Correction: Expansion of Cord Blood CD34+ Cells in Presence of zVADfmk and zLLYfmk Improved Their In Vitro Functionality and In Vivo Engraftment in NOD/SCID Mouse

Ectopic retroviral expression of homeobox B4 (HOXB4) causes an accelerated and enhanced regeneration of murine hematopoietic stem cells (HSCs) and is not known to compromise any program of lineage differentiation. However, HOXB4 expression levels for expansion of human stem cells have still to be established. To test the proposed hypothesis that HOXB4 could become a prime tool for in vivo expansion of genetically modified human HSCs, we retrovirally overexpressed HOXB4 in purified cord blood (CB) CD34+ cells together with green fluorescent protein (GFP) as a reporter protein, and evaluated the impact of ectopic HOXB4 expression on proliferation and differentiation in vitro and in vivo. When injected separately into nonobese diabetic–severe combined immunodeficient (NOD/SCID) mice or in competition with control vector–transduced cells, HOXB4-overexpressing cord blood CD34+ cells had a selective growth advantage in vivo, which resulted in a marked enhancement of the primitive CD34+ subpopulation (P = .01). However, high HOXB4 expression substantially impaired the myeloerythroid differentiation program, and this was reflected in a severe reduction of erythroid and myeloid progenitors in vitro (P < .03) and in vivo (P = .01). Furthermore, HOXB4 overexpression also significantly reduced B-cell output (P < .01). These results show for the first time unwanted side effects of ectopic HOXB4 expression and therefore underscore the need to carefully determine the therapeutic window of HOXB4 expression levels before initializing clinical trials.

Download Full-text

In vitro clinical-grade generation of red blood cells from human umbilical cord blood CD34+ cells

Transfusion ◽

10.1111/j.1537-2995.2008.01828.x ◽

2008 ◽

Vol 48 (10) ◽

pp. 2235-2245 ◽

Cited By ~ 62

Author(s):

Eun Jung Baek ◽

Han-Soo Kim ◽

Sinyoung Kim ◽

Honglien Jin ◽

Tae-Yeal Choi ◽

...

Keyword(s):

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Blood Cells ◽

Human Umbilical Cord Blood ◽

Human Umbilical Cord ◽

Clinical Grade ◽

Cd34 Cells ◽

Cord Blood Cd34

Download Full-text

Evaluation of Reliable Reference Genes for In Vitro Erythrocyte Generation from Cord Blood CD34+ Cells

DNA and Cell Biology ◽

10.1089/dna.2021.0185 ◽

2021 ◽

Author(s):

Lei Xu ◽

Zhan Gao ◽

Zhou Yang ◽

Mingyi Qu ◽

Huilin Li ◽

...

Keyword(s):

Cord Blood ◽

Reference Genes ◽

Cd34 Cells ◽

Cord Blood Cd34

Download Full-text

The αIIbβ3 integrin and GPIb-V-IX complex identify distinct stages in the maturation of CD34+cord blood cells to megakaryocytes

Blood ◽

10.1182/blood.v96.13.4169 ◽

2000 ◽

Vol 96 (13) ◽

pp. 4169-4177 ◽

Cited By ~ 51

Author(s):

Adeline Lepage ◽

Marylène Leboeuf ◽

Jean-Pierre Cazenave ◽

Corinne de la Salle ◽

François Lanza ◽

...

Keyword(s):

Cord Blood ◽

Messenger Rna ◽

Human Cord Blood ◽

Cd34 Cells ◽

Conditional Expression ◽

Multistep Process ◽

Cord Blood Cd34 ◽

Specific Proteins ◽

Cord Blood Cells

Abstract Megakaryocytopoiesis is a complex multistep process involving cell division, endoreplication, and maturation and resulting in the release of platelets into the blood circulation. Megakaryocytes (MK) progressively express lineage-restricted proteins, some of which play essential roles in platelet physiology. Glycoprotein (GP)Ib-V-IX (CD42) and GPIIb (CD41) are examples of MK-specific proteins having receptor properties essential for platelet adhesion and aggregation. This study defined the progressive expression of the GPIb-V-IX complex during in vitro MK maturation and compared it to that of GPIIb, an early MK marker. Human cord blood CD34+ progenitor cells were cultured in the presence of cytokines inducing megakaryocytic differentiation. GPIb-V-IX expression appeared at day 3 of culture and was strictly dependent on MK cytokine induction, whereas GPIIb was already present in immature CD34+ cells. Analysis by flow cytometry and of the messenger RNA level both showed that GPV appeared 1 day later than GPIb-IX. Microscopy studies confirmed the late appearance of GPV, which was principally localized in the cytoplasm when GPIb-IX was found on the cell surface, suggesting a delayed program of GPV synthesis and trafficking. Cell sorting studies revealed that the CD41+GPV+ population contained 4N and 8N cells at day 7, and was less effective than CD41+GPV− cells in generating burst-forming units of erythrocytes or MK colonies. This study shows that the subunits of the GPIb-V-IX complex represent unique surface markers of MK maturation. The genes coding for GPIb-IX and GPV are useful tools to study megakaryocytopoiesis and for tissue-specific or conditional expression in mature MK and platelets.

