scholarly journals Functional properties of cells obtained from human cord blood CD34+ stem cells and mouse cardiac myocytes in coculture

2008 ◽  
Vol 294 (4) ◽  
pp. H1541-H1549 ◽  
Author(s):  
Alessia Orlandi ◽  
Francesca Pagani ◽  
Daniele Avitabile ◽  
Giuseppina Bonanno ◽  
Giovanni Scambia ◽  
...  
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Functional Properties ◽  
Fluorescent Protein ◽  
Cell Populations ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Rt Pcr ◽  
Hematopoietic Stem ◽  
Human Specific

Prior in vitro studies suggested that different types of hematopoietic stem cells may differentiate into cardiomyocytes. The present work examined whether human CD34+ cells from the human umbilical cord blood (hUCB), cocultured with neonatal mouse cardiomyocytes, acquire the functional properties of myocardial cells and express human cardiac genes. hUCB CD34+ cells were cocultured onto cardiomyocytes following an infection with a lentivirus-encoding enhanced green fluorescent protein (EGFP). After 7 days, mononucleated EGFP+ cells were tested for their electrophysiological features by patch clamp and for cytosolic [Ca2+] ([Ca2+]i) homeostasis by [Ca2+]i imaging of X-rhod1-loaded cells. Human Nkx2.5 and GATA-4 expression was examined in cocultured cell populations by real-time RT-PCR. EGFP+ cells were connected to surrounding cells by gap junctions, acquired electrophysiological properties similar to those of cardiomyocytes, and showed action potential-associated [Ca2+]i transients. These cells also exhibited spontaneous sarcoplasmic reticulum [Ca2+]i oscillations and the associated membrane potential depolarization. However, RT-PCR of both cell populations showed no upregulation of human-specific cardiac genes. In conclusion, under our experimental conditions, hUCB CD34+ cells cocultured with murine cardiomyocytes formed cells that exhibited excitation-contraction coupling features similar to those of cardiomyocytes. However, the expression of human-specific cardiac genes was undetectable by RT-PCR.

Hoechst dye efflux reveals a novel CD7+CD34− lymphoid progenitor in human umbilical cord blood

Blood ◽  
2000 ◽  
Vol 96 (6) ◽  
pp. 2125-2133 ◽  
Author(s):  
Robert W. Storms ◽  
Margaret A. Goodell ◽  
Alan Fisher ◽  
Richard C. Mulligan ◽  
Clay Smith
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Large Population ◽  
Lymphoid Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract A novel Hoechst 33342 dye efflux assay was recently developed that identifies a population of hematopoietic cells termed side population (SP) cells. In the bone marrow of multiple species, including mice and primates, the SP is composed primarily of CD34−cells, yet has many of the functional properties of hematopoietic stem cells (HSCs). This report characterizes SP cells from human umbilical cord blood (UCB). The SP in unfractionated UCB was enriched for CD34+ cells but also contained a large population of CD34− cells, many of which were mature lymphocytes. SP cells isolated from UCB that had been depleted of lineage-committed cells (Lin− UCB) contained CD34+ and CD34− cells in approximately equivalent proportions. Similar to previous descriptions of human HSCs, the CD34+Lin− SP cells were CD38dimHLA-DRdimThy-1dimCD45RA−CD71−and were enriched for myelo-erythroid precursors. In contrast, the CD34−Lin− SP cells were CD38−HLA-DR−Thy-1−CD71−and failed to generate myelo-erythroid progeny in vitro. The majority of these cells were CD7+CD11b+CD45RA+, as might be expected of early lymphoid cells, but did not express other lymphoid markers. The CD7+CD34−Lin− UCB SP cells did not proliferate in simple suspension cultures but did differentiate into natural killer cells when cultured on stroma with various cytokines. In conclusion, the human Lin− UCB SP contains both CD34+ multipotential stem cells and a novel CD7+CD34−Lin− lymphoid progenitor. This observation adds to the growing body of evidence that CD34− progenitors exist in humans.

