Expansion of Human Hematopoietic Cells from Umbilical Cord Blood Using Roller Bottles in CO2and CO2-Free Atmosphere

2011 ◽  
Vol 20 (4) ◽  
pp. 593-598 ◽  
Author(s):  
Hera Andrade-Zaldívar ◽  
Marco A. Kalixto-Sánchez ◽  
Ana P. Barba de la Rosa ◽  
Antonio De León-Rodríguez
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Cells ◽  
Free Atmosphere ◽  
Roller Bottles

In vitrocell cycle dynamics of primitive hematopoietic cells from human umbilical cord blood

Hematology ◽  
2010 ◽  
Vol 15 (1) ◽  
pp. 11-20 ◽  
Author(s):  
Antonio Alvarado-Moreno ◽  
Antonieta Chávez-González ◽  
Arturo Cérbulo ◽  
Lourdes Arriaga ◽  
Hector Mayani
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord

Platelet-Derived Microparticels Stimulate Proliferation of Colony-Forming Unit Granulocyte-Macrophage of Umbilical Cord Blood Hematopoietic Stem Cells.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 4191-4191
Author(s):  
Bao-An Chen ◽  
Fei Fei ◽  
Cheng-Yin Huang ◽  
Cui-Ping Li ◽  
Xiao-Ping Pei ◽  
...  
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Cord Blood ◽  
Adhesion Molecules ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Cells ◽  
Control Group ◽  
Hematopoietic Stem ◽  
Hematopoietic Recovery

Abstract Umbilical Cord blood has become an important source of hematopoietic stem-progenitor cells for transplantation, however hematopoietic recovery after transplantation with umbilical cord blood is slower than with bone marrow or mobilized peripheral blood. Adhesion molecules on hematopoietic cells are involved in hematopoietic cells’ homing, which is considered the most important step of hematological recovery. Some articles indicated that expressions of adhesion molecules on CD34+ cells could predict the time to hematopoietic recovery after transplantation with bone marrow and peripheral blood of many adhesion molecules (such as CD62, CXCR4) are significantly lower on umbilical cord blood than on bone marrow. It is a possible reason for the difficulty in hematopoietic recovery after umbilical cord blood transplant. Platelet -derived microparticles (PMPs) are submicroscopic (<1 μm) membrane vesicles released from platelet if they are stimulated with agonists such as thrombin, collagen, or calcium ionophore A23187 or if exposed to high-stress shear forces. PMPs express several platelet-endothelium attachment receptors on their surface, for example, glycoprotein IIb/IIIa (CD41), Ib and IaIIa, and P-selectin (CD62P) and several other platelet relevant receptors such as CXCR4 and PAR-1. Some articles indicate that PMPs can affect the function of hematopoietic stem cells by increasing the adhesion of hematopoietic cells to fibrinogen, which suggests that PMP-transferred CD41 antigen plays an important role in this process. PMPs can also increase the survival of human hematopoietic cells including human CD34+ clonogenic progenitors. In our research, we observe the function of PMP to affect the cloning efficiency of colony-forming unit granulocyte-macrophage (CFU-GM). We adopt different concentrations of Thrombin (2U/ml, 1.5U/ml, 1.0U/ml and 0.5U/ml) to activate the platelet and acquire PMPs. Then PMPs were evaluated by using flow cytometry. Based on the result that stimulation of platelets by Thrombin (1U/ml) can acquire the best efficiency of PMPs, we used this concentration in all subsequent experiments. Umbilical cord mononuclear cells (MNCs) were obtained from healthy donors and isolate the MNCs by Ficoll-Hypaque density gradient centrifugation. Briefly, MNCs incubated with or without PMPs cultured in 2.7% methylcellulose. CFU-GM growth was stimulated with 30% umbilical cord serum, rhIL-3 and rh GM-CSF. Cultures were incubated at 37°C in a fully humidified atmosphere supplemented with 5% CO2. Colonies were counted under an inverted microscope after 7 or 10 days. The research was divided into four groups: 1. control group; 2. PMPs(10μg/ml); 3. PMPs(50μg/ml); 4. PMPs(100μg/ml). The colony formation was enhanced with PMPs and is dependently stimulated with PMPs. The number of colonies in the group of PMPs(100μg/ml) is more than that of other groups. The number of colonies in control group, PMPs(10μg/ml), PMPs(50μg/ml) and PMPs(100μg/ml) are 57.4±3.2, 65.6±5.6, 77.1±1.7 and 87.8±5.0 per 1×105 respectively. These increases in different groups were statistically significant when compared with control group(p<0.05). To sum up, PMPs can affect the cloning efficiency of CFU-GM of umbilical cord hematopoietic stem cells and the efficiencies are depended on the concentration of PMPs.

