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Enrichment of Umbilical Cord Blood Mononuclears with Hemopoietic Precursors in Co-Culture with Mesenchymal Stromal Cells from Human Adipose Tissue E. V. Maslova*, E. R. Andreeva*, I. V. Andrianova*,**, P. I. Bobyleva*, Yu. A. Romanov**, N. V. Kabaeva**, E. E. Balashova**, S. S. Ryaskina***, T. N. Dugina***, and L. B. Buravkova*
Translated from Kletochnye Tekhnologii v Biologii i Meditsine, No. 4, pp. 238-243, October, 2013 Original article submitted April 10, 2012 We demonstrated the possibility of enrichment of umbilical cord blood mononuclear fraction with early non-differentiated precursors under conditions of co-culturing with mesenchymal stromal cells from the human adipose tissue. It was established that umbilical cord blood mononuclear cells adhered to mesenchymal stromal cell feeder and then proliferate and differentiate into hemopoietic cells. In comparison with the initial umbilical cord blood mononuclear fraction, the cell population obtained after 7-day expansion contained 2-fold more CFU and 33.4±9.5 and 24.2±11.2% CD34+ and CD133+ cells, respectively, which corresponds to enrichment of precursor cell population by 148±60. The proposed scheme of expansion of hemopoietic cells from umbilical cord blood is economically expedient and can widely used in biology and medicine. Key Words: umbilical blood; hemopoietic stem cell, multipotent mesenchymal stromal cells; cell culture Umbilical cord blood (UCB) is a promising source of hemopoietic stem cells (HSC) and can be used for transplantation as an alternative to donor bone marrow [16]. More than 25,000 transplantations of UCB cells to patients with various congenital and acquired pathologies have been performed worldwide [10]. UCB offers substantial advantages such as universal availability, easy isolation procedure, safety for the donor, and lower (in comparison with the bone marrow) incidence and severity of “graft-versus-host” disease [9]. One of the main limitations of UBC application is insufficient content of nucleated cells and/or HSC in the transplant (for effective engraftment is should *Institute of Biomedical Problems, Russian Academy of Sciences; **Russian Cardiology Research-and-Production Complex, Ministry of Health of the Russian Federation; ***CryoCenter, Moscow, Russia. Address for correspondence:
[email protected]. E. V. Maslova.
contain not less that 3×107 nuclears or 105 CD34+ cells per 1 kg recipient body weight). For solving this problem, various approaches were proposed, in particular, doubling of UBC dose, combined transplantation with peripheral blood CD34+ cells, and preliminary in vitro expansion of HSC. To this end, the following models were used: enrichment of the initial UCB mononuclear fraction with CD34+ cells or cell culturing in the presence of cytokine cocktails and/or on a stromal cell feeder. It was found that the fraction enriched with CD34+ cells provided better results in further in vitro expansion of hemopoietic cells [3,5]. However, preparative procedures including immunoselection are associated with the loss of far not abundant HSC [14]. It should be also noted that immunoseparation requires a large volume of the initial mononuclear population; besides, the procedure is rather costly. Nevertheless,
0007-4888/14/15640584 © 2014 Springer Science+Business Media New York
E. V. Maslova, E. R. Andreeva, et al.
this approach allows acceleration, though insignificant, of hemopoiesis recovery under clinical settings [12,15]. Our aim was to develop methodical approaches for enrichment of UCB mononuclear fraction with early undifferentiated hemopoietic precursors by using co-culturing with multipotent mesenchymal stromal cells (MSC) from human adipose tissue.