Umbilical Cord Blood Stem Cell Transplantation

1993 ◽  
Vol 16 (5_suppl) ◽  
pp. 113-115 ◽  
Author(s):  
R. Miniero ◽  
U. Ramenghi ◽  
N. Crescenzio ◽  
L. Perugini ◽  
A. Busca ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Cord Blood Transplantation ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Alternative Source ◽  
Hematopoietic Stem

Human umbilical cord blood as an alternative source of hematopoietic stem cells for bone marrow reconstitution, has recently been demonstrated to yield successful HLA-matched placental blood grafts in children. It has been shown that cord blood contains sufficient progenitor cells to effect hematological reconstitution. Since then, more than 25 cord blood stem cells (CBSCs) transplants have been performed worldwide for the treatment of a variety of malignant and nonmalignant diseases. The majority of the grafts performed thus far have utilized CBSCs from HLA-identical siblings. However, much of the interest in this setting is devoted to the potential use of CBSCs for HLA-mismatched and unrelated transplants. Preliminary results suggest that allorecognition and graft-versus-host disease may be less intense in CBSCs transplants than in recipients of similarly compatible bone marrow. This review summarizes the results and potential future applications of cord blood transplantation.

Use of Chromatin Modifying Agents for Ex Vivo Expansion of Human Umbilical Cord Blood Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4208-4208
Author(s):  
Hiroto Araki ◽  
Nadim Mahmud ◽  
Mohammed Milhem ◽  
Mingjiang Xu ◽  
Ronald Hoffman
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Functional Properties ◽  
Ex Vivo ◽  
Fold Increase ◽  
Scid Mice ◽  
Human Umbilical Cord ◽  
Self Renewal ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Abstract The fixed number of hematopoietic stem cells (HSCs) within a single cord blood (CB) unit has limited the use of CB grafts for allogeneic transplantation in adults. Efforts to promote self-renewal and expansion of HSCs have been met with limited success. Using presently available ex-vivo culture techniques HSCs lose their functional properties in proportion to the number of cellular divisions they have undergone. We hypothesized that chromatin modifying agents, 5-aza-2′-deoxycytidine (5azaD) and histone deacetylase inhibitor, trichostatin A (TSA) could reactivate pivotal genes required for retaining the functional properties of dividing HSC. We have demonstrated previously that the fate of human bone marrow CD34+ cells could be altered by the addition of 5azaD/TSA (Milhem et al. Blood.2004;103:4102). In our current studies we hypothesized that in vitro exposure of CB CD34+ cells to chromatin modifying agents might lead to optimal HSC expansion to permit transplantation of adults. A 12.5-fold expansion was observed in the 5azaD/TSA treated CD34+CD90+ cell cultures containing SCF, thrombopoietin and FLT3 ligand (cytokines) in comparison to the input cell number. Despite 9 days of culture, 35.4% ± 5.8% (n = 10) of the total cells in the cultures exposed to chromatin modifying agents were CD34+CD90+ as compared to 1.40 % ± 0.32% in the culture containing cytokines alone. The 12.5-fold expansion of CD34+CD90+ cells was associated with a 9.8-fold increase in the numbers of CFU-mix and 11.5-fold expansion of cobblestone area-forming cells (CAFC). The frequency of SCID repopulating cells (SRC) was 1 in 26,537 in primary CB CD34+CD90+ cells but was increased to 1 in 2,745 CD34+CD90+ cells following 9 days of culture in the presence of 5azaD/TSA resulting in a 9.6-fold expansion of the absolute number of SRC. In contrast, the cultures lacking 5azaD/TSA had a net loss of both CFC/CAFC as well as SRC. The expansion of cells maintaining CD34+CD90+ phenotype was not due to the retention of a quiescent population of cells since all of the CD34+CD90+ cells in the culture had undergone cellular division as demonstrated by labeling with a cytoplasmic dye. CD34+CD90+ cells that had undergone 5–10 cellular divisions in the presence of 5azaD/TSA but not in the absence still retained the ability to repopulate NOD/SCID mice. 5azaD/TSA treated CD34+CD90+ cells, but not CD34+CD90- cells were responsible for in vivo hematopoietic repopulation of NOD/SCID assay, suggesting a strong association between CD34+CD90+ phenotype and their ability to repopulate NOD/SCID mice. We next assessed the effect of 5azaD/TSA treatment on the expression of HOXB4, a transcription factor which has been implicated in HSC self-renewal. A significantly higher level of HOXB4 protein was detected by western blot analysis after 9 days of culture in the cells treated with 5azaD/TSA as compared to cells exposed to cytokines alone. The almost 10-fold increase in SRC achieved using the chromatin modifying agents should be sufficient to increase the numbers of engraftable HSC within a single human CB unit so as to permit these expanded grafts to be routinely used for transplanting adult recipients.