In Vitro Large-Scale Expansion of Unfractionated Mononucleated Cells of Human Umbilical Cord Blood: Investigation for Clinical-Grade Generation of Cord Blood Hematopoietic Cells.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 3222-3222
Author(s):  
Ping Mao ◽  
Huaxin Duan ◽  
Caixia Wang ◽  
Tingfen Deng ◽  
Changru Luo ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Cord Blood ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Culture System ◽  
Large Scale ◽  
Hematopoietic Cells ◽  
Cell Number ◽  
Human Specific

Abstract Abstract 3222 Poster Board III-159 Previous studies of expansion for hematopoietic stem/progenitor cells showed progenitor cells could reach a great expansion but were mainly performed at small culture volume. The poor cell number of expanded cells even great folds of expansion in a no more than 10 ml culture system limited the application for clinic transplantation. Thereof, we expanded umbilical cord blood (UCB) hematopoietic cells at a large scale in 250 ml bioreactor culture system. The mononuclear cell isolated form fresh UCB samples were ex vivo expanded in 250 ml bioreactor with serum-free medium supplied with SCF, FL3 and TPO at concentration 20 ng/ml. Cell number, surface markers and colony forming potential after 7 days of expansion have been evaluated. The sub-lethally irradiated animal models were used to analyze engraftment capability of HSC by transplant of expanded cells in severe combined immunodeficient and nonobese diabetic (NOD/SCID) mice. The detection of human hematopoietic cells in the bone marrow of the mice was performed at 6 wk after the transplant. We have successfully expanded hematopoietic progenitor cells 5 times using the modified bioreactor. The cell viability showed no obvious variations during culture. Total cells increased from 1.76±0.50 ×108 (range 1.2-2.5) to 4.14±0.83×108 (range 3.7-5.6) after expansion, CD34+ cells from 1.25±0.31 ×106 (range 0.96-1.75) to 3.96±0.78×106 (range 2.88-5.04) and CD133+ cells from 1.55±0.69×106 (range 0.96-2.75) to 4.77±0.88×106 (range 4.00-5.04). The colonies per 105 cells of CFU-E /BFU-E, CFU-GM and CFU-Mix increased from 369.6±71.5 to 1648.5±504.3, 42.8±81.4 to 146.4±54.5, 39.1±10.3 to 144.7±38.8, respectively. The positive expression of CD34+ was 0.7%±0.28% at day 0 and 0.9%±0.34% after culture in bioreactor at day 7 and CD133+ cells was 0.8%±0.24% and 1.1%±0.35%. There was no significant survive rate difference between expanded and nonexpanded cells transplantation group. The analysis of multilineage hematopoiesis showed that transplanted human hematopoietic cells is represented in murine bone marrow cells by detection the percentage of human cells identified with human specific anti CD3/19/33/45/61/71 MoAb by FACS and the human specific gene Alu-1 and Cat-1 by PCR. Disclosures No relevant conflicts of interest to declare.

scholarly journals Optimisation of transfection conditions of CD34+ hematopoietic cells derived from human umbilical cord blood.

2002 ◽  
Vol 49 (3) ◽  
pp. 625-632 ◽  
Author(s):  
Tomasz Ołdak ◽  
Marcin Kruszewski ◽  
Eugeniusz Krzysztof Machaj ◽  
Agnieszka Gajkowska ◽  
Zygmunt Pojda
Keyword(s):  
Cell Viability ◽  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Mononuclear Cells ◽  
Transfection Efficiency ◽  
Hematopoietic Cells ◽  
Human Umbilical Cord Blood ◽  
Target Cells ◽  
Human Umbilical Cord

