Myeloid differentiation of human cord blood CD34⁺ cells during ex vivo expansion using thrombopoietin, flt3‐ligand and/or granulocyte‐colony stimulating factor

Abstract

Summary. We investigated the phenotypic changes of human umbilical cord blood (CB) CD34þ cells during ex vivo expansion using thrombopoietin (TPO), flt3-ligand (FL), and/or granulocyte-colony stimulating factor (G-CSF). During ex vivo expansion of CD34 þ cells isolated from human CB for up to 5 weeks, surface expression of molecules on the cultured cells including CD64 (FcgRI), CD32 (FcgRII), CD16 (FcgRIII), CD11b (MAC-1) and CD18 (b 2 -integrin) was analysed by flow cytometry along with simultaneous measurement of apoptosis by 7-aminoactinomycin D staining method. CD64, CD32 and/or CD18 expressing cells appeared in the cultures both with and without the addition of G-CSF until the tenth day. However, without G-CSF, CD16þ fractions did not appear and CD11b þ fractions were not maintained. With G-CSF, the CD16 þ or CD11b þ fractions appeared only from the second week. These results suggest that G-CSF is necessary for the late stage of myeloid maturation during which CD16 and CD11b are expressed.

Potential Mechanisms for Thrombocytopenia Development with Trastuzumab Emtansine (T-DM1)

“…Grade !3 anemia occurred in 2.7% of patients, grade !3 neutropenia occurred in 2.0% to 2.5%, and grade !3 leucopenia occurred in <2% of patients (6,8). These clinical data are consistent with an FcgRIIa-dependent mechanism because FcgRIIa is expressed at relatively low (or absent) levels in CD34 þ cells isolated from human cord blood (27) and in lymphocyte lineages in normal human bone marrow (28). However CD32 is expressed in myeloid lineages (28).…”

Decrease in Apoptosis and Increase in Polyploidization of Megakaryocytes by Stem Cell Factor During Ex Vivo Expansion of Human Cord Blood CD34 + Cells Using Thrombopoietin

“…CD34 + cells were purified using Ficoll‐Hypaque (density, 1.077; Pharmacia Biotech; Uppsala, Sweden; http://www.pnu.com) density centrifugation and immunomagnetic (Miltenyi Biotec; Bergisch Gladbach, Germany; http://www.miltenyibiotec.com) selection, as previously described [20]. The efficiency of purification, as verified by flow cytometry counterstaining with an anti‐human CD34 antibody conjugated with fluoroscein isothiocyanate (FITC; HPCA‐2; Becton Dickinson; Mountain View, CA; http://www.bd.com) was consistently more than 95%.…”

Leukotriene B4 pathway regulates the fate of the hematopoietic stem cells

“…The CD34 + cell fraction was isolated with a superparamagnetic microbead selection using monoclonal antibody (QBEND10) and miniMACS columns (Miltenyi Biotec; Bergisch Gladbach, Germany) (Kim, 2003). The efficiency of purification and immunophenotype were verified with flow cytometry and counterstained with a FITC-anti-human CD34 (HPCA-2; Becton Dickinson; Mountain View, California) as previously described (Yoo et al, 1999).…”

“…Earlier report of optimal culture condition for hematopoietic stem cells with combined supplementation of G-CSF, TPO and FL (Yoo et al, 1999) was used as a control culture and compared with the additional supplementation of LTB4. In the absence of exogenous cytokines, no viable cells were observed after 2 weeks of culture, regardless of whether or not LTB4 was present (data not shown).…”