Productive Infection of Human Cord Blood Stem Cells and Lineage Progenitors by Porcine Endogenous Retrovirus (PERV).

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4178-4178
Author(s):  
Doris A. Morgan ◽  
John Fitzsimmons ◽  
Takele Argaw ◽  
Carolyn Wilson
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Genetic Material ◽  
Endogenous Retroviruses ◽  
Productive Infection ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Hematopoietic Stem ◽  
Porcine Endogenous Retrovirus

Abstract Endogenous retroviruses were once considered “junk DNA” and merely residual evolutionary genetic material. In the human system, recent reports have suggested a link between activation of human endogenous retroviruses (HERV) and certain diseases. Activation may be restricted to an up-regulation of viral specific transcripts or may involve gene translation with the assembly of virus-like particles (Morgan, Exp.Hem., 2004). Against this background of a possible link between activated endogenous retroviruses and disease, the potential risks of porcine to human transmission in the context of xenotransplantation becomes more plausible. The principal focus of this report was to determine the susceptibility of human primary cells to porcine endogenous retroviruses (PERV). Expression of a receptor for PERV has been described to be widespread in human tissues (Ericsson,PNAS,2003) and was expressed in hematopoietic stem cells and progenitors of our study. We exposed human umbilical cord blood (UCB) stem cells and cytokine-induced erythroid, myeloid and megakaryocytic progenitors to retroviral pseudotypes composed of replication competent wildtype PERV-NIH virions that also carry an MLV-based retroviral vector genome encoding β-galactosidase (β-gal) (Wilson, J. Vir., 2000; Harrison, J. Vir., 2004). Three days after exposure to PERV, cells were washed in order to remove unbound virus and assayed for viral RNA and DNA using quantitative PCR and RT-PCR (Argaw, J. Gen. Vir. 2002). Cell pellets from stem cells as well as lineage-induced cells were consistently positive for viral DNA and RNA and persisted during a kinetic study 3,5,6,and 7 days post-exposure to PERV. Control cells that were not exposed to PERV were negative in all parameters. There were no detectable adverse effects of PERV infection on cell proliferation or on terminal maturation of the progenitors of all three lineages. PERV infection does not require lineage commitment and provides a mechanism by which self-renewing stem cells may serve as an in vivo reservoir of virus in human bone marrow. Of note, the natural course of disease (anemia) of a related retrovirus, feline leukemia virus, also relies upon infection of hematopoietic progenitors in the bone marrow.

scholarly journals Characterization of glycoprotein IIb/IIIa-positive cells in human umbilical cord blood: their potential usefulness as megakaryocyte progenitors

Blood ◽  
1992 ◽  
Vol 79 (2) ◽  
pp. 347-355
Author(s):  
D Zucker-Franklin ◽  
JS Yang ◽  
G Grusky
Keyword(s):  
Stem Cells ◽  
Cord Blood ◽  
Experimental Studies ◽  
Cell Types ◽  
Mononuclear Leukocytes ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord ◽  
Phenotypic Analysis ◽  
Human Cord Blood ◽  
Hematopoietic Stem

A need for hematopoietic stem cells, particularly cells destined to enter the megakaryocyte (MK) series, prompted phenotypic analysis of mononuclear leukocytes in human cord blood. To this end, immunohistochemical, flow cytometric, and ultrastructural techniques were used. The immunogold silver enhancement method (IGS) for the detection of the MK-specific glycoprotein (GP) IIb/IIIa epitopes combined with a monocyte-specific stain for alpha-naphthyl butyrate esterase proved to be superior to flow cytometry (FACS) for precise quantitation of cell types in each sample. Immunoelectron microscopy afforded a description of distinctions between precursors bearing GPIIb/IIIa epitopes and other stem cells of the myeloid series. The number of presumed MK progenitors was surprisingly high, averaging 1.8% +/- 1.3% (range, 0.2% to 4.6%) by IGS and 4.1% +/- 3.0% (range, 0.2% to 9.3%) by FACS analysis. The occurrence of GPIIb/IIIa-positive denuded MK nuclei in cord blood was of interest, but was too small to affect these data. These observations should advocate a greater use of cord blood for restitution of MK/platelet-lineage-depleted patients as well as for experimental studies concerned with MK differentiation.

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