MATERIALS AND METHODS Isolation of UCB mononuclear cell fraction (UCBMNC). UCB was collected from healthy women after obtaining informed consent at V. I. Kulakov Research Center of Obstetrics, Gynecology, and Perinatology. The blood was collected into blood transfusion system bags with CPDA-1 anticoagulant and was processes within 24 h. Nucleated cell concentrate was obtained by double centrifugation according to the previously registered medical technology (FS No. 2009/387, November 23, 2009). After erythrocyte sedimentation and plasma excess removal, the fraction of nucleated cells was resuspended in autologous plasma with 10% dimethyl sulfoxide (Sigma) and 1% dextran-40, transferred to cryostat tubes, and automatically frozen to a final temperature of -90oC according to the standard protocol of Stem Cell Bank. During quarantine storage in liquid nitrogen vapor, the samples were tested for blood-transmitted infections (AIDS-1/2, hepatitis B and C, herpes simplex virus 1 and 2, HTLV-1/2, cytomegalovirus, syphilis). Seropositive samples and cells that did not pass the tests for sterility were disposed according to the prescribed procedure. Suitable samples were transferred to liquid nitrogen and stored until use. On the day of the experiment, the cells were defrosted in a water bath at 37oC and washed free from the cryoprotector in a great volume of culture medium. After evaluation of cell viability in the trypan blue exclusion test, the cell concentration was brought to 1.5-2.5×106 cells/ml and used within 30 min. Isolation and culturing of MSC. MSC were isolated from the stromal-vascular fraction of human adipose tissue. The cells were isolated and cultured according to the standard protocol [23] with our modifications [1] in α-MEM (Gibco) supplemented with 10% FBS (HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin (BioloT) and subcultured after attaining 70-80% confluence. Passage 3-4 cells were used in the study. Before the experiment, the cells were incubated for 18 h with mitomycin C (1.5 μg/ml; Sigma-Aldrich) to arrest cell division, and then the cells were seeded at a density yielding 70-80% monolayer. MSC and UCB-MNC co-culturing (Fig. 1). Suspension of UCB-MNC was added to MSC in a concentration of 1.5-2.5×106 cells/ml. After 72-h culturing
585 (37oC, 5% CO2, humid atmosphere), nonadherent cells were removed and the medium was replaced with a fresh portion. Some cultures were used for controlling the number and count of adherent cells using simultaneous staining with fluorescein diacetate (Sigma) and propidium iodide (Invitrogen) in final concentrations of 0.05 and 300 μg/ml, respectively. Cell viability was analyzed under a fluorescent microscope Nikon Eclipse TiU (Nikon): live and dead cells were distinguished by green (fluorescein diacetate; λex/em=470/515 nm) and red (propidium iodide; λex/em=535/590 nm) fluorescence. At least 5 randomly selected 0.6-mm2 fields of view were analyzed in each culture. The images were processed using NIS-elements software (Nikon). The rest cells were cultured for the next 96 h (total time of culturing was 7 days). The formed suspension of UCB-MNC was collected and used for phenotyping and evaluation of colony-forming capacity in a semisolid medium. Analysis of UCB-MNC composition. The cell composition of UBC-MNC before and after co-culturing with MSC was determined by flow cytometry on EPICS XL (Beckman Coulter) and FACSCalibur (Becton Dickinson) cytometers. FITC-, phycoerythrin-, and PerCP-conjugated antibodies to СD45, СD34, and СD133 (BD Pharmingen; Miltenyi Biotec) were used in concentrations recommended by the manufacturer. Detection of CFU. The presence of CFU was evaluated by cell capacity to form colonies in semisolid medium containing a cocktail of cytokines and growth factors (MethoCult H4034; STEMCELL Technologies). UCB-MNC (3-5×104 cells per 35-mm Petri dish) were added to the medium and cultured according to manufacturer’s instruction. Initial (defrosted) UCBMNC in the same concentration served as the reference sample. The number and composition of colonies were analyzed in 14 days. The data were processed by Mann–Whitney test using Microsoft Excel 2000 and Statistica 7.0 software. The differences were significant at p<0.05.
RESULTS Characteristics of the initial UCB-MNC population. The yield of nucleated cells and HSC after isolation was 91.3±3.4 and 93.0±19.1%; lymphocytes and monocytes constituted 27.6±5.2 and 9.1±0.5%, respectively (Table 1). Total viability of isolated cells was 99.2±0.6%. After defrosting and washing from the cryoprotector, UCB-MNC viability remained high: 98.2±1.3 and 98.5±1.2% for lymphocytes and monocytes, respectively (flow cytometry with 7-AAD vital dye staining). Viability of HSC (CD45+/CD34+ cells) also remained practically unchanged and was 97.1±1.8%.
