Massive Clonal Selection and Transiently Contributing Clones During Expansion of Mesenchymal Stem Cell Cultures Revealed by Lentiviral RGB-Barcode Technology

Abstract

Mesenchymal stem (or stromal) cells (MSCs) have been used in more than 400 clinical trials for the treatment of various diseases. The clinical benefit and reproducibility of results, however, remain extremely variable. During the in vitro expansion phase, which is necessary to achieve clinically relevant cell numbers, MSCs show signs of aging accompanied by different contributions of single clones to the mass culture. Here we used multicolor lentiviral barcode labeling to follow the clonal dynamics during in vitro MSC expansion from whole umbilical cord pieces (UCPs). The clonal composition was analyzed by a combination of flow cytometry, fluorescence microscopy, and deep sequencing. Starting with highly complex cell populations, we observed a massive reduction in diversity, transiently dominating populations, and a selection of single clones over time. Importantly, the first wave of clonal constriction already occurred in the early passages during MSC expansion. Consecutive MSC cultures from the same UCP implied the existence of more primitive, MSC culture-initiating cells. Our results show that microscopically homogenous MSC mass cultures consist of many subpopulations, which undergo clonal selection and have different capabilities. Among other factors, the clonal composition of the graft might have an impact on the functional properties of MSCs in experimental and clinical settings.

Significance: Mesenchymal stem cells (MSCs) can easily be obtained from various adult or embryonal tissues and are frequently used in clinical trials. For their clinical application, MSCs have to be expanded in vitro. This unavoidable step influences the features of MSCs, so that clinical benefit and experimental results are often highly variable. Despite a homogenous appearance under the microscope, MSC cultures undergo massive clonal selection over time. Multicolor fluorescence labeling and deep sequencing were used to demonstrate the dynamic clonal composition of MSC cultures, which might ultimately explain the variable clinical performance of the cells.

Keywords: Clonal evolution; DNA barcoding; Mesenchymal stem cells; Reproducibility of results; Stem cell niche; Umbilical cord.

Figure 1.

Lentiviral RGB barcode vector and experimental procedure. (1–3): RGB barcode design with constant and variable (N) nucleotides (1) as part of a (2) lentiviral vector construct (2) driven by an SFFV promoter/enhancer used for the transduction of UCPs (3). (4): Outgrowth of primary MSC-EMs with exemplary microscopic pictures. (5, 6): Transfer of UCP to a new well for initiation of consecutive MSC-EM (5) or independent cultivation of MSC-EM resulting in clonal selection over time (6). (7): MSC-EM after several passages. (8): Next-generation sequencing to assess barcode complexity within DNA samples. Abbreviations: Δ, self-inactivating U3 region; B, barcode; LTR, long terminal repeat; MSC-EM, mesenchymal stem cell explant monolayer; R, repeat region; SFFV, spleen focus forming virus; U, unique region; UCP, umbilical cord piece; wPRE, woodchuck hepatitis virus postregulatory element.

Figure 2.

Histological and immunohistochemical proliferation staining of fresh and 5 weeks cultivated umbilical cord piece (UCP). The left column shows fresh and the right column 5 weeks cultivated UCP. The first row depicts the histological staining (H&E staining) and the second row the immunostaining of proliferating cells (anti-Ki-67 antibody). Nuclei of proliferating cells are highlighted with purple circles, and nuclei of nonproliferating cells are blue. The last row is a magnification of the Ki-67 staining.

Figure 3.

Fluorescent microscopic comparison of the transduction methods and the development of transgene expression in whole tissue pieces. Fresh and 5 weeks cultivated umbilical cord piece (UCP) were transduced with three viruses either coding for mCherry (red), Cerulean (blue), or Venus (green). Fresh pieces were additionally cut in half to increase the surface of the pieces. Alternatively, the virus was injected in UCP. Depicted is the midlayer of the UCP 1 week (left) or 10 weeks (right) posttransduction. Abbreviation: pTdx, posttransduction.

Figure 4.

Analysis of transduced umbilical cord piece (UCP)-derived mesenchymal stem cell explant monolayer (MSC-EM). Whole UCPs were transduced with three different viruses coding for fluorescent proteins and a genetic barcode. After 21 days, the first MSC-EM grew out. (A): Induction time between consecutive MSC-EMs. (B): Flow cytometric analysis of overall transgene positive cells in MSC-EM arranged by transduction method. (C): Amount of unique barcodes in MSC-EM arranged by transduction method. (D): Flow cytometry and unique barcodes in MSC-EM arranged by the order of induction. The data are represented by means, and error bars show the standard deviation. Abbreviations: P1–4, passage of UCP to new cell culture well.

Figure 5.

Clonal development of one exemplary MSC-EM. (A): Flow cytometric analysis of the clonal development. The graph displays the percentage of fluorescence-positive cells, and the color of the symbol indicates the color of the respective cell population (black indicates fluorescent-negative cells). The development of the mass culture during the first 12 passages (P1.01 to P1.12) of the primary MSC-EM culture is shown. (B): Vector copy number analysis of mass cultures by quantitative polymerase chain reaction. Data are displayed as means ± SD. (C): Shannon index describing the diversity of the barcodes in the mass culture. (D): Area plot of the barcode contribution. Each area represents an individual barcode from one of the vector constructs (different shades of the colors red, blue, and green refer to the RGB construct the barcode originated from). The percentages show the contribution of a barcode after the last passage in case it contributed >5% to the total sequencing reads. Abbreviations: MSC-EM, mesenchymal stem cell explant monolayer; P, passage; UCP, umbilical cord piece; VCN, vector copy number.

Figure 6.

Clonal development of consecutive MSC-EM. Analysis of MSC-EM of two individually transduced UCP (UCP 2, UCP 3). Colors of the symbols and areas indicate fluorescent proteins coded by the vectors. (A): Flow cytometric analysis of the first 13 passages (P1.1 to P1.13) of the primary MSC-EM of UCP 2. (B): Flow cytometric analysis of the first 3 passages (P2.1 to P2.3) of the secondary MSC-EM of UCP 2. (C): Flow cytometry analysis of the first 5 passages (P1.1 to P1.5) of the primary MSC-EM of UCP 3. (D): Flow cytometry analysis of the first 3 passages (P2.1 to P2.3) of the secondary MSC-EM of UCP 3. (E): Area plot of the barcode distribution of the first 5 passages (P1.1 to P1.5) of the primary MSC-EM of UCP 3. (F): Area plot of the barcode contribution of the first 3 passages (P2.1 to P2.3) of the secondary MSC-EM of UCP 3. The percentage show the contribution after the last passage, if it was >5%. The two dominant barcodes for the primary and secondary MSC-EM of UCP 3 were identical. Abbreviations: MSC-EM, mesenchymal stem cell explant monolayer; P, passage; UCP, umbilical cord piece.

Figure 7.

Tube formation assay with different fluorescence sorted clones. A freshly initiated mesenchymal stem cell explant monolayer was first expanded, before cells were sorted by the present colors green, yellow, and negative. A tube formation assay was performed in triplicate with the different sorted populations. Depicted is microscopic fluorescence overlay after 4 hours. The yellow and green population had a strongly reduced tube formation capability compared with cells from the fluorescence negative population. Magnification, ×5.