The in vitro growth of a cord blood–derived cell population enriched for CD34+ cells is influenced by its cell cycle status and treatment with hydroxyurea

Abstract Transplanted human bone marrow (BM) CD34+ cells remain mitotically quiescent up to 72h following their homing to the BM of irradiated NOD/SCID mice (Blood2002;99(5):1585). To investigate whether umbilical cord blood (UCB)-derived CD34+ cells behave similarly and to assess the impact of the BM microenvironment on this observed temporary cell cycle arrest, we examined the cell cycle status of BM- and UCB-derived CD34+ cells recovered from the BM of irradiated (325 cGy) and non-irradiated NOD/SCID IL2Rγnull (NS2) mice 20h after transplantation (AT). To understand the molecular control of this sustained, but short-lived induced quiescence, expression of cell cycle-related proteins among BM-homed cells was examined by real-time quantitative PCR. Freshly isolated CD34+ cells were stained with CFSE-1 and transplanted into normal or conditioned NS2 mice or cultured in vitro with and without SCF, Flt3, GM-CSF, and IL-3. BM was harvested from NS2 recipients 20h AT and CFSE+ cells were recovered by cell sorting. Cell cycle status was assessed by PI staining in a fraction of recovered CFSE+ and cells cultured in vitro and mRNA was isolated from the remaining cells in both groups. The proportion of cells in G0/G1 phase of cell cycle among BM CD34+ cells was maintained in recipient BM 20h AT regardless of whether the microenvironment was irradiated or not (fresh: 83.7% ± 4.4%, n = 14; irradiated: 82.7% ± 5.3%, n = 6; non-irradiated: 84.4% ± 4.1%, n = 10), suggesting that exit of BM-homed cells from G0/G1 is either a strictly cell intrinsic property or is a phenomenon mediated by microenvironmental modulators present in both intact and injured BM. However, cultured BM CD34+ cells cycled efficiently such that after 20h, only 75.5% ± 5.7%, n = 12 remained in G0/G1. Surprisingly, BM-homed UCB CD34+ cells did not remain quiescent and at 20h AT, only 84.85% ± 13.1% (n = 5) were in G0/G1 compared to 97.0% ± 1.8% (n = 14) for freshly isolated cells, suggesting that different cell cycle regulatory mechanisms control BM versus UCB CD34+ cells. UCB CD34+ cells recovered from the BM of irradiated recipient mice 20h AT had increased levels of Bcl-2 and CDKN1B (p27) while those of CDKN1C (p57) and p53 were decreased relative to those isolated from non-irradiated recipients. These findings suggest that at least in the case of UCB CD34+ cells, different cell cycle regulatory networks may impact cell cycle progression of these cells in an irradiated versus intact BM microenvironment. Compared to freshly isolated BM CD34+ cells, those recovered from the marrow of non-irradiated recipients 20h AT had significantly elevated levels of mRNA for CDKN1a (p21), CDKN1B (p27), p53, and N-cadherin, while the mRNA levels for these molecules in comparable cells cultured in vitro for 20h was relatively unchanged. Taken together, these data illustrate that human CD34+ cells from different tissues behave differently during the first few hours following their homing to the BM and that this behavior may be partially regulated by the status of the microenvironment. Furthermore, data from BM-derived CD34+ cells suggest that following homing, active repression of cell cycle progression may be mediated by induced upregulation of p21 and p27. Mechanisms leading to the upregulation of these cell cycle regulatory molecules remain to be investigated.

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Isolation of small, primitive human hematopoietic stem cells: distribution of cell surface cytokine receptors and growth in SCID-Hu mice

Blood ◽

10.1182/blood.v86.2.512.bloodjournal862512 ◽

1995 ◽

Vol 86 (2) ◽

pp. 512-523 ◽

Cited By ~ 45

Author(s):

JE Wagner ◽

D Collins ◽

S Fuller ◽

LR Schain ◽

AE Berson ◽

...