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TABLE 1. Characteristics of UCB-MNC after Isolation and Cryogenic Storage Viability, % % of total population
Population
after isolation
after defrosting
Lymphocytes
27.6±5.2
99.8±0.4
98.2±1.3
Monocytes
9.1±0.5
99.8±0.1
98.5±1.2
44.2±5.3
98.9±1.1
80.1±8.5
0.48±0.21
99.8±0.2
97.1±1.8
80.7±4.5
–
–
Granulocytes +
+
HSC (CD45 /CD34 ), including (% of total HSC) +
+
–
late (CD45 /CD34 /CD133 ) +
+
+
medium (CD45 /CD34 /CD133 )
14.3±3.8
–
–
early (CD45+/CD34–/CD133+)
3.8±2.3
–
–
Effect of co-culturing on proliferation and cell composition of UCB-MNC. Over 24 h and subsequent 2 days after the start of co-culturing, some cells of UCB-MNC population adhered to the MSC monolayer. After removal of nonadherent cells and medium replacement, the number of adherent UCB-MNC in cultures from different donors varied insignificantly and was on average 363±77 cells/mm2. Typical photographs of the cultures are presented in Figure 2, a. Cell viability was high: 76±14% (Fig. 2, b). During co-culturing, a suspension fraction appeared above UCB cells adherent to the MSC feeder (Fig. 2, c). The number of floating nucleated cells increased and attained 144±47×103 cells/ml by day 7 of co-culturing. The cell composition of this population was assayed by flow cytometry. Leukocyte common antigen CD45, as well as CD34 and CD133 antigens, markers of HSC of different maturity [4], were used as the markers. In the newly formed suspension fraction, CD45 +/CD34 + and CD45 +/CD133 +cells constituted 33.4±9.5 and 24.2±11.2%, respectively (Fig. 3, a), which confirmed 148±60-fold enrichment of the cell suspension with undifferentiated precursors (Fig. 3, b). Effect of co-culturing on CFU content. The capacity to colony formation in semisolid medium in the presence of exogenous inductors was used for func-
Fig. 1. Experimental protocol of co-culturing of MSC and UCB-MNC.
tional characterization of HSC in the initial UCB-MNC samples and in the suspension fraction formed after 7-day co-culturing. The tests demonstrated a 2.3-fold increase in the total number of CFU in comparison with the initial population of UCB-MNC (Fig. 3, c). There are a number of approaches for in vitro culturing of hemopoietic cells, but the problem of optimization of culturing conditions for HSC expansion is not yet solved. The main attention is focused on the choice of components for the growth media and techniques of isolation of low-differentiated cells. Unfortunately, the majority of current models of HSC culturing underestimate the role of local microenvironment, in particular interactions of hemopoietic with stromal cells, extracellular matrix components, and paracrine factors. In vitro expansion of HSC depends on a number of factors. The first important moment is the choice between the use of non-fractionated UCB-MNC or their selection. It was found that enriched UCB-MNC fraction provided better in vitro expansion results [3,5]. However, this approach has certain drawbacks: it requires large amounts of initial cells and is accompanied by loss of hemopoietic cells during isolation [14]. Here we used initial (non-separated) population of UCB-MNC that was cultured for 72 h on the feeder
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Fig. 2. Cultured UCB-MNC. a, b) 72-h co-culturing: a) phase contrast; b) the same field of view, evaluation of cell viability: live cells (fluorescein diacetate) – green fluorescence; dead cells (propidium iodide) – red fluorescence; c) newly formed cell suspension, 7-day co-culturing.
