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Ann Thorac Surg 2008;85:1361-1366. doi:10.1016/j.athoracsur.2007.12.006
© 2008 The Society of Thoracic Surgeons
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Original Articles: Adult Cardiac

Characteristics and Function of Cryopreserved Bone Marrow–Derived Endothelial Progenitor Cells

Shigetoshi Mieno, MD, Richard T. Clements, PhD, Munir Boodhwani, MD, Neel R. Sodha, MD, Basel Ramlawi, MD, Cesario Bianchi, MD, PhD, Frank W. Sellke, MD*

Division of Cardiothoracic Surgery, Beth Israel Deaconess Medical Center, Boston, Massachusetts

Accepted for publication December 3, 2007.

* Address correspondence to Dr Sellke, Division of Cardiothoracic Surgery, Harvard Medical School, Beth Israel Deaconess Medical Center, 110 Francis St, Suite 2A, Boston, MA 02215 (Email: fsellke{at}caregroup.harvard.edu).


   Abstract
Top
 Abstract
Introduction
 Material and Methods
 Results
 Comment
 References
 
Background: This study examined ex vivo expansion of bone marrow–derived endothelial progenitor cell (EPC) from cryopreserved bone marrow–derived mononuclear cells, and evaluated proliferation and migration function of the cryopreserved EPC (Cryo-EPC).

Methods: Bone marrow samples were taken from swine iliac bone (n = 6). Isolated bone marrow–derived mononuclear cells were cultured or cryopreserved at –80°C for 2 to 3 months. After cell culture for 4 days, attached cells, EPCs with or without cryopreservation, were collected. Direct fluorescent staining by acetylated low-density lipoprotein, isolectin B4, and 4',6-diamidino-2-phenylindole were performed to confirm the attached cells as EPC. Endothelial progenitor cell proliferation by vascular endothelial growth factor was evaluated by the tetrazolium method. Endothelial progenitor cell migration in response to stromal-derived factor-1{alpha} was also evaluated by using a Boyden chamber assay.

Results: The percentage of cells positively stained by direct fluorescent staining by acetylated low-density lipoprotein and isolectin B4 was similar between fresh and Cryo-EPC (EPC = 96.0 ± 0.42 versus Cryo-EPC = 95.2 ± 1.2; p = 0.21). Vascular endothelial growth factor increased proliferation activity in fresh and Cryo-EPC (p < 0.01). Stromal-derived factor-1{alpha} increased migration activity in fresh and Cryo-EPC (p < 0.01). There was no difference in proliferation and migration activity between fresh and Cryo-EPC.

Conclusions: Ex vivo expansion by cell culture was a useful method for collection of bone marrow–derived EPC from cryopreserved mononuclear cells. Proliferation and migration function of EPC is preserved after cryopreservation.


   Introduction
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 Abstract
 Introduction
Material and Methods
 Results
 Comment
 References
 
Transplantation of endothelial progenitor cells (EPC) enhances myocardial angiogenesis, leading to functional improvement in ischemic territories. The beneficial effect of EPC transplantation is demonstrated in patients with acute myocardial infarction as well as in acute ischemic animal models [1, 2]. However, the therapeutic effects of EPC transplantation in clinical trials are limited as compared with those in animal experimental studies. Recently, it has been reported that diabetes mellitus, hypercholesterolemia, smoking, and advanced age could be risk factors for reduction in the circulating number and function of EPC [3–5]. In addition, the reduced functional capacity of EPC is associated with impaired recovery and higher mortality in patients with chronic post-infarction heart failure [6]. The growing evidence suggests that reduction in function as well as the number of EPC contribute to the limited effects of angiogenesis in patients as compared with animal experimental models.

Ex vivo expansion after cryopreservation of EPC could be a promising strategy to overcome the clinical problem of limited cell number. Multiple collections may allow sufficient numbers of EPC to be obtained. Ex vivo expansion may be able to enhance or restore impaired EPC function through in vitro manipulation including pharmacologic treatments, gene therapy, or small interfering RNA (siRNA) techniques. Although human bone marrow (BM)–derived mesenchymal stem cells have pluripotent capabilities and maintain viability and osteogenic potential even after ex vivo expansion and cryopreservation [7], the freezing and thawing process induces cell and membrane damage. However, little is known concerning the direct effects of cryopreservation on EPC viability and function. Furthermore, methods to isolate and collect EPC from cryopreserved mononuclear cells (MNCs) have not been established. In this study, we isolated BM-derived EPC from cryopreserved MNCs and evaluated the efficacy of this method. Furthermore, we evaluated proliferation and migration function of the cryopreserved EPC.


