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Table of Contents
RESEARCH ARTICLE
Year : 2019  |  Volume : 4  |  Issue : 4  |  Page : 116-120

Isolation and culture of vascular wall-resident cd34+ stem/progenitor cells


1 Department of Physiology, School of Basic Medicine Science, Hubei University of Medicine; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine, Shiyan, Hubei, China
2 Department of Physiology, School of Basic Medicine Science, Hubei University of Medicine; Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine; Department of Cardiology, Institute of Clinical Medicine, Renmin Hospital, Hubei University of Medicine, Shiyan, Hubei, China

Date of Submission16-Sep-2019
Date of Acceptance15-Nov-2019
Date of Web Publication31-Dec-2019

Correspondence Address:
Jun-Ming Tang
Department of Physiology, School of Basic Medicine Science, Hubei University of Medicine, Hubei, 442000, China, Hubei Key Laboratory of Embryonic Stem Cell Research, Hubei University of Medicine Shiyan, Hubei 442000; Department of Cardiology, Institute of Clinical Medicine, Renmin Hospital, Hubei University of Medicine, Hubei 442000
China
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/cp.cp_19_19

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  Abstract 


Objective: The aim of this study is to observe the maintenance of stem cell properties of purified CD34-positive cells in vessel walls during in vitro expansion. Materials and Methods: Cells that migrated from the adventitial tissues of rat were collected and purified by microbead selection method to obtain CD34+ vascular wall-resident stem (VRS)/progenitor cell (PC). Those purified CD34+ VRS/PCs were evaluated by flow cytometry and immunofluorescent staining. The CD34+ VRS/PCs were continuously cultured until passage P3. Each passaged cell was evaluated by flow cytometry with anti-CD34. Results: After microbead selection, the CD34+ cells reached 88.07% ± 4.36% and these cells expressed neither endothelial (CD31) nor mature smooth muscle cell (smooth muscle-myosin heavy chain and SM22α) markers. Incubation of the purified CD34+ VRS/PCs at a density of 1.5 × 105 cells/100-mm dish, resulted in a gradual reduction of CD34-positive traits when passaged in vitro, starting at P1. Interestingly, the purified primary CD34+ VRS/PCs at a density of 1.0 × 104 cells per 100-mm dish show the traits of colony form growth, and P1 passaged cells were 79.2% ± 2.15% positive for CD34, then gradually lost the traits of CD34-positive cells when passaged in vitro. Conclusions: High purity CD34+ VRS/PCs can be obtained by magnetic bead screening. In vitro, low cell densities contribute to the maintenance of CD34+ VRS/PC traits.

Keywords: CD34+ vascular wall-resident stem/progenitor cells, culture, magnetic bead screening


How to cite this article:
Wu Y, Zhang RN, Sen-Zhao, Tang JM. Isolation and culture of vascular wall-resident cd34+ stem/progenitor cells. Cardiol Plus 2019;4:116-20

How to cite this URL:
Wu Y, Zhang RN, Sen-Zhao, Tang JM. Isolation and culture of vascular wall-resident cd34+ stem/progenitor cells. Cardiol Plus [serial online] 2019 [cited 2020 Apr 2];4:116-20. Available from: http://www.cardiologyplus.org/text.asp?2019/4/4/116/274571




  Introduction Top


Neointimal formation is a key pathological process in the early development of various vascular occlusive diseases, such as atherosclerosis, postangioplasty restenosis, and transplant vasculopathy.[1],[2],[3] Traditional “phenotypic modulation hypothesis” suggests that the proliferating neointimal cells are derived from mature medial smooth muscle cells (SMCs) which have undergone a phenotypic modulation as a response to injury stimuli.[4],[5],[6],[7],[8],[9] However, this long-standing and widely accepted theory was recently challenged by evidence that the vascular wall-resident stem/progenitor cells (VRS/PCs) exist in the blood vessel walls, particularly noting that adventitia contributes a significant percentage of neointimal SMCs during vascular repair.[10],[11],[12],[13] A number of studies, particularly evidence from Tang et al. have shown that the vascular remodeling is at least partly attributable to the proliferation and differentiation of the VRS/PCs.[14]

