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 Table of Contents  
Year : 2017  |  Volume : 42  |  Issue : 2  |  Page : 45-51

Development of ex-vivo expanded megakaryocyte progenitors for platelet recovery

1 Department of Clinical Pathology, Faculty of Medicine, Suez Canal University, Suez Canal, Egypt
2 Department of Physiology, Faculty of Medicine, Suez Canal University, Suez Canal, Egypt

Date of Submission30-Dec-2016
Date of Acceptance26-Feb-2017
Date of Web Publication6-Oct-2017

Correspondence Address:
Hassan M Amany
Department of Clinical Pathology, Faculty of Medicine, Suez Canal University, Suez Canal
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Source of Support: None, Conflict of Interest: None

DOI: 10.4103/ejh.ejh_42_16

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Context Megakaryocytes (MKs) can be produced using umbilical cord mononuclear cells and CD34 cells in the same cocktail media.
Aim The aim of this study was to produce human MKs and platelets through ex-vivo culturing of human cord blood.
Patients and methods Cord blood samples were collected under sterile conditions, separated using Ficoll–Hypaque technique for mononuclear separation, followed by magnetic separation for CD34+ cells; both types of cells were cultured in the same cocktail media and followed up for 14 days. This prospective ex-vivo study was conducted to determine the differentiation potential of haematopoietic stem cells isolated from cord blood towards megakeryopoiesis lineage. Cord blood was collected in the obstetric emergency room of Suez Canal University Hospital. Stem cell separation and induction of megakeryopoiesis were carried out in the Tissue Culture Unit, Physiology Department, Faculty of Medicine, Suez Canal University.
Results Both types of cells showed success towards megakaryopoiesis; however, mononuclear cells showed better results compared with CD34 cells.
Conclusion Umbilical cord stem cell culture could be a new easy method for ex-vivo MK progenitor production and final platelet recovery. This success would encourage further studies in ex-vivo megakeryopoiesis and in using it in different medical aspects.

Keywords: CD34 cells, magnetic separation, mononuclear cells, stem cell culture

How to cite this article:
Naglaa AF, Yasser EM, Amany HM, Omar DF, Hagar KA. Development of ex-vivo expanded megakaryocyte progenitors for platelet recovery. Egypt J Haematol 2017;42:45-51

How to cite this URL:
Naglaa AF, Yasser EM, Amany HM, Omar DF, Hagar KA. Development of ex-vivo expanded megakaryocyte progenitors for platelet recovery. Egypt J Haematol [serial online] 2017 [cited 2022 Aug 13];42:45-51. Available from: http://www.ehj.eg.net/text.asp?2017/42/2/45/216117

  Introduction Top

Because of the increasing demand for platelet transfusion and the associated risks besides the difficulties to find suitable donors, especially for immune-compromised patients or patients with platelet refractoriness, many ongoing trials are being held to obtain platelets from ex-vivo culture of stem cells to expand megakaryocyte (MK) progenitor for platelet recovery [1].

Hematopoietic stem cells (HSCs) and MKs are both extremely rare cell types, representing ∼0.05 and 0.4% of whole marrow, respectively. As a consequence of their low frequency, these cells are often studied after enrichment using various cell surface antigens. For instance, MKs are commonly enriched through the selection of CD41a+ (GPIIb) or CD61+ (GPIIIa) cells. Conversely, MKs at varying levels of purity can be generated in larger numbers using simple culture protocols. Culture systems are commonly used to study the development of human MKs, their maturation and platelet biogenesis. Most key events occurring during MK maturation appear to be well-replicated ex vivo, except for the regulatory activities normally provided by the micro-osteoblastic and microvascular niches on immature and mature MKs, respectively [2],[3].

MKs and platelets can now be produced ex vivo [4] from various sources, such as cell lines [5], embryonic stem cells [6],[7], and HSCs [8] maintained in suspension cultures [9] or with feeder layers [6],[8],[10] or in bioreactors [11].