   Material and Methods
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 Abstract
 Introduction
 Material and Methods
Results
 Comment
 References
 
Cell Culture Enrichment of Endothelial Progenitor Cells
Yorkshire miniswine (approximately 20 kg) were anesthetized with ketamine (10 mg/kg, intramuscular injection) and thiopental (5 to 10 mg/kg intravenous injection) and maintained with a gas mixture of oxygen at 1.5 to 2.0 L/min and isoflurane at 0.75% to 3.0%. The animals were intubated and mechanically ventilated at 12 to 20 breathes/min. Bone marrow cells were aspirated from the iliac bone. All animals were euthanized by removal of the heart, which was used for a separate experiment. Bone marrow MNCs were isolated from fresh BM samples by Histopaque 1077 (Sigma-Aldrich, St. Louis, MO) density-gradient centrifugation of buffy coats. After isolation of MNCs, we counted the number of MNCs by 0.2% trypan blue staining. Then, 1.3 x 105/cm2 of MNCs were plated on fibronectin-coated culture dishes and maintained in endothelial cell basal medium-2 (EBM-2; Clonetics, San Diego, CA) supplemented with EGM-2 (Clonetics, San Diego, CA) microvascular single aliquots and 5% fetal bovine serum (FBS). After 4 days, we counted cells by 0.2% trypan blue staining and calculated the concentration of MNCs in the total BM samples. MNCs were also preserved at –80°C at a concentration of 1.0 x 107/mL by use of Bambanker (Wako, Richmond, VA) according to the manufacturer’s instructions. Two to three months later, cryopreserved MNCs were thawed and mixed into EBM cell culture media. Mononuclear cells were collected by centrifugation, and the media changed to EPC cell culture media (EBM + EGM + 5% of FBS) and were cultured on fibronectin-coated tissue plates to isolate EPC.

Fluorescent Staining of Endothelial Progenitor Cells
To confirm isolated cells were EPC, direct fluorescent staining was performed using fluorescein Griffonia simplicifolia lectin I, isolectin B4 (Vector Laboratories, Burlingame, CA), acylated low-density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3'-3'-tetramethylindo-carbocyanine perchlorate (DiI-acLDL; Biomedical Technologies, Stoughton, MA), and 4',6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma Chemical Co, St. Louis, MO) with a fluorescent microscope, and adherent cells that stained positive by isolectin B4, DiI-acLDL, and DAPI were considered EPCs. The percentage of EPC was calculated by isolectin B4-, DiI-acLDL-, and DAPI-positive cells divided by DAPI-positive cells.

Proliferation Assay
The proliferative activity of EPC with or without cryopreservation was examined by the use of Cell Titer 96 nonradioactive cell proliferation assay (Promega, Madison, WI) according to the manufacturer’s instructions. Briefly, 10,000 cells per well were reseeded onto 96-well flat-bottomed plates with 100 µL of control medium, cell culture medium (EBM-2 + EGM-2 + 5% FBS), and 100 ng/mL of human recombinant vascular endothelial growth factor (VEGF; R&D Systems, Minneapolis, MN), respectively. A final concentration 0.2% FBS in EBM-2 was used as a control media. Cell culture medium and recombinant VEGF were added to the control media. The cells were then incubated for 72 hours at 37°C. After mixture of the kit reagent, Cell Titer 96 Aqueous one solution (Promega, Madison, WI), the absorbance at 490 nm wavelength was recorded with the use of a 96-well enzyme-linked immunosorbent assay plate reader (Bionetics Laboratory, Kensington, MD).

Migration Assay
Endothelial progenitor cell migratory function was evaluated using a modified Boyden chamber (Neuro Probe, Inc, Gaithersburg, MD). A polycarbonate filter with 8-µm pore size (Neuro Probe, Inc) was placed between the upper and lower chambers. Cell suspensions (5 x 104 cells per well) were placed in the upper chamber, and the lower chamber was filled with medium containing 50 ng/mL of human recombinant stromal-derived factor-1{alpha} (SDF-1{alpha}; R&D Systems). The chamber was incubated for 6 hours at 37°C and 5% CO2. The migrated cells on the lower side of the filter were fixed and stained with Diff-Quick (Dade Behring, Liderbach, Germany). Migration activity was evaluated by the number of cells in 100x power fields.

