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Critical Parameters for the Isolation of Mesenchymal Stem Cells from Umbilical Cord Blood
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     Institute of Transfusion Medicine and Immunology, German Red Cross Blood Service of Baden-Württemberg-Hessen, Germany; University of Heidelberg, Faculty of Clinical Medicine Mannheim, Germany

    Key Words. Mesenchymal stem cells ? Umbilical cord blood ? Isolation ? Multilineage differentiation

    Correspondence: Karen Bieback, Ph.D., Institute of Transfusion Medicine and Immunology, German Red Cross Blood Service of Baden-Württemberg-Hessen, Friedrich-Ebert-Strasse 107, D-68167 Mannheim, Germany. Telephone: 49-621-3706-8216; Fax: 49-621-3706-851; e-mail: k.bieback@blutspende.de

    ABSTRACT

    Mesenchymal stem cells (MSCs) comprise a rare population of multipotent progenitors capable of both supporting hematopoiesis and differentiating into at least the osteogenic, adipogenic, and chondrogenic lineages . These characteristics make MSCs very promising candidates to develop new cell-based therapeutic strategies, such as the treatment of mesenchymal tissue injuries or the supportive application in the context of hematopoietic stem cell (HSC) transplantation . Currently, bone marrow (BM) represents the main source of MSCs for both experimental and clinical studies . As the number of MSCs and their differentiation capacity decline with age , their therapeutic potential might be diminished as well. Therefore, it can be argued that cells with both extensive potency of proliferation and differentiation would represent an optimal tool for future cell-based therapeutic applications.

    Umbilical cord blood (UCB) has turned out to be an excellent alternative source of HSCs for clinical-scale allogeneic transplantation . Essential preclinical studies proved a higher percentage of CD34+CD38– cells in UCB compared with BM, suggesting that more primitive progenitors may be abundant in neonatal blood . The same might apply for the presence of MSCs or progenitor cells. However, previous attempts to isolate MSCs from UCB have either failed or have demonstrated a low frequency of mesenchymal progenitors in UCB of full-term deliveries . Therefore, the first goal of the present study was to verify whether cells with MSC traits can be isolated from full-term UCB units. Second, those cells were to be compared with BM-derived MSCs to assess potential similarities or differences. In addition, we intended to optimize MSC isolation protocols to increase the efficacy towards levels that are applicable for therapeutic approaches by modifying cell culture conditions as well as by defining quality criteria for UCB units.

    MATERIALS AND METHODS

    Generation of Primary Cultures from Adherent UCB Cells

    In total, 59 UCB units entered this study and were analyzed regarding their capacity to generate cultures of MSCs.

    After plating the MNCs, only a few cells attached to the plastic culture dishes and formed adherent cells within 3 weeks. Most of those cells were monocytes, which fused to form osteoclast-like cells . These appeared in 80%–90% of the wells uncoated with FCS and reached a subconfluent condition within 3 to 4 weeks, as shown in Figure 1A.

    Figure 1. Morphology of UCB-derived adherent cells displaying an osteoclast-like or fibroblastoid morphology. (A): Morphology of UCB-derived osteoclast-like cells in primary culture at day 16. UCB-derived fibroblastoid cells were identified as distinct colonies at days 14 through 16 after initial plating. They displayed either a consistent spindle-shaped (B, D) or a more spherical, less spindle-shaped morphology (C). The clone shown in (D) maintained a homogenous phenotype until passage 8 or 19.3 population doublings. (D): The colony at passage 0, day 16 before split. (F, G): The same clone at passage 3, day 37 (4 days after split, approximately eight population doublings) and passage 8, day 90 (4 days after split, 19.3 population doublings). BM-derived MSCs are shown as controls in (H) and (I), with (H) representing cells at passage 0, reaching a confluent stage at day 14. The MSCs depicted in (I) present an altered morphology and a stop in proliferation already at passage 4 (day 31, 7 days after split, 4.5 population doublings). These are representative examples of 29 clones of UCB-derived fibroblastoid cells and six processed BM aspirates, shown at x 100 magnification. (G): Mean values of the cumulative population doublings, determined at each subcultivation, with UCB-derived cells shown in black (n = 12) and BM-derived MSCs shown in gray (n = 5). Abbreviations: BM, bone marrow; MSC, mesenchymal stem cell; UCB, umbilical cord blood.

    Rare events displayed fibroblastoid cells, which expanded rapidly (MSC-like cells). In total, we obtained 29 colonies in 17 of the 59 processed UCB units. The onset of colony formation could be observed at first after 14 to 16 days. Cell confluence was reached after approximately 20 days of culture. In contrast, BM-derived MNCs formed clusters of spindle-shaped MSCs within 5 to 10 days and cell confluence after 2 to 3 weeks. But the UCB-derived MSC-like cells were of clonal origin, because at most one colony developed per well. Therefore, the mean (± SD) frequency of MSC-like cells was calculated from all tested UCB units to be 0.7 ± 0.2 clone-forming units per 1 x 108 MNCs of full-term UCB, ranging from 0 to 2.3 per 1x 108 MNCs. In units that gave rise to MSC-like cells, the frequency was variable and ranged from 0.2 to 2.3 clones per 1 x 108 MNCs (mean, 1.1 ± 0.2).