Download Full-text

miR-17 promotes expansion and adhesion of human cord blood CD34+ cells in vitro

Stem Cell Research & Therapy ◽

10.1186/s13287-015-0159-1 ◽

2015 ◽

Vol 6 (1) ◽

Author(s):

Yuxia Yang ◽

Saifeng Wang ◽

Zhenchuan Miao ◽

Wei Ma ◽

Yanju Zhang ◽

...

Keyword(s):

Cord Blood ◽

Human Cord Blood ◽

Cd34 Cells ◽

Cord Blood Cd34

Download Full-text

Effect of testosterone and hypoxia on the expansion of umbilical cord blood CD34+ cells in vitro

Experimental and Therapeutic Medicine ◽

10.3892/etm.2017.5026 ◽

2017 ◽

Cited By ~ 2

Author(s):

Liping Zhou ◽

Xiaowei Zhang ◽

Panpan Zhou ◽

Xue Li ◽

Xuejing Xu ◽

...

Keyword(s):

Cord Blood ◽

Umbilical Cord ◽

Umbilical Cord Blood ◽

Cd34 Cells ◽

Cord Blood Cd34

Download Full-text

Human NK cell development in NOD/SCID mice receiving grafts of cord blood CD34+ cells

Blood ◽

10.1182/blood-2002-07-2024 ◽

2003 ◽

Vol 102 (1) ◽

pp. 127-135 ◽

Cited By ~ 30

Author(s):

Christian P. Kalberer ◽

Uwe Siegler ◽

Aleksandra Wodnar-Filipowicz

Keyword(s):

Cord Blood ◽

Nk Cells ◽

Scid Mouse ◽

Nk Cell ◽

Cell Development ◽

Scid Mice ◽

Differentiation Potential ◽

Ifn Γ

Abstract Definition of the cytokine environment, which regulates the maturation of human natural killer (NK) cells, has been largely based on in vitro assays because of the lack of suitable animal models. Here we describe conditions leading to the development of human NK cells in NOD/SCID mice receiving grafts of hematopoietic CD34+ precursor cells from cord blood. After 1-week-long in vivo treatment with various combinations of interleukin (IL)–15, flt3 ligand, stem cell factor, IL-2, IL-12, and megakaryocyte growth and differentiation factor, CD56+CD3- cells were detected in bone marrow (BM), spleen, and peripheral blood (PB), comprising 5% to 15% of human CD45+ cells. Human NK cells of NOD/SCID mouse origin closely resembled NK cells from human PB with respect to phenotypic characteristics, interferon (IFN)–γ production, and cytotoxicity against HLA class 1–deficient K562 targets in vitro and antitumor activity against K562 erythroleukemia in vivo. In the absence of growth factor treatment, CD56+ cells were present only at background levels, but CD34+CD7+ and CD34-CD7+ lymphoid precursors with NK cell differentiation potential were detected in BM and spleen of chimeric NOD/SCID mice for up to 5 months after transplantation. Our results demonstrate that limitations in human NK cell development in the murine microenvironment can be overcome by treatment with NK cell growth–promoting human cytokines, resulting in the maturation of IFN-γ–producing cytotoxic NK cells. These studies establish conditions to explore human NK cell development and function in vivo in the NOD/SCID mouse model. (Blood. 2003;102:127-135)

Download Full-text

Recombinant Tpo Stimulates Maturation of Megakaryocytes Derived from Adult Peripheral Blood- but Not Cord Blood-CD34+ Cells.