  Patients and methods Top

Informed written consent was obtained from all pregnant women who participated in the study. They were informed about the details of the procedure.
  1. Agreements from the responsible authorities:

    This is the first most important consideration as the study topic, aims, methodology and measurements are politically accepted and also to facilitate any problem.
  2. Socially acceptable methodology and measurements.
  3. Confidentiality of the collected data:
    1. Only for research use.
  4. Good communication channel between researchers and target population by:
    1. Giving background about aim of study
    2. Stress on confidentiality of data.
  5. Acceptance of all the participants will be obtained by written informed consent

    Consent includes:
    1. Aims of study and methods to be used.
    2. The source of funding: .
    3. The institutional affiliation of the researcher: Faculty of medicine, Suez Canal University.
    4. The potential risks: no suspected risk.
    5. Finger print a will be used with illiterate participants.

Inclusion criteria

  1. Full-term neonates of normal pregnant women undergoing elective cesarean section with no medical history of chronic illness such as hypertension and diabetes, with normal viral profile and normal hematological parameters during labor.
  2. Samples obtained maximally 3–5 min after delivery.

Exclusion criteria

  1. Carrying any infectious diseases such as HIV, hepatitis B virus and hepatitis B virus.
  2. Known hematological disease.
  3. Placental birth.
  4. Multiple pregnancy.

Umbilical cord collection and handling

Cord blood samples were collected in sterile 50 ml graded plastic Falcon tubes (Greiner Bio-One, North America Insurance, Germany) containing 10 ml of citrate phosphate dextrose anticoagulant. Citrate phosphate dextrose was added under laminar flow to ensure sterility. The umbilical cord blood (UCB) was collected when the placenta was still in utero. The maternal end of the umbilical cord was sterilized with ethyl alcohol (70%), and the blood was aspirated using 50 ml sterile syringe to avoid formation of blood clots. The blood was freshly processed (maximum within 2 h), or stored at 4°C until processing. UCB samples were transported to the work tissue culture unit using a sterile transporter bag to ensure optimum sterility and temperature.

Laboratory preparation

Place preparation

Tissue culture laboratory components (laminar flow, centrifuge, CO2 incubator, vortex, pinches and laboratory floor) were subjected to cleaning by means of decontamination, followed by washing using detergent, and finally sterilization using chlorine (effervescent HAZ tablet: chlorine release tablet) and alcohol 70%. Pipettes and sample racks were swabbed with chlorine followed by alcohol 70%. The ultraviolet light of the laminar flow was switched on 12 h before sample processing, and all sterile materials such Pasteur pipettes, automated pipettes, tips, falcon tubes, magnetic columns, and gloves were placed inside to ensure sterility.

Chemical material preparation

Chemical materials such as PBS and RPMI were placed inside fridge at 4°C. Before sample processing, these materials were removed from the fridge and left 10 min at room temperature and followed by swabbing using cholorine and alcohol 70%, and finally the materials were placed inside laminar follow to ensure it is sterility. Culture media and fetal bovine serum (FBS) were first thawed and transferred into sterile falcon tubes and then frozen at −20°C. Before sample processing, these materials were placed at room temperature for about 20 min, followed by swabbing with cholorine and alcohol 70%, and finally placed inside laminar air flow.

Cord blood processing

Mononuclear cells separation

PBS, enriched placental media RPMI 1640 (Bio Whittaker), sterile filter, glutamine and 25 mmol/l HEPES (rpm), FBS (GIBCO 10270 FBS), and mononuclear cell separation medium Ficoll–Hypague (Histopaque-1077; Sigma-Adrich corporation, St Loius, Mo, USA) were used.

Each cord blood sample was diluted 1 : 1 with isolation buffer in a sterile container. A volume of 5.0 ml of Ficoll was placed in a 15 ml falcon tube, and 7 ml of diluted blood was carefully layered onto Ficoll and centrifuged at 400g (1500 rpm) for exactly 20 min at room temperature. After centrifugation, the upper layer was carefully aspirated with sterile Pasteur pipette to within 0.5 cm of the opaque interface containing mononuclear cells. The buffy coat was carefully transferred to a sterile falcon tube with a Pasteur pipette. Isolation buffer was added to this tube and then mixed by means of gentle aspiration in the ratio 2 : 1 between isolation buffer and mononuclear cells, and then centrifuged at 400g (1500 rpm) for 5 min. The supernatant was aspirated and discarded. The cell pellet was suspended with isolation buffer in the same ratio 2 : 1 and mixed by means of gentle aspiration with a Pasteur pipette, and then centrifuged at 400g (1500 rpm) for 5 min. The quantity of the isolated cells was assessed using an automated cell counter.

