (1.5 x 106 final CD34+cells/kg) issued from the coculture of unirradiated (20%) and 2.5-Gy in vitro irradiated (80%) CD34+ cells on an allogeneic stromal cell layer in the presence of 4F. Further preclinical research needs to be performed to clearly establish this therapeutic approach that could be optimized by the early administration of antiapoptotic cytokines.     

Human CD34+ HLA-DR- bone marrow cells contain progenitor cells capable of self-renewal, multilineage differentiation, and long-term in vitro hematopoiesis.

Human bone marrow cells expressing CD34 but not HLA-DR were isolated by immunofluorescence flow cytometric cell sorting. These cells contained a hematopoietic cell (CFU-B1) capable of producing, in an in vitro semisolid culture system, blast-cell-containing colonies, which possessed the capacity for self-renewal and commitment to multipotential differentiation. In addition, CD34+ HLA-DR- marrow cells contained primitive megakaryocyte progenitor cells, the burst-forming unit-megakaryocyte (BFU-MK). A subset of CD34+ HLA-DR- marrow cells lacking the expression of CD15 and CD71 was obtained by flow cytometric cell sorting and was capable of sustaining in vitro hematopoiesis in suspension culture for up to 8 weeks in the absence of a preestablished adherent marrow cell layer. The combination of IL-3 + IL-1 alpha and IL-3 + IL-6 sustained proliferation of these cells for 8 weeks, induced maximal cellular expansion, and increased the numbers of assayable progenitor cells. These studies demonstrate that human CD34+ HLA-DR- marrow cells and their subsets contain primitive multipotential hematopoietic cells capable of self-renewal and of differentiation into multiple hematopoietic lineages.     

Human T-cell leukemia virus infection of human hematopoietic progenitor cells: maintenance of virus infection during differentiation in vitro and in vivo.

Human T-cell leukemia virus type I (HTLV-1) is the etiologic agent of adult T-cell leukemia and lymphoma and HTLV-1-associated myelopathy-tropical spastic paraparesis. We examined whether HTLV could productively infect human hematopoietic progenitor cells. CD34+ cells were enriched from human fetal liver cells and cocultivated with cell lines transformed with HTLV-1 and -2. HTLV-1 infection was established in between 10 and >95% of the enriched CD34+ cell population, as demonstrated by quantitative PCR analysis. HTLV-1 p19 Gag expression was also detected in infected hematopoietic progenitor cells. HTLV-1-infected hematopoietic progenitor cells were cultured in semisolid medium permissive for the development of erythbroid (BFU-E), myeloid (CFU-GM), and primitive progenitor (CFU-GEMM, HPP-CFC, or CFU-A) colonies. HTLV-1 sequences were detected in colonies of all hematopoietic lineages; furthermore, the ratio of HTLV genomes to the number of human cells in each infected colony was 1:1, consistent with each colony arising from a single infected hematopoietic progenitor cell. Severe combined immunodeficient mice engrafted with human fetal thymus and liver tissues (SCID-hu) develop a conjoint organ which supports human thymocyte differentiation and maturation. Inoculation of SCID-hu mice with HTLV-1-infected T cells or enriched populations of CD34+ cells established viral infection of thymocytes 4 to 6 weeks postreconstitution. Thymocytes from two mice with the greatest HTLV-1 proviral burdens showed increased expression of the CD25 marker and the interleukin 2 receptor alpha chain and perturbation of CD4+ and CD8+ thymocyte subset distribution profiles. Hematopoietic progenitor cells and thymuses may be targets for HTLV infection in humans, and these events may play a role in the pathogenesis associated with infection.     

人造血干／祖细胞多态性的研究：：Ⅺ.人正常骨髓CD34^＋造血细胞周 …

DNA, RNA and PRO comprise the bulk of macromolecules in cells, which have been proven to play an important role in regulating cell cycle transverse capacity, cell division, growth, and size. Simultaneous analysis of these moieties could provide more comprehensive and accurate information on cell cycle kinetics. In this study, DNA, RNA and PRO contents related to cell cycle kinetics in CD34+ hematopoietic cells of human bone marrow were measured to understanding the cell cycle kinetic features in CD34+ hematopoietic cells. For this reason, CIMS-100 immunomagnetic isolator, a novel isolation system, was used to enrich efficiently CD34+ hematopoietic cells from human bone marrow. The purity of enriched CD34+ hematopoietic cells determined by both FACS and APAAP staining is ranging from 90%-95%. Cellular DNA, RNA and PRO were stained with fluorochromes propidium iodide, pyronin Y and fluorescein isothiocyante respectively The fluorescence intensities reflecting the DNA, RNA and PRO content of individual cell were analyzed in FACS can by different excitation wavelengthes. DNA, RNA and PRO contents in CD34+ hematopoietic cells were far lower than these of bone marrow mononuclear cells, only being 34 +/- 3% (DNA), 48 +/- 21% (RNA) and 62 +/- 14% (PRO) of BMMNCs respectively. Collectively, these data combined with previous results from both ours and others indicated that CD34+ hematopoietic cells are indeed an unique cell population, not only in reconstitute of hematopoietic and immunological functions, but also in cell cycle kinetics. This is, to our knowledge, the first detailed report on the analysis of DNA, RNA and PRO contents related to cell cycle kinetics in CD34+ hematopoietic cells. And the results provide more direct evidence that the majority of CD34+ hematopoietic cells are in resting state.