Recent studies in our laboratory and others have demonstrated that CD34+, CD31- VRS/PCs exist in the adventitia of human and rat blood vessel walls.[15],[16],[17],[18] To better understand the traits and role of CD34+, CD31 VRS/PCs in the process of physiology and pathological condition, the specific VRS/PCs in vitro should be cultured and identified before their biology effects. Here, we provide general and detailed methods for CD34+ VRS/PCs using microbead selection and flow cytometry. This protocol will enable the application of CD34+ VRS/PCs to clinical and scientific research.


  Materials and Methods Top


Animal

The animal experiment protocols were approved by the Institutional Animal Care Committee of Wuhan University, PR. China.

Materials

Antibody against CD34 (AF4117; R and D Systems; Alaska; USA), Donkey Anti-Goat IgG Mag Beads (11-RBaGT-11; pluriSelect Life Science; Leipzig, Germany), fetal calf serum (10091-148; Gibco; Australia Origin), antibody against CD31 (ab64543), antibody against SM22 (ab14106), and smooth muscle myosin heavy chain (SM-MHC) (ab53219) were bought from Abcam (Hong Kong, China).

CD34+ vascular wall-resident stem/progenitor cells isolation and purification

To isolate the CD34+ cells, the media layer was carefully stripped off the aseptically harvested fresh aortas from young animals (2 months old). The remaining adventitial tissues were cut into 0.5 mm3 pieces and explanted onto the bottom of a culture bottle. The bottle was inverted in a CO2 incubator at 37°C for 3 h before adding the stem cell growth medium (Dulbecco's Modified Eagle Medium [DMEM]/F12 with 10% fetal calf serum of charcoal adsorption, 0.1 mmol/L 2-mercaptoethanol and 100 U/ml penicillin/streptomycin) which contains 10 ng/ml leukemic inducible factor (LIF) to promote VRS/PCs proliferation and suppress their differentiation.[19] After 5–7 days of incubation, the cells that migrated from the adventitial tissues were enzyme-resuspended and collected for purification. Usually, 15 aortas were used each time to obtain 1 × 106 CD34+ cells. A microbead selection with anti-CD34 was performed to purify the isolated adventitial cells according to the manufacturer's instructions. The purity of selected CD34+ cells was evaluated using both flow cytometry with anti-CD34, and immunofluorescent microscopy with anti-CD34 and SM MHC, SM22, or CD31.

CD34+ vascular wall-resident stem/progenitor cells culture and subculture

To get more CD34+ VRS/PCs we amplified cell growth in vitro. Purified CD34+ VRS/PCs were seeded in the stem cell growth medium and cultured at 37°C in 5% CO2. Nonadherent cells were removed by replacing the media at 24 h after plating and every 3 days thereafter. Once the cells had reached 80% confluence, the adherent CD34+ VRS/PCs were detached with 0.25% trypsin and passaged at a density of either 1.0 × 104 or 1.5 × 105 cells per 100-mm dish. The CD34+ VRS/PCs were continuously cultured until passage P3. Each passage cell was evaluated by flow cytometry with anti-CD34.

Fluorescence-activated cell sorting analysis

To observe the positive rate of CD34+ VRS/PCs, each passage cell was evaluated by flow cytometry. The cultured cells in suspension were labeled with PE-conjugated monkey anti-goat IgG antibody to determine by fluorescence-activated cell sorting analysis. For each treatment, 106 cells were examined, and the experiment repeated three times. Data were analyzed using WinMDI software (The Scripps Research Institute, Flow Cytometry Core Facility, BD, Beckman Coulter, American).

Immunofluorescent staining

Briefly, cultured cells were labeled with goat mAbs against CD34, mouse mAbs against CD31, rabbit mAbs against SM22, and rabbit mAbs against SM MHC antibody, and visualized with anti-goat, rabbit, or mouse IgG conjugated with fluorescein isothiocyanate (Dakopatts) or Cy3.