Selection of CD34+ progenitor cells (magnetic cell labeling) (Miltenyi Biotec protocol)

MiniMACS buffer, microbeads for CD34 selection, MiniMACS columns [sterile; Miltenyi Biotec, GmbH, Bergisch,,Gladbach, Germany] were used.

Cell suspensions were centrifuged at 1500 rpm for 5 min (to pellet cells without clump formation). The supernatants were removed, and the pellets were suspended in 300 µl of buffer ‘for each 108 cells’. (The tube tip was flicked to ensure dissolving of the cells and to avoid clumps.) According to the cell count, 100 µl of FcR blocking reagent and 100 µl of CD34 microbeads were added for each 108 cells. The samples were mixed well and kept in refrigerator (4–8°C) for 30 min; 2–5 ml of buffer was added to the cell microbead suspension, and then centrifuged at 1500 rpm for 10 min. The supernatants were removed and the pellets were resuspend in 500 µl of buffer. About 30 µm nylon mesh was soaked in 500 µl buffer. The 500 µl of cell suspension was passed through the wet nylon mesh. The MACS separation unit (magnet) was placed in the multistand of the MiniMACS. The MS column was also placed in the MACS separation unit, with its lips facing the outer surface. (The tip of the column should not be touched.) Tube rack was prepared with two tubes, one for unlabeled cells (marked as negative cells), and the other for labeled cells (marked as positive cells). The tip of the MS column was placed in the negative cells tube. MS column was soaked with 500 µl buffer and allowed to drip in the negative tube; 500 µl of cell suspension was added (one push with no bubbles), which allows drops to flow in negative tube. After ensuring that the MS column reservoir is empty, 500 µl of buffer (three times) was added, and allowed to drop in negative tube to ensure flow of all unlabeled cells. The MS column was removed from the magnet and the tip was placed in the positive tube; 1 ml of buffer was added to the column, followed immediately by placing the plunger in the MS column and pushing it forcefully and rapidly.

Cord blood mononuclear/CD34+ cells culture

For cord blood mononuclear/CD34+ cells culture the following were used: flask T-25 Greiner culture flask, tissur culture treated, sterile Pasteur pipettes, sterile falcon tube, culture media (selective media − stem MACS HSC×media×f HSC expansion media XF, that enables robust expansion of hematopoietic stem cells from cord blood, bone marrow, or peripheral blood. With stable glutamine; Biotec GMBH, USA) containing thrombopiotein (TPO), interleukin-3, interleukin-6, stem cell factor and FLT3, FBS, antibiotics cocktail (100× − penicillin 10 000 U/ml+streptomycin 10 000 mg/ml+amphotericin B25 mg/ml; BioWhittaker, Lnza, USA).

The cells were cultured in T-25 cm2 culture flasks at a density of 1×106 mononuclear cells/ml, for CD34+ 3×105 cell\ml. The cells were suspended in culture medium containing 1% antibiotic/antimycotic. Cultures were incubated at 37°C, humidified atmosphere containing 5% CO2. The culture medium was changed every 3 days and cellular growth was assessed under an inverted microscope. As metabolic waste can affect platelet integrity, we replaced the culture medium on day 10 or 11. The culture was taken and centrifuged at 1500 rpm for 20 min. Cell pellets were taken and assessed with light microscope and flow cytometer for glycoprotein marker expression − CD41 and CD61 expression on the platelets [12].

Culture cell analysis

Microscopic analysis was performed on day 3 to assess the appearance of the colony-forming cell, and on day 7 to assess micro-MKs, and finally on day 14 to assess the appearance of fully mature MK and platelets. Inverted microscope together with direct flask culture analysis was used to assess different cellular maturations. Light microscope was used to examine the stained form of different cellular stages. The samples were centrifuged for 10 min at 1500 rpm, and cell pellets were aspirated with a pipette, spread onto glass slide, dried on hot plate, and then stained with Leishman’s stain [13].