Statistical Analysis
Results are reported as means ± standard error of the mean. Comparisons of positive cells after fluorescent staining between two groups were analyzed by Student’s t test. Differences between two groups and response to VEGF and SDF-1{alpha} were analyzed by repeated measurement two-way analysis of variance. Probability values less than 0.05 were considered statistically significant. Statistical analyses were conducted using JMP 5.0 (SAS institute Inc, Cary, NC) and Graph Pad Prism 4 (Graph Pad Software Inc, San Diego, CA).


   Results
Top
 Abstract
 Introduction
 Material and Methods
 Results
Comment
 References
 
Endothelial Progenitor Cell Isolation From Bone Marrow Sample
We collected 23.7 ± 5.2 mL of BM from the iliac bone by multiple aspirations; 3.6 ± 0.6 x 108 MNCs were isolated from the BM sample. The concentration of MNCs in the BM samples was 2.0 ± 1.0 x 107 cells/mL. Four days after culture, the percentage of attached cells (EPC) was 0.83% ± 0.18%. Based on these data, 9.45 ± 1.56 x 104 EPC were present in 1 mL of BM sample. The recovery rate of MNCs after cryopreservation was 53.9% ± 7.2%. After ex vivo expansion of cryopreserved MNCs, the percentage of attached EPC was 0.83% ± 0.22%. The percentage of EPC in the total MNCs was similar with or without cryopreservation (p = 0.79).

Endothelial Progenitor Cell Fluorescent Staining
To confirm the attached cells were characteristic of EPC, we stained the attached cells 4 days after cell culture by isolectin B4, DiI-acLDL, and DAPI. Attached cells after MNC isolation were stained by isolectin B4 (Fig 1-IA), DiI-acLDL (Fig 1-IB), and DAPI (Fig 1-IC). Merge pictures are shown in Figure 1-ID. As DAPI is a marker for cell nuclei, we calculated the EPC displaying positive staining for isolectin B4, DiI-acLDL, and DAPI as a percentage of total DAPI-staining cells. The percentage of EPC was 96.0% ± 0.42% in attached cells from freshly isolated MNCs (Fig 1-ID). The percentage of EPC was 95.2% ± 1.2% in attached cells after cryopreservation (Fig 1-IID). There was no significant difference in the percentage of isolated EPC between the two groups (p = 0.21).


Figure 1
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  		Fig 1. Direct fluorescent staining of endothelial progenitor cells (EPC) from fresh mononuclear cells (MNCs) (I) and cryopreserved mononuclear cells (II). (A) Isolectin B4 staining. (B) Acetylated low-density lipoprotein labeled with 1,1'-dioctadecyl-3,3,3'-3'-tetramethylindo-carbocyanine perchlorate staining. (C) 4',6-diamidino-2-phenylindole dihydrochloride staining. (D) Merged images of all three staining techniques.

 
Endothelial Progenitor Cell Proliferation Response
Proliferation in cell culture media including fibroblast growth factor, VEGF, insulin-like growth factor-1, epidermal growth factor, and FBS increased in EPC with or without cryopreservation (p < 0.01; Fig 2).


Figure 2
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  		Fig 2. Proliferation activity in cell culture media. White column, endothelial progenitor cells from fresh mononuclear cells; black column, endothelial progenitor cells from cryopreserved mononuclear cells. Asterisk and sharp indicate significance compared with white and black columns of control, respectively.

 
Vascular endothelial growth factor increased proliferation activity of EPC with or without cryopreservation (Fig 3; p < 0.01). Cryopreservation does not affect EPC proliferation response to VEGF as compared with EPC without cryopreservation (p = 0.56).


Figure 3
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  		Fig 3. Vascular endothelial growth factor (VEGF)–induced proliferation activity of endothelial progenitor cells. White column, endothelial progenitor cells from fresh mononuclear cells; black column, endothelial progenitor cells from cryopreserved mononuclear cells. Asterisk and sharp indicate significance compared with white and black column of control, respectively.