The proliferation of the CD34+ VRS/PCs CCK8 method was adopted. P1 generation isolated CD34+ VRS/PCs were selected as experimental subjects. The CD34+ VRS/PCs were cultured for 7 days in the stem cell growth medium. The optical density value of 450 nm was detected on the automatic microplate at day 1, 3, 5, and 7.

CD34+ vascular wall-resident stem/progenitor cells multipotency identification

To verify the multipotency of the isolated CD34+ VRS/PCs, an induction medium (Phenol red-free DMEM/F12, 10% fetal calf serum of charcoal adsorption, 0.1 mmol/L 2-mercaptoethanol, 100 U/ml penicillin/streptomycin) containing either 20 ng/ml platelet-derived growth factor -BB (for the induction of SMC differentiation) or 50 ng/ml vascular endothelial growth factor (for induction of endothelial differentiation) was used. The cells (5 × 104) were incubated in a 100-mm culture dish with either SMC or endothelial cell induction medium. After 7 days of induction, the cells were routinely fixed and immunofluorescently stained for SMC maker SM MHC, or endothelial maker CD31.

Statistical analysis

All data were statistically analyzed using the software SPSS 17.0 (SPSS Inc., Chicago, IL, USA) with the Student-Newman–Keuls multiple comparison test after a one-way ANOVA or Student's t-test. A difference was considered statistically significant when P < 0.05.


  Results Top


CD34+ vascular wall-resident stem/progenitor cells isolation and purification

To isolate the CD34+ cells, the adventitial tissue was allowed to attach onto the bottom of a culture dish containing the stem cell growth medium. After 5–7 days of incubation, the cells migrated from the adventitial tissues were 18.5% ± 2.18% positive for CD34 as detected by flow cytometry, and after microbead selection, the CD34+ cells reached up to 88.07% ± 4.36% [Figure 1]a. Immunocytochemistry demonstrated that these cells expressed neither endothelial (CD31) nor mature SMC (SM-MHC and SM22α) markers [Figure 1]b.
Figure 1: Identification of isolated CD34+ vascular wall-resident stem/progenitor cells. (a) Representative flow cytometric plots (left) and histograms of percentages of CD34+ cells in isolated adventitial cells before or after microbead selection. *P < 0.05, compared to migrated adventitial cells before microbead selection (N = 9). (b) Representative immunofluorescent micrographs of isolated CD34+ vascular wall-resident ste /progenitor cells

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Identification of CD34+ vascular wall-resident stem/progenitor cells in different passages

Since different stem cells numbers could affect stem cell differentiation we examined the effect of cell density on cell phenotype. We compared the changes of CD34 positive ratio of VRS/PCs when either 1.0 × 104 or 1.5 × 105 cells were incubated in per 100-mm dish. After 3–5 days of incubation of the purified CD34+ VRS/PCs with 1.5 × 105 cells per 100-mm dish, the cultured P1 cells could not colonially grow [Figure 2]b, and rapidly reached 90% confluence in vitro, showing 44.0% ± 2.13% positive for CD34. Furthermore, the high cell-density decreased the positive ratio of CD34–17.7% ± 2.21% at P2 and 2.5% ± 1.02% at P3 [Figure 3]a. This indicates that the CD34+ VRS/PCs gradually lost the traits of CD34 positive cells when passaged with high cell-density in vitro. Interestingly, the purified primary CD34+ VRS/PCs at a density of 1.0 × 104 cells per 100-mm dish showed the traits of colony form growth [Figure 2]a, and after almost every passage these cells following colony form growth were 79.2% ± 2.15% positive for CD34 [Figure 3]b. Taken together, low cell densities contributed to the maintenance of CD34+ VRS/PC traits.
Figure 2: The growth state of isolated CD34+ vascular wall-resident stem/progenitor cells in different planted density by microscope. (a) Representative microscope images of colony form of CD34+ cells planted at density of 1 × 10^4 cells per 100-mm dish. (b) Representative microscope images of uncolony form of CD34+ cells planted at density of 1 × 10^5 cells per 100-mm dish