Blood ◽

10.1182/blood.v104.11.1191.1191 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1191-1191

Author(s):

Karen M. Pastos ◽

William B. Slayton ◽

Lisa M. Rimsza ◽

Martha C. Sola

Keyword(s):

Cord Blood ◽

Peripheral Blood ◽

Developmental Differences ◽

Ploidy Levels ◽

Cd34 Cells ◽

Cord Blood Cd34 ◽

Adult Bone ◽

Adult Peripheral Blood ◽

Cells Cultured

Abstract Umbilical cord blood (CB) is a valuable source of stem cells for transplantation. However, platelet engraftment is slow, taking approximately 70 days for CB transplants versus 20 days for mobilized adult peripheral blood (PB) transplants. This time is not significantly shortened by the administration of recombinant thrombopoietin (rTpo). The cause for the delay in platelet engraftment following CB transplant is unknown. We hypothesized that developmental differences in size and ploidy of neonatal versus adult megakaryocytes (MKs) contribute to this delay. To mimic these two types of transplant in vitro, we compared CB to PB CD34+ cells cultured in adult bone marrow stromal-conditioned media (CM) or unconditioned media (UCM) for 14 days. Increasing doses of rTpo were added to the CM, and the resulting MK maturation was compared with that of UCM with maximal rTpo concentration. MK number and ploidy were determined by flow cytometry using CD41-FITC and propidium iodide, respectively. Increased ploidy levels were expressed as percentage of MKs with a ploidy ≥ 8N. Results represent an average of three independent experiments. Figure Figure As seen in the figure, PB-derived MKs reached highest ploidy levels in the presence of UCM + 100 ng/ml rTpo. When cultured in CM, they exhibited lower ploidy levels, regardless of Tpo concentration. In contrast, CB-derived MKs exhibited higher ploidy levels in response to CM with either 0 or 0.1 ng/ml (physiologic concentration) of rTpo, as compared to higher rTpo concentrations or UCM + 100 ng/ml rTpo. MK numbers increased in response to rTpo in a dose-response manner, regardless of the source of the MKs (data not shown). These results indicate that intrinsic differences between CB- and PB-derived megakaryocytes exist, and that maturation is regulated differently in neonatal versus adult MKs. While Tpo is a potent stimulator of MK maturation in PB-derived MKs, it appears to inhibit this process in CB-derived MKs. These differences may be relevant to understanding the delayed platelet engraftment following CB transplants.

Download Full-text

Ex Vivo Production of Transfusable Red Blood Cells at Large Scale from Cord Blood CD34+ Cells Using a Coculture System with Human Telomerase Catalytic Subunit (hTERT)-Transfected Human Stromal Cells at Early Phase and with Acitivated Macrophage from Cord Blood CD34+ Cells at Late Phase.

Blood ◽

10.1182/blood.v104.11.2701.2701 ◽

2004 ◽

Vol 104 (11) ◽

pp. 2701-2701

Author(s):

Akihito Fujimi ◽

Takuya Matsunaga ◽

Masayoshi Kobune ◽

Yutaka Kawano ◽

Ikuta Tanaka ◽

...

Keyword(s):

Blood Loss ◽

Cord Blood ◽

Stromal Cells ◽

Blood Cells ◽

Large Scale ◽

Acute Blood Loss ◽

Cd34 Cells ◽

Cord Blood Cd34 ◽

Human Stromal Cells

Abstract New sources of red blood cells (RBC) would improve the transfusion capacity of blood centers. Several investigators have previously reported that erythroblasts could be obtained from hematopoietic stem cells including those of cord blood (CB) by in vitro culture. However, transfusion of erythroblasts may not be suitable for supplementation of acute blood loss because it should need some time lag until hemoglobin (RBC) boost in circulation due to the fact that transfused erythroblasts once lodged at bone marrow where they undergo maturation into RBCs which are bound to be released into circulation. We have developed a culture system for producing large quantity of enucleated RBCs (e-RBCs) as well as erythroblasts from CB in vitro: one unit e-RBCs (2 x 1012 RBCs) was obtained from one standard CB unit (corresponding to 2 x 106 CD34+ cells) using a coculture system with hTERT-transfected human stromal cells at early phase followed by with activated macrophage in liquid culture (American Society of Hematology 45th Annual Meeting, SanDiego, 2003). In the present study, we first analyzed the function of those manufactured e-RBCs in comparison of that of adult peripheral blood RBCs (PB-RBCs) or that of eryhthroblasts. The hemoglobin (Hb) content of the e-RBCs quantified by photometric determination was almost equivalent to that of adult PBRBC. A Hb A/Hb F ratio of e-RBC analyzed by high-performance liquid chromatography (HbA: HbF = 35: 65) was between those of CB RBCs (10: 90) and adult PB-RBC (99: 1). Oxygen dissociation curves of e-RBCs measured by Hemox-Analyzer was comparable to that of fresh adult PB-RBCs. The erythroblasts showed adhesive property to stromal cells in vitro but e-RBC did not. When we injected e-RBCs into NOD/SCID mice, they were detectable in circulation while erythroblasts were not. In conclusion, the e-RBCs produced by large-scale culturing system from CB CD34+ cells may be useful for acute blood loss.

Download Full-text