 
Endothelial Progenitor Cell Migration Response to Stromal-Derived Factor-1{alpha}
Stromal-derived factor-1{alpha} increased migration activity of EPC with or without cryopreservation (Fig 4; p < 0.01). Cryopreservation did not affect EPC migration response to SDF-1{alpha} as compared with EPC without cryopreservation (p = 0.63).


Figure 4
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  		Fig 4. Migration activity in response to stromal-derived factor-1{alpha} (SDF-1{alpha}). White column, endothelial progenitor cells from fresh mononuclear cells; black column, endothelial progenitor cells from cryopreserved mononuclear cells Asterisk and sharp indicate significance compared with white and black column of control, respectively.

 

   Comment
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
References
 
Cryopreservation is widely used for storage of various types of cells. In this study we collected EPC by ex vivo expansion from cryopreserved BM-derived MNCs. We found that EPC characteristics and function were preserved after cryopreservation. This conclusion is based on the following results: (1) the percentage of EPC in MNC after ex vivo expansion was similar with or without cryopreservation; (2) no difference was found in the percentage of positive cells stained by ac-LDL and isolectin B4 after ex vivo expansion; (3) VEGF increased EPC proliferation activity in fresh EPC, and the activity was similar between EPC with or without cryopreservation; and (4) SDF-1{alpha} increased the migration response of EPC, and this response was maintained in cryopreserved EPC.

Mononuclear cell storage using rapid cryopreservation has been performed for many years [8, 9]. In a recent study using human cord blood MNC, regulation of cooling speed in the cryopreservation allowed more efficient cell recovery, showing that the cooling speed at 1°C/min, 5°C/min, and 10°C/min results in 44%, 76%, and 93% recovery rates, respectively, of MNC after thawing [10]. More recently, Jang and colleagues [11] have shown a high recovery rate with more than 90% of cryopreserved human cord blood–derived MNCs. They achieved this efficient cryopreservation using a controlled-rate freezer at –196°C. Little is reported about cryopreservation of BM-derived EPC. In this study, the recovery rate of MNCs after cryopreservation was 54%. We preserved MNCs at –80°C, and did not use a controlled-rate freezer in this study. The difference in cooling speed and final temperature for cell storage may be associated with the lower recovery rate of cryopreserved MNCs. Furthermore, the percentage of CD34+ cells in human cord blood MNCs does not change after cryopreservation [10]. The percentage of EPC in MNCs after ex vivo expansion was similar with or without cryopreservation in this study. This may indicate that the freezing and thawing process involved in cryopreservation is not cell type specific, damaging EPC, lymphocytes, and monocytes. Although the efficiency of ex vivo expansion of EPC after cryopreservation needs to be improved, we found that EPC could be cryopreserved and isolated by ex vivo expansion from BM-derived MNCs.

Current freezing protocols for cells generally involve use of additives and cytoprotectants for increased viability and recovery after cryopreservation [12]. In addition, use of the biologic antioxidant catalase and the membrane stabilizer trehalose in addition to conventional freezing medium improves the functional capacity of human hematopoietic cells [13]. In addition, Jang and colleagues [11] also showed the more than 90% viability of thawed human cord blood cells using 10% dimethylsulfoxide in autologous plasma. The cell-freezing medium we used in this study is completely free from biologic products and toxicity seen with serum albumin and dimethylsulfoxide. Although this negatively impacted cell recovery, this freezing medium may be applicable for clinical use. However, future studies altering conditions such as freezing speed and other additives should be conducted to obtain cryopreserved EPC with high efficiency and functional quality.

Endothelial progenitor cells are defined as adherent cells derived from peripheral and BM-derived MNCs demonstrating Dil-AcLDL uptake and isolectin B4 binding capacity [14]. This observation has been reported in human [3], mouse [15, 16], and canine [17]. Similar to previous reports, Dil-AcLDL and isolectin B4 double-positive cells were also reactive in swine EPC even after cryopreservation in this study. Endothelial progenitor cells are known to express different lineage markers depending on their state of differentiation in vitro [18]. At 4 days after culture, EPC can be categorized as early EPC and express hematopoietic stem cell markers such as AC133, CD34, VEGFR2, and CD117, as shown in human and mouse studies [14]. Recently Romagnani and associates [19] showed that CD34-positive cells possess the capability of proliferation in response to stem cell growth factors and can differentiate into endothelial cells. However, it is difficult to evaluate the EPC characterization by stem cell lineage markers in swine owing to a lack of commercially available anti-swine antibodies. Effects of cryopreservation on changes in EPC lineage markers should be investigated in further studies.