Keyword(s):

Cell Cycle ◽

Cell Surface ◽

Cell Population ◽

Size Fraction ◽

Cell Surface Antigens ◽

Inhibitory Protein ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Cell Fractions

Human CD34+ cells were subfractionated into three size classes using counterflow centrifugal elutriation followed by immunoadsorption to polystyrene cell separation devices. The three CD34+ cell fractions (Fr), Fr 25/29, Fr 33/37, and Fr RO, had mean sizes of 8.5, 9.3 and 13.5 microns, respectively. The majority of cells in the large Fr RO CD34+ cell population expressed the committed stage antigens CD33, CD19, CD38, or HLA-DR and contained the majority of granulocyte- macrophage colony-forming units (CFU-GM), burst-forming units-erythroid (BFU-E), and CFU-mixed lineage (GEMM). In contrast, the small Fr 25/29 CD34+ cells were devoid of committed cell surface antigens and lacked colony-forming activity. When seeded to allogeneic stroma, Fr RO CD34+ cells produced few CFU-GM at week 5, whereas cells from the Fr 25/29 CD34+ cell population showed a 30- to 55-fold expansion of myeloid progenitors at this same time point. Furthermore, CD34+ cells from each size fraction supported ontogeny of T cells in human thymus/liver grafts in severe combined immunodeficient (SCID) mice. Upon cell cycle analyses, greater than 97% of the Fr 25/29 CD34+ cells were in G0/G1 phase, whereas greater proportions of the two larger CD34+ cell fractions were in active cell cycle. Binding of the cytokines interleukin (IL)-1 alpha, IL-3, IL-6, stem cell factor (SCF), macrophage inhibitory protein (MIP)-1 alpha, granulocyte colony- stimulating factor (G-CSF), and granulocyte-macrophage (GM)-CSF to these CD34+ cell populations was also analyzed by flow cytometry. As compared with the larger CD34+ cell fractions, cells in the small Fr 25/29 CD34+ cell population possessed the highest numbers of receptors for SCF, MIP1 alpha, and IL-1 alpha. Collectively, these results indicate that the Fr 25/29 CD34+ cell is a very primitive, quiescent progenitor cell population possessing a high number of receptors for SCF and MIP1 alpha and capable of yielding both myeloid and lymphoid lineages when placed in appropriate in vitro or in vivo culture conditions.

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Immune regulation of in vitro murine megakaryocyte development. Role of T lymphocytes and Ia antigen expression.

Journal of Experimental Medicine ◽

10.1084/jem.162.6.2053 ◽

1985 ◽

Vol 162 (6) ◽

pp. 2053-2067 ◽

Cited By ~ 12

Author(s):

M W Long ◽

D N Shapiro

Keyword(s):

Cell Cycle ◽

T Cell ◽

T Lymphocytes ◽

Progenitor Cells ◽

Immune Modulation ◽

Immune Recognition ◽

Activated T Cells ◽

Ia Antigen ◽

Cell Cycle Status

Mitogen-activated murine T lymphocytes or T cell hybridomas produce an activity (megakaryocyte [Mk] potentiator activity) that enhances the in vitro growth and development of Mk colonies. This activity was found in optimal concentrations (2.5%) in T cell hybridoma-conditioned medium, and was also produced by feeder layers of concanavalin A-activated T cells. A subpopulation of murine Mk progenitor cells (colony-forming units; CFU-Mk) bears the Ia antigen. Separate experiments indicated that T cell products stimulate CFU-Mk by increasing their basal levels of Ia expression as well as the frequency of cells actively synthesizing DNA. The hypothesis that the expression of this antigen was related to the cell cycle status of these progenitor cells was confirmed in studies that indicated that ablation of actively cycling cells in vivo abrogated the cytotoxic effects of anti-Ia monoclonal antibodies. The interdependence of T cell lymphokine regulation of both Ia expression and cell cycle status was also seen in in vitro experiments in which Ia+ progenitor cells were eliminated by complement-dependent cytotoxicity. The removal of Ia+ cells prevented 5-hydroxyurea-mediated inhibition of cells in S phase. We hypothesize that immune modulation of megakaryocytopoiesis occurs via soluble T cell products that augment Mk differentiation. Further, the mechanism of immune recognition/modulation may occur via Ia antigens present on the surface of these progenitor cells.

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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

PLoS ONE ◽

10.1371/journal.pone.0092493 ◽

2014 ◽

Vol 9 (3) ◽

pp. e92493

Author(s):

Keyword(s):

Cord Blood ◽

Scid Mouse ◽

Cd34 Cells ◽

Cord Blood Cd34

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In vitro evidence that LHRH stimulates the recruitment of prolactin mRNA-expressing cells during the postnatal period in the rat

Journal of Molecular Endocrinology ◽

10.1677/jme.0.0120107 ◽

1994 ◽

Vol 12 (1) ◽

pp. 107-118 ◽

Cited By ~ 21

Author(s):

A Van Bael ◽

R Huygen ◽

B Himpens ◽

C Denef

Keyword(s):