Flow cytometry analysis was carried out at the end of MKs culturing (day 14) using fluorochrome-conjugated antibodies against specific platelet surface glycoprotein markers, CD61 and CD41.

Analysis was performed in a dot-plot acquisition window on Accuri C6 from BD, equipped with two lasers (four colors). FCS file analysis was performed using FlowJo software (TreeStar, California, USA). The forward scatter (FSC) and side scatter (SSC) were plotted on the x-axis and the y-axis, respectively. We plotted CD61 (using an APC antibody, FL4 channel) and CD41 (using an FITC antibody, FL1 channel) on the y-axis and the x-axis, respectively, to analyze MKs and platelets obtained on day 14 of culture of mononuclear cord blood cells and CD34 cord blood cells.

Further analysis of platelets produced ex vivo was performed using fluorescent histograms and mean channel fluorescent intensity to compare the ex-vivo platelet production obtained from mononuclear cells and platelet production from CD34 cord blood cells [12].

Statistical analysis

Collected data were processed using SPSS version 15. Quantitative data were expressed as mean±SD, and qualitative data were expressed as numbers and percentages.

Student’s t-test was used to test significance of difference for quantitative variables, and the χ2-test was used for qualitative variables. A P-value of less than 0.05 was considered statistically significant.

  Results Top

The 18 UCB units were analyzed as regards their capacity to generate UCB-derived nonadherent MK progenitor cells in short-term culture. Totally, 15 of the 18 UCB samples generated nonadherent MK progenitor cells, giving a success rate of 81.8% (n=15/18) for positive UCB samples ‘successful MK progenitor-like cell isolation’ in relation to 18.1% (n=3/18) for negative UCB samples that failed to generate MKs because of unavoidable causes.

[Table 1] describes the criteria of MNC and CD34 as regards fetal sex: 40% were male and 60% were female for CD34 samples compared with 80% male and 20% female in MNC samples. In addition, [Table 1] shows that both maternal and gestational age was equally distributed in both MNC and CD34 samples. CD34 samples show that 40% of samples were cultured in less than 6 h and the rest (60%) were cultured in more than 6 h, compared with MNC samples in which 66.7% were cultured in less than 6 h and 33.3% were cultured in more than 6 h.
Table 1 Description of umbilical cord MNC and CD34 sampling criteria

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[Table 2] shows there was a statistically significant difference between MNC and CD34 and the culture yield throughout culture time [colony-forming unit, Megakaryocyte (MEG), and platelet glycoproteins], and Mononuclear cells (MNC) shows higher yield outcome compared with CD34.
Table 2 Relation between culture yield and umbilical cord MNC and CD34

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[Figure 1] shows ex-vivo formation of the colony-forming cells, characterized by small round cell with round eccentric nucleus and basophilic cytoplasm.
Figure 1 (a) Inverted microscope pictures of ex-vivo colony-forming unit by mononuclear cord blood cells on day 3. (b) Light microscope pictures of ex-vivo colony-forming unit by mononuclear cord blood cells on day 3. Magnification ×100

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[Figure 2] shows that some of the colony-forming cells start to produce immature MK (megakeryoblast), characterized by large cell with poorly differentiated nucleus and abundant basophilic cytoplasm.
Figure 2 (a) Inverted microscope picture of micromegakaryocytes produced ex vivo by mononuclear cord blood cells on day 7 (b) Light microscope picture of micromegakaryocytes produced ex vivo by mononuclear cord blood cells on day 7. Magnification ×100

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[Figure 3] shows fully mature MK characterized by fully mature lobulated nucleus, surrounded by scatter platelets; it also shows few platelet aggregations.
Figure 3 Mature megakaryocyte and platelets produced ex vivo by mononuclear cord blood cells on day 14. The picture was obtained using light microscope ×100

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Analysis of platelets produced ex vivo using flow cytometer

Phenotypic analysis by means of flow cytometry was carried out at the end of MKs culturing (day 14), using fluorochrome-conjugated antibodies against specific platelet surface glycoprotein markers, CD61 and CD41. Platelets were enumerated as PI-negative CD41 and CD61 events with scatter properties similar to those of control platelets prepared from peripheral blood. Platelet-gating strategy and cytometer settings were validated with normal platelet sample that could be easily isolated from a small blood sample. We used fresh PRP platelets to ensure proper adjustments of the various settings of the flow cytometer.