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Figure 3: Identification of isolated CD34+ vascular wall-resident stem/progenitor cells in different passages and planted density. (a) Representative flow cytometric plots of percentages of CD34+ cells in isolated adventitial cells after microbead selection from P1 to P3. (b) Representative histograms of percentages of CD34+ cells in isolated adventitial cells after microbead selection from P1 to P3. *P < 0.05, compared to planted density of 1 × 10^5 cells per 100-mm dish (N = 9)

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The proliferation of CD34+ vascular wall-resident stem/progenitor cells

To observe the proliferation of CD34+ VRS/PCs, the cells were cultured in the stem cell growth medium for 7 days. CCK8 kits test [Figure 4] showed that at day 3 of culture when the cells were in active logarithmic growing phase, the cells did not continuously increase at day 5–7 because the cultures had already reached confluence.
Figure 4: Proliferation of CD34+ vascular wall-resident stem/progenitor cells growing in Stem cell growth medium

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Multipotency of isolated CD34+ vascular wall-resident stem/progenitor cells

To verify the multipotency of the isolated CD34+ VRS/PCs, the cells were incubated in the induction medium for endothelial or SMC differentiation. As shown in [Figure 5], after induction numerous cells expressing mature SMC (SM MHC and SM22) or endothelial (CD31) markers could be observed.
Figure 5: Representative immunofluorescent micrographs of isolated CD34+ vascular wall-resident stem/progenitor cells after in vitro induction

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  Discussion Top


The CD34+ VRS/PCs in the adventitia of the blood vessel wall were first reported by Hu et al. in 2004.[11] The existence of the cells in the human vessel adventitia and atherosclerotic lesions were verified by a number of independent laboratories.[17],[20],[21] In the present study, CD34+ cells isolated from the adventitial tissue were negative for markers of mature endothelial cell and vascular SMC, only showing differentiation into these two vascular cell types in vitro. When planted at low density, these CD34+ cells appeared to have colonial form growth. This indicates that they are the VRS/PCs.

When cultured in stem cell growth medium (which contains LIF that stimulates the cells to proliferate but suppresses their differentiation[11]), the CD34+ VRS/PCs retained the ability to proliferate. However, we found CD34+ VRS/PCs also have the ability to differentiate, and the differentiation rate was higher with more passages. The finding in the present study provides evidence that CD34+ VRS/PCs cannot retain their “stem cell character” in cultures. To study CD34+ VRS/PCs better, we needed to select primary purified CD34+ VRS/PCs. The number of primary purified CD34+ VRS/PCs is often low because CD34+ VRS/PCs are low in the source adventitial tissue.[11] To get more CD34+ VRS/PCs, we passaged the purified primary CD34+ VRS/PCs at different densities. When the density is lower, the CD34+ VRS/PCs appeared to display colony form growth and about 80% of the cells are CD34+. We posit that P1 CD34+ VRS/PCs planted at lower density from primary purified cells can be used for further study. As for why planted density affected the positive rate of CD34+ VRS/PCs, we hypothesize that higher cell density may lead to the rapid fusion of cells which secrete cytokines, which promotes the differentiation and depresses the proliferation of stem cells.[22],[23]

In summary, because CD34+ VRS/PCs are not abundant in adventitia, it is difficult to obtain enough use in scientific research. It is necessary to amplify the purified CD34+ VRS/PCs in vitro. However, in the process of culturing CD34+ VRS/PCs, two factors must be paid attention to: CD34+ VRS/PCs should be cultivated using stem cell culture medium to prevent differentiation, and CD34+ VRS/PCs can only be amplified one passage with lower density, otherwise CD34+ VRS/PCs will differentiate. This method provides a potential solution to the problem of insufficient stem cells in scientific research.


  Conclusion Top


This study introduced how to obtain high-purity rat vascular wall c-kit+ cells by magnetic bead sorting and found low cell densities contribute to maintenance of CD34+ VRS/Pc traits in vitro, which provides support and guarantee for the further application of c-kit+ cells in experimental studies.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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    Figures

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



 

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