of human adipose tissue MSC followed by removal of nonadherent cells. Over this short co-culturing period, MSC feeder promoted adhesion and expansion of low-differentiated hemopoietic cells. We conclude that skipping the preliminary immunoseparation procedure precludes potential cell damage caused by the great number of laboratory manipulations (centrifugation, resuspending, etc.). The second point is the choice of growth factor source for expansion of hemopoietic cells. Human and animal blood sera containing natural cocktail of growth factors, adhesion molecules, mineral compounds, lipids, and hormones are usual components for culturing of the majority of human cells, including HSC. There is no consensus about the possibility of their use for fabrication of cell products for clinical purposes. The main shortcomings are difficulties in standardization of serum composition and risk of viral contamination and immunization of the recipient with foreign proteins [19,20]. For this reason, some researches give preference to cytokine cocktails over sera [3,5]. There are a great number of soluble factors affecting HSC proliferation and differentiation.
Different combinations of these factors can determine the rate and degree of expansion of cultured cells for subsequent transplantation. However, we cannot exclude the presence of some minor components in the serum, whose action cannot be fully compensated by serum-free media. In the present study we performed successful expansion of UCB-MNC on the feeder of human adipose tissue MSC in a standard medium without additional transmitters. The use of stromal cells as the feeder for in vitro expansion of HSC is a common approach [2,6,7,11,13,17,18]. It was found that stromal cells produce a number of cytokines and chemokines involved in hemopoiesis regulation [7]. Moreover, they can directly interact with hemopoietic cells via cell– cell contacts. Thus, stromal cells can form microenvironment corresponding to that in the bone marrow. When modeling in vitro the conditions of bone marrow “niche”, researchers use MSC from different sources: bone marrow, UCB, adipose tissue [11,18,21]. It is known that MSC from the stromal-vascular fraction of the adipose tissue is a good alternative to bone marrow MSC and an easy available source for clinical
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Fig. 3. Identification of non-differentiated hemopoietic precursors. a) expression of surface markers; b) content of CD34+ cells; c) number of CFU per 10 cm2.
practice [8]. Like bone marrow MSC, MSC from human adipose tissue can maintain hemopoiesis in vitro [6]. Despite close relations, MSC from different tissues are not identical. For instance, MSC from the bone marrow can maintain primitive hemopoietic precursors less effectively than MC from other sources [21]. Similar results were obtained in other studies: the yield of CD34+ cells after co-culturing of HSC with adipose tissue MSC was lower than after co-culturing with bone marrow MSC [7,12]. On the other hand, adipose tissue MSC promoted HSC differentiation and more effectively supported differentiated hemopoietic precursors [7,17]. In vivo studies have demonstrated that human adipose tissue MSC transplanted with HSC considerably facilitated homing of hemopoietic cells and decreased mortality of lethally irradiated mice after transplantation [17]. In our study, short-term culturing of initial UCBMNC on the feeder of MSC from human adipose tissue promoted adhesion and further expansion of hemopoi-
etic precursors. The content of CFU in the cell population obtained after this expansion 2-fold surpassed that in the initial UCB-MNC suspension and the content of CD34+ was by more than two orders of magnitude higher than in the initial suspension. Similar results (more than 100-fold expansion) were demonstrated for CD45+/CD133+ precursor cells constituting, according to our data and published reports [22], vast majority of CD34+ HSC. These findings clearly demonstrate the possibility of considerably increasing the content of low-differentiated hemopoietic precursors in comparison with the initial population of UCB-MNC, which confirms appropriateness of this approach for UCBMNC enrichment. Thus, the proposed approach is justified from physiological viewpoint, because it allows us to skip the procedure of preliminary enrichment of initial UCBMNC with CD34+ cells and provides the possibility of using bioactive factors produced by cells equivalent to
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hemopoietic microenvironment. We believe that this scheme of expansion will be economic and applicable in experimental and clinical studies. Further studies in this field can be aimed at non-using the components of animal origin for cell culturing The study was performed within the framework of Agreement on Scientific Collaboration with CryoCenter Company and supported by the Russian Foundation for Basic Research (grant No. 13-04-00791).
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