    Twenty-two UCB-derived clonal cell populations were spindle-shaped, but the other seven were more spherical (Figs. 1B–1D). However, they maintained this phenotype even in between subsequent passages (Figs. 1D–1F). Most clones could be cultured for at least eight passages and approximately 20 population doublings (Fig. 1G) (maximum up to now passage 12, with approximately 27 population doublings, n = 3), showing neither changed morphology nor reduced proliferation (n = 21). In contrast, all BM-derived MSCs displayed a change in morphology and markedly reduced proliferation already after approximately five population doublings (passages 3 through 4), as shown in Figures 1G–1I.

    Characterization of MSC-Like Cells from UCB

    Immune Phenotype ? The expression of cell-surface antigens by flow cytometry was evaluated on 12 clones of MSC-like cells and 4 BM-derived MSC preparations, each at passage 4. Neither the cells derived from UCB nor from BM expressed the hematopoietic markers CD14, CD34 and CD45, and CD133 (Fig. 2). Similar to MSCs from BM, UCB-derived clones were strongly positive for CD29, CD44, and CD73. In addition, cells from both sources stained positive for HLA-class I and negative for HLA-class II. However, only 53.6 ± 4.3% of the UCB-derived cells expressed CD105 (also known as SH2) compared with 84.4 ± 2.8% of BM MSC (p = .02). In contrast, 72.4 ± 3.2% of the UCB cells stained positive for CD106 compared with 42.4 ± 6.5% of BM MSC (p = .04). In addition, although most UCB-derived and BM-derived cells expressed CD90 (95.4% and 99.9%, respectively), there was a significant difference in the intensity of antigen expression. The MSC-like cells displayed a mean fluorescence intensity of 416.4 ± 186.5, whereas BM MSCs showed a significantly higher expression with 2193.9 ± 254.8 (p = .03).

    Figure 2. Immune phenotype of MSC-like cells from UCB and BM MSCs. Fibroblastoid cells from UCB and BM at passage 4 were trypsinized, labeled with antibodies against the indicated antigens, and analyzed by flow cytometry. The respective isotype control is shown as a dotted line. Arepresentative example of 12 UCB clones (—) and 4 BM harvests (—) is shown. Values represent the mean percentage of all assessed cells positively stained by the respective antibodies in the flow cytometry analyses. Abbreviations: BM, bone marrow; MSC, mesenchymal stem cell; UCB, umbilical cord blood.

    Differentiation Potential ? The differentiation potential of all 29 UCB-derived clones was compared with BM MSCs. Cells from passage 1 or 2 were cultured under conditions that are favorable for an osteogenic, adipogenic, or chondrogenic differentiation, respectively.

    First, osteogenic differentiation was induced in all BM MSC preparations and all MSC-like cell clones but not in fibroblasts (not shown), as shown by increased activity of alkaline phosphatase (Figs. 3A–3D) and enhanced mineralization defined by von Kossa staining (Figs. 3E–3H). This capacity was retained in all analyzed UCB clones (n = 8, passages 5 and 7).

    Figure 3. Differentiation capacity of MSC-like cells from UCB and MSCs from BM. Cultured cells from UCB-derived clones and BM harvests were exposed in vitro to differentiation medium to induce osteogenic, adipogenic, and chondrogenic differentiation, respectively. UCB-derived fibroblastoid cells were shown to differentiate appropriately to the osteogenic lineage by enhancement of alkaline phosphatase activity (B) and calcium mineralization detected by von Kossa stain (F) . Correspondingly, the BM MSCs used as a control are shown in (C), (D), (G), and (H) (magnification x 100). Adipocytes could solely be detected by Oil Red O stain in the adipogenic cultures of UCB MSC-like cells after intense induction for at least 5 weeks . In contrast, BM MSC-derived adipocytes could easily be induced by cyclic changes of adipogenic induction and maintenance medium for 3 weeks. [(K): not induced; (L): induced; magnification x 200). Chondrogenesis was shown by Safranin O staining in cryosections from both the UCB-derived clonal cells in (M) and the BM MSC in (N) (magnification x 100). A representative example of 29 UCB clones (five for adipogenic differentiation) and six BM harvests at passage 1 is shown. Abbreviations: BM, bone marrow; MSC, mesenchymal stem cell; UCB, umbilical cord blood.