[Figure 4] shows that most of the platelet events have reduced FSC and SSC properties as shown in the figure. The platelet region is used as a gate to select platelet events and it was validated by using fresh platelets.
Figure 4 Unstained platelet distribution event with forward scatter and side scatter on the x-axis and y-axis, respectively

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[Figure 5] shows that dot-plot analysis of the platelets produced ex vivo by mononuclear cord blood cells depicted 55.6% of the total events with coexpression of CD61/CD41 platelet markers. In a small population, 12% of the events was positive for CD61, whereas 22.4% of the events demonstrate single CD41+.
Figure 5 Dot-plot analysis of platelets produced ex vivo by mononuclear cord blood cells using CD41 (FITC) on the forward scatter and CD61 (APC)

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[Figure 6] illustrates dot-plot analysis of the platelet produced ex vivo from umbilical cord CD34 cells. The CD41/CD61 coexpression was demonstrated on 19.3 of the total events. The majority of the platelets (55.1%) express single marker CD41 and 1.87% express single marker CD61.
Figure 6 Shows dot plot analysis of ex vivo produced platelets by CD34 cord blood using CD41(FITC) on the FSC and CD61(APC)

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  Discussion Top

UCB is a promising source of stem cells for research and clinical applications. It is ethically sound and its abundant supply, immunological immaturity, and high plasticity make it superior to other sources of stem cells. UCB has been utilized in many different clinical trials aiming to treat a wide range of diseases and disorders. Although still at early stages, preliminary results from these clinical trials demonstrated high potential and hope toward developing effective therapies for various diseases and disorders for which current mode of therapy is inadequate [14].

The precultural factors in our research consist of three main items, maternal factors (gestational and maternal age) and neonatal sex; these factors showed no statistical significance with culture yield examined using microscope and flow cytometry.

There is still debate among researchers as to which is better, HSC isolation from MNC or from CD34, as MNC source is considered a simple and easy method with low commercial price [15], compared with its isolation from CD34+ cells aiming to debulk the HUCB and obtain only colony genic cells [16].

Our results show that both types of umbilical cord stem cells (MNC and CD34) succeeded to proliferate into MKs progenitors cells and mature MKs with final platelet recovery, but umbilical cord mononuclear stem cells showed statically significantly better results (P>0.002) compared with umbilical cord CD34 cells in the proliferation of MK progenitors cells in the three cellular stages examined using inverted and light microscope. These cellular stages are as follows: colony-forming unit formation on day 3, micro-MKs on day 7, and final mature MKs with platelet recovery on day 14. Moreover, umbilical cord mononuclear stem cells showed more significant results using flow cytometer analysis of platelet glycoprotein expression (CD41 and CD61), compared with umbilical cord CD34.

Thus, we can explain that the better yield of MNC compared with CD34+ cell may be attributed to MNC proliferation to give both HMSC and CD34+ cells in which HMSC act as adherent stromal-like feeder layer for the proliferation of CD34+ cells and only provides nutrients that are essential for its proliferation and maturation.

Moreover, we may lose CD34+ cells during magnetic cells separation. Finally, this may be attributed to the small amount of blood compared with other studies, which used a large amount of UCB.

Our results show that there was successful production of MKs progenitors and final platelet recovery from both mononuclear and CD34 stem cells, but there was a higher ability of MNC compared with CD34 to produce ex-vivo MK progenitors cells and final platelets recovery assessed microscopically and by means of flow cytometery. Moreover, our results showed no effect of neonatal or maternal factors (maternal and gestational age) on the ability of both umbilical cord stem cells in ex-vivo MKs progenitors cell production and final platelet recovery.

Thus, we could conclude that umbilical cord stem cell culture could be a new easy method for ex-vivo MK progenitor’s production and final platelet recovery. This success would encourage further studies in ex-vivo megakaryopoiesis and using it in different medical aspects.

Financial support and sponsorship


Conflicts of interest

There are no conflicts of interest.