Proliferation and migration functions are important factors for angiogenesis. We demonstrated that functional activity of proliferation and migration were preserved in cryopreserved EPC as well as fresh EPC in this study. It is known that VEGF signaling is involved in EPC proliferation [20]. Vascular endothelial growth factor receptor 1 and 2 are expressed on EPC during ex vivo expansion and play a critical role in EPC proliferation and differentiation, leading to angiogenesis [21]. Vascular endothelial growth factor consistently induced proliferation in EPC in this study. We demonstrated that the proliferation response to VEGF was also preserved in cryopreserved EPC as compared with fresh EPC. Furthermore, the migration response to SDF-1{alpha} was preserved in cryopreserved EPC as well as fresh EPC. Stromal-derived factor-1{alpha} increases migration of ex vivo expanded human EPC through interactions with its receptor, CXCR4 [22]. This growing evidence suggests that the receptor function and the downstream signaling pathways responsible for EPC proliferation and migration may be preserved after cryopreservation.

In this study, we demonstrate that ex vivo function of EPC is maintained after cryopreservation as compared with noncryopreserved cells. The optimal number of EPC required for functional improvement in large animal models and patients is still controversial. Ex vivo expansion of EPC from cryopreserved MNCs should allow increased yields of available EPC, through multiple collection procedures as well as ex vivo expansion. Furthermore, the circulating number and function of EPC are reduced in patients with diabetes mellitus, hypercholesterolemia, and smoking. The ability to increase the number of EPC as well as modulate EPC characteristics and function of cells during ex vivo expansion in these patient populations could be a valuable therapeutic tool. Although outside the scope of the current investigation, future studies should evaluate dosing strategies and the in vivo efficacy of cryopreserved and ex vivo expanded EPC. However, the current results support the idea that this could likely be an effective therapeutic strategy as cryopreservation did not alter ex vivo EPC function.

In this study, we demonstrated that ex vivo expansion after cryopreservation is a useful method to preserve and isolate EPC, maintaining proliferation and migration capability. These results suggest ex vivo expansion of EPC after cryopreservation could be an option to overcome clinical limitations of EPC transplantation in patients.


   References
Top
 Abstract
 Introduction
 Material and Methods
 Results
 Comment
 References
 

  1. Schachinger V, Assmus B, Britten MB, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOPCARE-AMI Trial J Am Coll Cardiol 2004;44:1690-1699.[Abstract/Free Full Text]
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  6. Assmus B, Fischer-Rasokat U, Honold J, et al. Transcoronary transplantation of functionally competent BMCs is associated with a decrease in natriuretic peptide serum levels and improved survival of patients with chronic postinfarction heart failure: results of the TOPCARE-CHD Registry Circ Res 2007;100:1234-1241.[Abstract/Free Full Text]
  7. Kotobuki N, Hirose M, Machida H, et al. Viability and osteogenic potential of cryopreserved human bone marrow-derived mesenchymal cells Tissue Eng 2005;11:663-673.[Medline]
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  16. Hamada H, Kim MK, Iwakura A, et al. Estrogen receptors alpha and beta mediate contribution of bone marrow-derived endothelial progenitor cells to functional recovery after myocardial infarction Circulation 2006;114:2261-2270.[Abstract/Free Full Text]
  17. Wu Y, Zhang J, Gu Y, et al. Expansion of canine bone marrow-derived endothelial progenitor cells and dynamic observation Ann Vasc Surg 2006;20:387-394.[Medline]
  18. Murohara T, Ikeda H, Duan J, et al. Transplanted cord blood-derived endothelial precursor cells augment postnatal neovascularization J Clin Invest 2000;105:1527-1536.[Medline]
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Invited Commentary
Dan Spiegelstein, Christopher Kim, and Terrence M. Yau
Ann. Thorac. Surg. 2008 85: 1366. [Extract] [Full Text] [PDF]



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Ann. Thorac. Surg., April 1, 2008; 85(4): 1366 - 1366.
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