Differential Expression Of CD34 Research Articles

The Tunica Adventitia of Human Arteries and Veins As a Source of Mesenchymal Stem Cells

We previously demonstrated that human pericytes, which encircle capillaries and microvessels, give rise in culture to genuine mesenchymal stem cells (MSCs). This raised the question as to whether all MSC are derived from pericytes. Pericytes and other cells defined on differential expression of CD34, CD31, and CD146 were sorted from the stromal vascular fraction of human white adipose tissue. Besides pericytes, CD34+ CD31- CD146- CD45- cells, which reside in the outmost layer of blood vessels, the tunica adventitia, natively expressed MSC markers and gave rise in culture to clonogenic multipotent progenitors identical to standard bone marrow-derived MSC. Despite common MSC features and developmental properties, adventitial cells and pericytes retain distinct phenotypes and genotypes through culture. However, in the presence of growth factors involved in vascular remodeling, adventitial cells acquire a pericytes-like phenotype. In conclusion, we demonstrate the co-existence of 2 separate perivascular MSC progenitors: pericytes in capillaries and microvessels and adventitial cells around larger vessels.

Atrial natriuretic peptide and CD34 overexpression in human idiopathic dilated cardiomyopathies

Idiopathic dilated cardiomyopathy (IDCM) is a primary myocardial disease of unknown cause characterized by ventricular chamber enlargement with impaired contractile function. In familial forms of IDCM, mutations of genes coding for cytoskeletal proteins related to force transmission, such as dystrophin, cardiac actin, desmin, and delta-sarcoglycan, have been identified. Here, we report the data of a retrospective investigation carried out to evaluate the expression of atrial natriuretic peptide (ANP), CD34, troponin T and nestin in the myocardium of patients affected with IDCM. Formalin-fixed and paraffin-embedded consecutive tissue sections from the ventricular wall of 10 human normal hearts (NH) following forensic autopsy and 22 IDCM (living explanted hearts) were studied using primary monoclonal antibodies against ANP, CD34, troponin T and nestin by immunohistochemistry. Myocardial fibers were counted independently by three pathologists. Statistics included analysis of variance, log-rank test for Kaplan-Meier analysis, and kappa assessment for intra- and inter-observer variability. ANP and CD34 were significantly overexpressed in IDCM compared to NH (p<0.05). Conversely, troponin T and nestin expression levels did not show significant variation. Inter-observer kappa statistics showed a value of 0.87 and intra-observer kappa statistics a value of 0.98. Evaluation of the marker distribution in the myocardium of patients with IDCM CD34 expression curve was similar to that of troponin T (p<0.0001), although two groups could be identified. Patients with a difference of more than 20 myocardial fibers in expression of CD34 and troponin T had a somewhat less favorable survival although the difference was not significant. The analysis of cells positive for troponin T resulted in a similar number of cardiac fibers between NH and IDCM. This is in agreement with cardiac enlargement present in IDCM, which is due to ventricular dilatation rather than increased number of myocytes. Moreover, the expression of nestin, a marker of activation of myocardial precursors, did not change either, and this may confirm that there are no hyperplastic phenomena in the IDCM pathogenesis. The increase in ANP-positive cells in IDCM could be a consequence of neurohormonal activation due to a decline in the impaired myocyte contractility. Furthermore, since it was already shown that ANP could be important in the control of vascular remodeling, we postulated that the increase in CD34-positive cells might be functionally correlated with the increase in ANP production. Differential expression of CD34 and troponin T might be used in future studies to evaluate their prognostic value.

Elucidation of the Phenotypic, Functional, and Molecular Topography of a Myeloerythroid Progenitor Cell Hierarchy

The major myeloid blood cell lineages are generated from hematopoietic stem cells by differentiation through a series of increasingly committed progenitor cells. Precise characterization of intermediate progenitors is important for understanding fundamental differentiation processes and a variety of disease states, including leukemia. Here, we evaluated the functional in vitro and in vivo potentials of a range of prospectively isolated myeloid precursors with differential expression of CD150, Endoglin, and CD41. Our studies revealed a hierarchy of myeloerythroid progenitors with distinct lineage potentials. The global gene expression signatures of these subsets were consistent with their functional capacities, and hierarchical clustering analysis suggested likely lineage relationships. These studies provide valuable tools for understanding myeloid lineage commitment, including isolation of an early erythroid-restricted precursor, and add to existing models of hematopoietic differentiation by suggesting that progenitors of the innate and adaptive immune system can separate late, following the divergence of megakaryocytic/erythroid potential.