Human umbilical cord blood is frequently used as a source of transplantable hematopoietic cells and more recently as a target of gene therapy - a new approach for treatment of various disorders. The aim of our study was optimisation of the transfection conditions of cord blood-derived CD34(+) hematopoietic cells. Mononuclear cells fraction was isolated from cord blood samples by density gradient centrifugation. Subsequently, CD34(+) hematopoietic cells were separated on immunomagnetic MiniMACS columns. Pure population of CD34(+) cells was incubated in a serum free medium supplemented with thrombopoietin, stem cell factor and Flt-3 ligand for 48 h and then transfected with plasmid DNA carrying the enhanced version of green fluorescent protein (EGFP) as a reporter gene. We studied the influence of various pulse settings and DNA concentrations on the transfection efficiency, measured by flow cytometry as the fluorescence of target cells due to the expression of EGFP. The optimal settings were as follows: 4 mm cuvette, 1600 microF, 550 V/cm, and 10 microg of DNA per 500 microl. With these settings we obtained a high transfection frequency (41.2%) without a marked decrease of cell viability. An increase of the pulse capacitance and/or of DNA concentration resulted in a greater electroporation efficiency, but also in a decrease of cell viability. In conclusion, the results described here allow one to recommend electroporation as an efficient method of gene delivery into CD34(+) hematopoietic cells derived from human umbilical cord blood.

scholarly journals The scientific and practical use of umbilical cord blood hematopoietic cells

1995 ◽  
Vol 11 (2) ◽  
pp. 5-14 ◽  
Author(s):  
A. P. Kukcharenko ◽  
G. I. Kogut ◽  
A. D. Shved
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Cells

scholarly journals High efficiency retroviral mediated gene transduction into single isolated immature and replatable CD34(3+) hematopoietic stem/progenitor cells from human umbilical cord blood.

1993 ◽  
Vol 178 (6) ◽  
pp. 2089-2096 ◽  
Author(s):  
L Lu ◽  
M Xiao ◽  
D W Clapp ◽  
Z H Li ◽  
H E Broxmeyer
Keyword(s):  
Gene Therapy ◽  
Cord Blood ◽  
Single Cell ◽  
Progenitor Cells ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
High Efficiency ◽  
Hematopoietic Cells ◽  
Hematopoietic Stem ◽  
Gm Csf

Umbilical cord blood is rich in hematopoietic stem and progenitor cells and has recently been used successfully in the clinic as an alternative source of engrafting and marrow repopulating cells. With the likelihood that cord blood stem/progenitor cells will be used for gene therapy to correct genetic disorders, we evaluated if a TK-neo gene could be directly transduced in a stable manner into single isolated subsets of purified immature hematopoietic cells that demonstrate self-renewed ability as estimated by colony replating capacity. Sorted CD34(3+) cells from cord blood were prestimulated with erythropoietin (Epo), steel factor (SLF), interleukin (IL)-3, and granulocyte-macrophage colony stimulating factor (GM-CSF) and transduced with the gene in two ways. CD34(3+) cells were incubated with retroviral-containing supernatant from TK-neo vector-producing cells, washed, and plated directly or resorted as CD34(3+) cells into single wells containing a single cell or 10 cells. Alternatively, CD34(3+) cells were sorted as a single cell/well and then incubated with viral supernatant. These cells were cultured with Epo, SLF, IL-3, and GM-CSF +/- G418. The TK-neo gene was introduced at very high efficiency into low numbers of or isolated single purified CD34(3+) immature hematopoietic cells without stromal cells as a source of virus or accessory cells. Proviral integration was detected in primary G418-resistant(R) colonies derived from single immature hematopoietic cells, and in cells from replated colonies derived from G418R-colony forming unit-granulocyte erythroid macrophage megakaryocyte (CFU-GEMM) and -high proliferative potential colony forming cells (HPP-CFC). This demonstrates stable expression of the transduced gene into single purified stem/progenitor cells with replating capacity, results that should be applicable for future clinical studies that may utilize selected subsets of stem/progenitor cells for gene therapy.

scholarly journals Human Umbilical Cord Blood Progenitors: The Potential of These Hematopoietic Cells to Become Neural

Stem Cells ◽  
2005 ◽  
Vol 23 (10) ◽  
pp. 1560-1570 ◽  
Author(s):  
Ning Chen ◽  
Jennifer E. Hudson ◽  
Piotr Walczak ◽  
Iwona Misiuta ◽  
Svitlana Garbuzova-Davis ◽  
...  
Keyword(s):  
Cord Blood ◽  
Umbilical Cord ◽  
Umbilical Cord Blood ◽  
Hematopoietic Cells ◽  
Human Umbilical Cord Blood ◽  
Human Umbilical Cord
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