  References Top

Tse W, Laughlin MJ. Umbilical cord blood transplantation: a new alternative option. Hematology Am Soc Hematol Educ Program 2005; XX:377–383.  Back to cited text no. 1
Avecilla ST, Hattori K, Heissig B, Tejada R, Liao F, Shido K et al. Chemokine-mediated interaction of hematopoietic progenitors with the bone marrow vascular niche is required for thrombopoiesis. Nat Med 2004; 10:64–71.  Back to cited text no. 2
Dunois-Larde C, Capron C, Fichelson S, Bauer T, Cramer-Borde E, Baruch D et al. Exposure of human megakaryocytes to high shear rates accelerates platelet production. Blood 2009; 114:1875–1883.  Back to cited text no. 3
Reems JA, Pineault N, Sun S. In vitro megakaryocyte production and platelet biogenesis: state of the art. Transfus Med Rev 2010; 24:33–43.  Back to cited text no. 4
Gandhi MJ, Drachman JG, Reems JA, Thorning D, Lannutti BJ. A novel strategy for generating platelet-like fragments from megakaryocytic cell lines and human progenitor cells. Blood Cells Mol Dis 2005; 35:70–73.  Back to cited text no. 5
Fujimoto TT, Kohata S, Suzuki H, Miyazaki H, Fujimura K. Production of functional platelets by differentiated embryonic stem (ES) cells in vitro. Blood 2003; 102:4044–4051.  Back to cited text no. 6
Takayama N, Nishikii H, Usui J, Tsukui H, Sawaguchi A, Hiroyama T et al. Generation of functional platelets from human embryonic stem cells in vitro via ES-sacs, VEGF-promoted structures that concentrate hematopoietic progenitors. Blood 2008; 111:5298–5306.  Back to cited text no. 7
Matsunaga T, Tanaka I, Kobune M, Kawano Y, Tanaka M, Kuribayashi K et al. Ex-vivo large-scale generation of human platelets from cord blood CD34+ cells. Stem Cells 2006; 24:2877–2887.  Back to cited text no. 8
Ungerer M, Peluso M, Gillitzer A, Massberg S, Heinzmann U, Schulz C et al. Generation of functional culture-derived platelets from CD34+ progenitor cells to study transgenes in the platelet environment. Circ Res 2004; 95:e36–e44.  Back to cited text no. 9
Nishikii H, Eto K, Tamura N, Hattori K, Heissig B, Kanaji T et al. Metalloproteinase regulation improves in vitro generation of efficacious platelets from mouse embryonic stem cells. J Exp Med 2008; 205:1917–1927.  Back to cited text no. 10
Sullenbarger B, Bahng JH, Gruner R, Kotov N, Lasky LC. Prolonged continuous in vitro human platelet production using three-dimensional scaffolds. Exp Hematol 2009; 37:101–110.  Back to cited text no. 11
Robert A, Cortin V, Garnier A, Pineault N. Megakaryocyte and platelet production from human cord blood stem cells. Methods Mol Biol 2012; 788:219–247.  Back to cited text no. 12
Mattia G, Vulcano F, Milazzo L, Barca A, Macioce G, Giampaolo A, Hassan HJ. Different ploidy levels of megakaryocytes generated from peripheral or cord blood CD34+ cells are correlated with different levels of platelet release. Blood 2002; 99:888–897.  Back to cited text no. 13
Sangeetha V, Kale VP, Limaye LS. 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 2010; 5:e12221.  Back to cited text no. 14
Yao CL et al. A systematic strategy to optimize ex-vivo expansion medium for human hematopoietic stem cells derived from umbilical cord blood mononuclear cells. Exp Hematol 2004; 32:720–727.  Back to cited text no. 15
Köhler T, Plettig R, Wetzstein W, Schaffer B, Ordemann R, Nagels H-O et al. Defining optimum conditions for the ex-vivo expansion of human umbilical cord blood cells. Influences of progenitor enrichment, interference with feeder layers, early-acting cytokines and agitation of culture vessels. Stem Cells 1999; 17:19–24.  Back to cited text no. 16


  [Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]

  [Table 1], [Table 2]


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