Genetic Evidence of a Novel Blood Differentiation Pathway from Lympho-Myeloid Hematopoietic Stem/Progenitor Cells, Independent of Common Myeloid Progenitors.

We have recently identified three novel subsets of multipotent hematopoietic stem/progenitor cells (HSCs) in the Lin−Sca-1+c-Kithi (LSK) compartment of adult murine bone marrow based on differential expression of CD34 and the cytokine tyrosine kinase receptor Flt3. Long-term HSCs (LT-HSCs) lack CD34 and Flt3 expression (LSKCD34-flt3-), whereas short-term HSCs (ST-HSCs) are LSKCD34+flt3−. A third LSK population is characterized by co-expression of CD34 and Flt3 (LSKCD34+flt3+) and possess a combined myeloid (granulocyte and monocyte) and lymphoid (B and T cell) differentiation potential, but surprisingly lack megakaryocytic (Mk) and erythroid (E) potential in vitro and in vivo. These findings implicate an alternative road map for blood lineage development distinct from the classical model in which the first lineage commitment step of HSCs is thought to result in a strict separation into myelopoiesis and lymphopoiesis. In the current study we sought genetic evidence in further support of this new model through genetic profiling of these three HSC subpopulations, using affymetrix chips, quantitative (Q)-PCR and single cell PCR. In contrast to the pluripotent LSKCD34−Flt3− LT-HSCs and LSKCD34+Flt3− ST-HSCs, LSKCD34+Flt3+ cells downregulated or turned of genes critically involved in promoting Mk and E lineage development, such as the Epo and Tpo receptors as well as the transcription factor Gata-1. In contrast, the gene for Il-7rα, critically involved in early B and T cell development was upregulated in LSKFlt3+ cells, but absent in LT-HSCs and ST-HSCs. However, in agreement with their sustained ability to produce granulocytes and monocytes, G-CSFR and Pu.1 expression was sustained from LT-HSC through the LSKCD34+flt3+ stage Particularly noteworthy, single cell PCR demonstrated that a fraction of single LSKCD34+Flt3+ cells upregulating Il-7rα gene expression, sustained and co-expressed G-CSFR expression. Thus, genetic profiling at the single cell level provide further and compelling evidence for a novel road map for blood lineage development, independent of the common myeloid progenitor (CMP). Our biological and gene expression data rather supports the existence of a pathway in which HSCs lose their lineage potentials one by one, starting with the Mk and E lineages.

A Novel Road Map for Blood Lineage Commitment and Development: Identification of a Lympho-Myeloid Stem/Progenitor Cell Lacking Erythro-Megakaryocytic Potential.

The recent identification of common myeloid and lymphoid progenitors (CMPs and CLPs, respectively) lends support to the classical and currently prevailing model for hematopoietic commitment and blood lineage development, suggesting that the first and decisive lineage commitment step of adult hematopoietic stem cells (HSCs) results in an immediate and complete separation of myelopoiesis and lymphopoiesis. Virtually all of multipotent (lympho-myeloid) stem and progenitor cells in adult mouse bone marrow (BM) reside in the small Lin−Sca1+kithi (LSK) compartment. We now present data demonstrating that the LSK compartment in adult BM, can be separated into three phenotypically and functionally distinct HSC subsets, based on differential expression of CD34 and flt3. Long-term HSCs (with extensive self-renewing potential) reside in the LSKCD34−flt3− fraction. The LSKCD34+ short-term HSC compartment consists of LSKCD34+flt3− cells fulfilling all criteria of short-term HSCs, being highly enriched in CFU-S activity, and capable of rapidly reconstituting myelo-erythropoiesis and thereby rescuing myeloablated mice. The short-term LSKCD34+flt3− HSCs give upon transplantation rise to LSKCD34+flt3+ cells, that although sustaining a combined lymphoid (B and T cell) and myeloid (granulocyte and monocyte) developmental potential at the single cell level, loose their ability to adapt megakaryocytic and erythroid fates in vitro as well as in vivo. These evidence for the existence of LSKCD34+flt3+ cells with granulocyte, monocyte, B and T cell, but not megakaryocyte and erythroid development potentials, are not compatible with the first lineage commitment step of HSCs resulting in strict separation of common lymphoid and common myeloid differentiation pathways. Based on the present findings we rather propose an alternative road map for blood lineage development in which LSKCD34+flt3− short-term HSCs genereate megakaryocyte-erythroid progenitors and LSKCD34+flt3+ cells upon asymmetric cell divisions.