    Second, the adipogenic induction was apparent in MSCs from BM by the cellular accumulation of lipid-rich vacuoles that stained with Oil Red O (Fig. 3L). However, the fibroblasts (not shown) and, most strikingly, all 29 MSC-like cell clones never generated adipocytes under standard differentiation conditions. Adipogenic differentiation could solely be induced in MSC-like cells cultured continuously in adi-pogenic induction medium for at least 5 weeks (Fig. 3J).

    Finally, in chondrogenic differentiation assays, MSC-like cells, BM MSC, and fibroblasts consolidated within 1 day, forming aggregates that dislodged to float freely in the suspension culture. Cryosections of the aggregates stained with Safranin O showed a condensed structure with chondrocyte-like lacunae (Figs. 3M and 3N, fibroblasts not shown). Also, UCB-derived MSC-like cells at later passages maintained the ability to form cartilage (n = 8).

    Optimization of Culture Conditions for the Isolation of MSC-Like Cells

    Initially, the frequency of MSC-like cells and the percentage of UCB units generating MSC-like cells was quite low, with one of eight processed UCB units (12.5%). Thus, we intended to improve the efficacy at first by modulating the culture conditions. Because no fibroblastoid cells could be visualized in wells showing osteoclast-like cells, our first approach was to prevent the adherence of monocytes as osteoclast precursors. To dispose the monocytes, we cultured the MNC in FCS-coated wells and removed the nonadherent cells after overnight adherence. Analyzing six UCB units in parallel by plating one half of the MNC fraction in FCS-precoated wells and the other half in uncoated wells, we observed a significantly higher percentage of monocytes harvested in the non-adherent fraction after seeding MNCs in FCS-coated wells (Table 1). This indicated that fewer monocytes adhered to the coated plates than to the uncoated plates. In addition, significantly fewer adherent cells and osteoclast-like cells were achieved in the wells precoated with FCS. In consequence, we received four colonies of MSC-like cells from three units plated in FCS-precoated wells compared with two colonies from two units seeded in uncoated wells (Table 1). Therefore, although the impact of FCS coating on displaying MSCs was not significant in those six units, we proceeded using pre-coated wells for the isolation and gained 12 units with 22 MSC-like cell clones out of 48 UCB units.

    Table 1. Effect of FCS coating on the percentage of monocytes in the nonadherent fraction after plating and on the amount of adherent osteoclast-like cells and mesenchymal stem cell (MSC)–like cells by plating one half of the mononuclear cells in coated and the other half in uncoated plates

    In a second approach, we tested different commercially available media known to promote the growth of MSCs from BM. Using MSCGM, based on DMEM, 29 colonies of MSC-like cells could be generated from 53 UCB units. Using MesenCult, based on McCoys, we succeeded in isolating MSCs from BM (n = 4) but not from UCB (n = 6; p = .04).

    Criteria for UCB Units for Efficient Isolation of MSC-Like Cells

    Even after adjusting the culture conditions to optimal levels, we were only able to isolate MSC-like cells from one third of the processed UCB units (Table 2).

    Table 2. Success rate in isolating mesenchymal stem cell (MSC)–like cells after recalculating umbilical cord blood (UCB) units fulfilling special criteria

    To establish criteria to predict an efficient isolation, we analyzed various quality aspects of the processed UCB units. Because it has been shown that cord blood from preterm deliveries can contain MSCs , we tried to correlate the presence of MSCs with either the gestational age or the mode of delivery. The gestational age ranged from 37 to 42 weeks, with a mean of 39.6 ± 1.5 weeks. Forty out of 59 processed units (67.8%) were derived from vaginal deliveries, and 19 were from cesarean sections. However, the presence of MSC-like cell clones correlated neither with the gestational age (p = .50) nor with the kind of delivery (p = .38).

    However, both the time from collection to isolation and the volume of the UCB units were crucial for MSC-like cell generation (Table 2). Units stored for more than 15 hours failed to establish any MSC-like cells (n = 15). The mean storage time of all processed UCB units was 10.2 ± 5.7 hours, ranging from 3 to 22 hours. Furthermore, a net volume of more than 33 ml placental blood and a MNC count greater than 1 x 108 were critical for isolation. Mean net volume was 42.8 ± 17.2 ml (range, 13 to 108 ml), and mean MNC count was 1.5 ± 0.9 x 108 (range, 0.3 to 4.5). Three of the processed UCB units showed signs of coagulation, and six units showed signs of hemolysis. These units failed to generate MSC-like cells. By taking into account only those units having optimal quality, we were able to enhance the success rate from 29% to 63% (Table 2).

    DISCUSSION

    We gratefully acknowledge the collaborators in the obstetric departments in Mannheim, especially Dudi Vuthaj, Dr. J. St?ve from the Orthopedic Department of the University Hospital Mannheim for supplying BM harvests, and Daniela Griffiths for editing the manuscript. This work was supported by Forschungsfonds der Fakult?t für Klinische Medizin Mannheim. Karen Bieback and Susanne Kern contributed equally to the results.

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