Cell Cycle ◽

Cell Population ◽

Blot Hybridization ◽

Postnatal Period ◽

S Phase ◽

Female Rats ◽

Mrna Levels ◽

Dot Blot ◽

Total Cell Population

ABSTRACT We have studied the effect of LHRH and neuropeptide Y (NPY) on prolactin (PRL) mRNA levels in pituitary reaggregate cell cultures from 14-day-old female rats, by means of in situ hybridization and Northern blot analysis. As estimated by computer-image analysis, addition of LHRH on day 5 in culture for 40 h resulted in a 37% increase in the total cytoplasmic areas of cells containing PRL mRNA, visualized using a digoxigenin-labelled PRL cRNA. The size of individual PRL-expressing cells was not influenced, nor was the content of PRL mRNA per cell. A similar effect of LHRH was found by dot blot hybridization of extracted RNA. PRL mRNA levels were not affected by NPY. LHRH induced a 29% increase in the number of PRL mRNA-expressing cells processing through the S phase of the cell cycle, visualized by the incorporation of [3H]thymidine ([3H]T) into DNA over 16 h. The fraction of [3H]T-labelled cells was 10–12% of the total cell population. NPY did not influence the number of [3H]T-positive cells expressing PRL mRNA, but completely blocked the effect of LHRH on the latter population. The present data suggest that LHRH, probably via a paracrine action of gonadotrophs, stimulates the recruitment of new lactotrophs, an action which is negatively modulated by NPY. Since the magnitude of this effect was the same in the total pituitary cell population as in cells processing through the S phase of the cell cycle and presumably mitosis, recruitment of lactotrophs seems to be based on differentiation of progenitor or immature cells into PRL-expressing cells, rather than on a mitogenic action on pre-existing lactotrophs alone.

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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

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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

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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.

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Effects of Cytokines on Platelet Production From Blood and Marrow CD34+ Cells

Blood ◽

10.1182/blood.v91.3.830 ◽

1998 ◽

Vol 91 (3) ◽

pp. 830-843 ◽

Cited By ~ 96

Author(s):

Françoise Norol ◽

Natacha Vitrat ◽

Elisabeth Cramer ◽

Josette Guichard ◽

Samuel A. Burstein ◽

...

Keyword(s):

Cell Population ◽

Culture System ◽

Simple Liquid ◽

Similar Response ◽

Platelet Production ◽

Liquid Culture System ◽

Cd34 Cells ◽

Proplatelet Formation ◽

And Function

Abstract The late stages of megakaryocytopoiesis, consisting of the terminal processes of cytoplasmic maturation and platelet shedding, remain poorly understood. A simple liquid culture system using CD34+ cells in serum-free medium has been developed to study the regulation of platelet production in vitro. Platelets produced in vitro were enumerated by flow cytometry. A truncated form of human Mpl-Ligand conjugated to polyethylene glycol (PEG-rHuMGDF) played a crucial role in both proplatelet formation and platelet production. A combination of stem cell factor (SCF), interleukin-3 (IL-3), and IL-6 was as potent as PEG-rHuMGDF for the growth of megakaryocytes (MKs). However, the number of proplatelet-displaying MKs and platelets was increased 10-fold when PEG-rHuMGDF was used. Peripheral blood mobilized CD34+ cells gave rise to a threefold augmentation of platelets compared with marrow CD34+ cells. This finding was related to the higher proliferative capacity of the former population because the proportion of proplatelet-displaying MKs was similar for both types of CD34+ cells. The production of platelets per MK from CD34+ cells was low, perhaps because of the low ploidy of the cultured MKs. This defect in polyploidization correlated with the degree of proliferation of MK progenitors induced by cytokines. In contrast, ploidy development closer to that observed in marrow MKs was observed in MKs derived from the low proliferative CD34+CD41+ progenitors and was associated with a twofold to threefold increment in platelet production per MK. As shown using this CD34+ CD41+ cell population, PEG-rHuMGDF was required throughout the culture period to potently promote platelet production, but was not involved directly in the process of platelet shedding. IL-3, SCF, and IL-6 alone had a very weak effect on proplatelet formation and platelet shedding. Surprisingly, when used in combination, these cytokines elicited a degree of platelet production which was decreased only 2.4-fold in comparison with PEG-rHuMGDF. This suggests that proplatelet formation may be inhibited by non-MK cells which contaminate the cultures when the entire CD34+ cell population is used. Cultured platelets derived from PEG-rHuMGDF– or cytokine combination-stimulated cultures had similar ultrastructural features and a nearly similar response to activation by thrombin. The data show that this culture system may be useful to study the effects of cytokines and the role of polyploidization on platelet production and function.

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