Differential expression of CD34 in normal colorectal tissue, peritumoral inflammatory tissue, and tumour stroma

Aims—To investigate the role of CD34 positive stromal cells, namely dendritic interstitial cells, in the desmoplastic stroma formation of malignant epithelial neoplasms the distribution of CD34 positive stromal cells was...

Differential expression of CD34 and Ki-M1p in pleomorphic fibroma and dermatofibroma with monster cells.

Pleomorphic fibroma (PF) and dermatofibroma with monster cells (DFMC) are characterized by the presence of numerous cells with large atypical nuclei. Despite cytologic similarities, the two entities are likely to be unrelated, but their histogenesis is poorly understood. In this study, we examined six cases of PF and eleven cases of DFMC by immunohistochemistry using antibodies against vimentin, alpha-smooth muscle actin, S-100 protein, CD34, factor XIIIa, and the pan-monocytic marker Ki-M1p. Strong vimentin expression was seen in all tumors, whereas none of them expressed S-100 protein. PF consistently exhibited CD34 staining but appeared to be depleted of Ki-M1p positive cells compared with the surrounding normal skin. Conversely, all cases of DFMC contained numerous Ki-M1p positive cells including atypical multinucleate cells, but virtually no CD34 reactivity was observed. A weak staining for alpha-smooth muscle actin was occasionally seen in a subset of the cells of both entities. Our results indicate that PF and DFMC are histogenetically distinct entities that may arise from two different types of dermal dendritic cells defined by their reactivity for CD34 and Ki-M1p, respectively. Immunohistochemistry using these two antibodies permits an easy and reliable discrimination between PF and DFMC.

Characterization and phenotypic analysis of differentiating CD34+ human bone marrow cells in liquid culture.

Our current understanding of human haematopoietic stem cell biology is based in part on the characterization of human CD34+ bone marrow cell differentiation in vitro. CD34 is highly expressed on early stem cells and haematopoietic progenitor cells with clonogenic potential and is gradually lost during differentiation and commitment. However, CD71 (transferrin receptor) is expressed at low levels on early stem cells and generally increases during haematopoietic progenitor cell proliferation. We reasoned that the combination of these surface markers would provide a useful framework for the simultaneous analysis of multiple lineage differentiation of CD34+ haematopoietic progenitor cells in liquid culture. In this report, we identify the phenotype of distinct subpopulations of myeloid, erythroid and lymphoid cells in liquid suspension culture using differential expression of CD34 vs. CD71 in combination with specific lineage markers. Freshly isolated human CD34+ bone marrow cells were introduced into suspension culture and monitored over a 6-d period using 3-colour flow cytometry. This is the first demonstration that differential expression of CD34 vs. CD71 can be used to simultaneously monitor differentiation of multiple haematopoietic cell lineages in liquid suspension culture, facilitating the study of cytokine-, drug- or chemical-induced alterations in haematopoietic progenitor cell differentiation in vitro.

Thrombopoietin Is Synthesized by Bone Marrow Stromal Cells

AbstractWe have previously characterized stromal progenitor cells contained in fetal bone marrow by fluorescence-activated cell sorting (FACS) using the differential expression of CD34, CD38, and HLA-DR, and found that a small number were contained within the CD34+ cell fraction. In the present study, the frequency of stromal progenitors in both the CD34+ and CD34− subpopulations from samples of fetal and adult bone marrow was approximately one in 5,000 of the mononuclear cell fraction. Using multiparameter single-cell sorting, one in 20 fetal bone marrow cells with the CD34+, CD38−, HLA-DR−, CDw90+ phenotype were clonogenic stromal progenitors, whereas greater than one in five single cells with the CD34−, CD38−, HLA-DR−, CDw90+ phenotype formed stromal cultures. We found that cultures initiated by hematopoietic and stromal progenitors contained within the CD34+ fraction of bone marrow cells formed mixed hematopoietic/stromal cell cultures that maintained the viability of the hematopoietic progenitor cells for 3 weeks in the absence of added hematopoietic cytokines. We characterized some of the hematopoietic cytokines synthesized by stromal cultures derived from either CD34+ or CD34− bone marrow cells using reverse transcriptase–polymerase chain reaction (RT-PCR) amplification of interleukin-3 (IL-3), stem cell factor (SCF), CD34, Flt3/Flk2 ligand (FL), and thrombopoietin (TPO) mRNA sequences. We found ubiquitous expression of TPO mRNA in greater than 90% of stromal cultures initiated by either CD34+ or CD34− cells, and variable expression of SCF, FL, and CD34 mRNA. In particular, SCF and CD34 mRNA were detected only in stromal cultures initiated by CD34+ bone marrow cells, although the differences between CD34+ and CD34− stromal cells were not statistically significant. IL-3 mRNA was not found in any stromal cultures. An enzyme-linked immunosorbent assay (ELISA) of soluble SCF and TPO present in culture supernatants demonstrated that biologically significant amounts of protein were secreted by some cultured stromal cells: eight of 16 samples of conditioned media from stromal cultures initiated by fetal and adult bone marrow contained more than 32 pg/mL SCF (in the linear range of the ELISA), with a median value of 32 pg/mL (range, 9 to 230), while 13 of 24 samples of conditioned media had more than 16 pg/mL TPO (in the linear range of the ELISA), with a median of 37 pg/mL (range, 16 to 106). Our data indicate that stromal cultures initiated by single bone marrow cells can make FL, SCF, and TPO. Local production of early-acting cytokines and TPO by stromal cells may be relevant to the regulation of hematopoietic stem cell self-renewal and megakaryocytopoiesis in the bone marrow microenvironment.

Expression and function of receptors for stem cell factor and erythropoietin during lineage commitment of human hematopoietic progenitor cells

The aim of the present study was to determine whether stem cell factor (SCF) and erythropoietin (EPO) act differently on defined subsets of progenitor cells, and if potential differences correlate with the receptor density on each subset. To investigate this possibility directly, we optimized conditions for the identification and purification of homogeneous progenitor cell subpopulations from human bone marrow. Populations containing 40% and 44% colony forming cells (CFCs) with 99% and 95% purity for the granulomonocytic and erythroid lineage, respectively, were sorted on the basis of differential expression of CD34, CD64, and CD71. In addition, a population containing 67% CFCs, of which 29-43% were CFU-MIX, was sorted from CD34hi CD38loCD50+ cells. Purified progenitor cell subsets were compared directly for responsiveness to SCF and EPO using a short-term proliferation assay. Expression of the receptors for SCF and EPO were then examined on each subset using a flow cytometer modified for high-sensitivity fluorescence measurements. The results show that EPO induces extensive proliferation of erythroid progenitor cells, but has no effect on the proliferation or survival of primitive or granulomonocytic progenitors, even when used in combination with other cytokines. The majority of erythroid progenitor cells furthermore stained positively with anti-EPO receptor (EPO-R) monoclonal antibodies, whereas other progenitor cells were negative. SCF alone induced extensive proliferation of erythroid progenitor cells, and had a stronger synergistic effect on primitive than on granulo-monocytic progenitors. In spite of these differences in SCF activity, there were no significant differences in SCF-R expression between the progenitor subsets. These results suggest that the selective action of EPO on erythropoiesis is determined by lineage-restricted receptor expression, whereas there are additional cell-type specific factors that influence progenitor cell responses to SCF.

Differential expression of factor XIIIa and CD34 in cutaneous mesenchymal tumors

The histogenetic relationship amongst various dendritic cells of the dermis which may express markers including factor XIIIa (FXIIIa) or CD34 remains unclear. In this study we utilized a sensitive indirect immunoperoxidase staining technique to identify CD34 and FXIIIa, as well the monocyte/macrophage markers KP-1 and MAC 387 expression in a variety of cutaneous dermal tumors of mesenchymal origin to see if differential expression of CD34 vs FXIIIa exists. Tumors studied included dermatofibroma (DF) (N = 10), keloid (N = 9), atypical fibroxanthoma (AFX) (N = 3), and dermatofibrosarcoma protuberans (DFSP) (N = 7). DF were all composed of FXIIIa+ spindle-shaped and stellate tumor cells (mean score = 4.9 or > or = 75% FXIIIa+) as previously reported, but these cells rarely (< 10%) expressed CD34. Six of 7 DFSP were found to be > 75% CD34+ and FXIIIa negative, while one DFSP was negative for both CD34 and FXIIIa. In all DFSP, there were trapped FXIIIa+ cells which were distinct from the spindle-shaped tumor cells. AFX showed sparse populations of FXIIIa+ cells in the stroma (mean score = 1.33 or 10-25% positive), which were distinct from the atypical giant cells characteristic of these tumors. Keloid similarly contained trapped FXIIIa+ cells (mean score = 0.44 or < 5% positive) that were distinct from the spindle-shaped fibroblasts of the tumor mass. Dendritic and spindle-shaped cells within these tumors were consistently both KP-1 and Mac-387 negative, while all lesions studied were characterized by scattered round, histiocytic cells which were KP-1+ and/or Mac-387+ irrespective of tumor cell type.(ABSTRACT TRUNCATED AT 250 WORDS)