Method to Convert Stem Cells into Cancer Stem Cells

The cancer stem cell (CSC) hypothesis suggests that tumors are sustained exclusively by a small population of the cells with stem cell properties. CSCs have been identified in most tumors and are responsible for the initiation, recurrence, and resistance of different cancers. In vitro CSC models will be of great help in revisiting the mechanism of cancer development, as well as the tumor microenvironment and the heterogeneity of cancer and metastasis. Our group recently described the generation of CSCs from induced pluripotent stem cells (iPSCs), which were reprogrammed from normal cells, and/or embryonic stem cells (ESCs). This procedure will improve the understanding of the essential niche involved in cancer initiation. The composition of this cancer-inducing niche, if identified, will let us know how normal cells convert to malignant in the body and how, in turn, cancer prevention could be achieved. Further, once developed, CSCs demonstrate the ability to differentiate into endothelial cells, cancer-associated fibroblasts, and other phenotypes establishing the CSC niche. These will be good materials for developing novel cancer treatments. In this protocol, we describe how to handle mouse iPSCs/ESCs and how to choose the critical time for starting the conversion into CSCs. This CSC generation protocol is essential for understanding the role of CSC in cancer initiation and progress.


Introduction
Cancer remains a major cause of mortality all over the world. Statistics have predicted new cancer cases more than 20 million worldwide each year by 2030 [1]. Therefore, early diagnosis and treatment of cancer are currently the most critical issue for cancer prevention.
Understanding the mechanism of cancer initiation will provide us with critical clues in finding ways to treat cancer. However, the mechanisms underlying cancer initiation appear extremely complicated, with various phenotypes and numerous signaling pathways involved.
Many studies have defined the functional role of cancer stem cells (CSCs) in cancer initiation [2]. CSCs are considered to critically influence tumorgenicity [3] and metastasis [4] as well as provide resistance against therapeutic agents [5]. CSCs are postulated to be originate from normal stem cells [6]. This conversion may result from chronic inflammation, which continuously imbalances homeostasis, resulting in the generation of an irregular microenvironment surrounding progenitor cells or stem cells.
Induced pluripotent stem cells (iPSCs)/embryonic stem cells (ESCs) could acquire the characteristics of CSCs when cultured in the presence of conditioned medium (CM) prepared from various cancer cell lines without transducing foreign genes and/or mutagens [6][7][8][9][10][11][12][13][14][15]. The CSCs established from iPSCs through epigenetic regulations have been shown to have the capacity of self-renewal, differentiation, and malignant tumorigenicity. Based on our data, we summarize the detailed procedure and the critical conditions step-by-step.

Experimental Design
This protocol describes how to generate CSCs from iPSCs/ESCs in the presence of microenvironments conferred by different cancer cell lines. The protocol shows detailed experimental steps related to the conversion of stem cells into CSCs. First of all, CM was collected from a confluent culture of cancer cell lines. Then, iPSCs were converted into CSCs in the presence of CM, as summarized in Figure 1. Afterwards, the surviving cells were suspended in Hank's balanced salt solution (HBSS) and injected into BALB/c nude mice. The surgical procedure was performed under sterile conditions. Almost one month later, malignant tumors developed in mice injected with the surviving cells, while teratoma developed in mice injected with mouse iPSCs/ESCs. Primary cultures from tumors were assessed for sphere formation to confirm characteristics of CSCs. The differentiation potential was confirmed by the appearance of a phenotype that was adhesive to the dishes. Ethanol (70%) was used for surgical instruments to maintain sterile conditions during the procedure. All animal experiments were reviewed and approved by the ethics committee for animal experiments of Okayama University under the ID OKU-2018078.

Conditioned Medium (CM) Preparation for Mouse iPSC/ESC Conversion
In our protocol, we used conditioned medium (CM) derived from cancer cell lines to convert stem cells to CSCs.

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Take of a vial of each cancer cell line from liquid nitrogen storage.

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Passage cells again after a couple of days, when the cells are 70% confluent.

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Prior to collecting conditioned medium, change the medium to 5% FBS at 80% confluency.
Critical: Cells should be at least 80% confluent.
• After incubation for 48 h, collect the conditioned medium (CM) from Huh7 cells from confluent dishes.
Critical: Precaution should be taken to avoid overgrowth.

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Separate the supernatant in a new tube and filters using 0.22 mm filter. • Remove 2 mL CM then add this to a 3.5 cm dish overnight to confirm there are no surviving cancer cells in CM.

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Store CM at −20 • C for later experiments.

Plating Mitomycin C-Treated Mouse Embryonic Fibroblasts (MEFs)
Our group revived iPSCs/ESCs on mitomycin C-treated mouse embryonic fibroblast (MEF) feeder cells for the maintenance the undifferentiated state of iPSCs/ESCs.

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Add 2 mL sterile 0.1% gelatin to cover the bottom of 6 dishes.

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Incubate the gelatin-coated dishes for at least 30 min at 37 • C.

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Remove the MEF vial from the liquid nitrogen and thaw quickly in a 37 • C water bath.

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Remove the vial from the water bath after the vial is half-thawed.  Change the medium twice a week.

Plating Mouse iPSCs/ESCs
Our group grew iPSCs/ESCs on MEF feeder cells using iPSCs complete medium in the presence of leukemia inhibitory factor (LIF). It is important that iPSCs/ESCs be subcultured every 4 days at a low density to maintain their growth in the exponential phase. Under carefully monitored iPSCs/ESCs culture conditions, iPSCs/ESCs maintain pluripotency and self-renewing capacity. After transferring iPSCs/ESCs to a gelatin-coated dish, cells should be monitored until forming separate colonies without differentiation. The time at which colonies become 70% confluent is considered to be the critical time for starting conversion. • Prepare iPSCs complete media as described above.

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Pre-warm mouse iPSC/ESC medium in a 37 • C water bath for 30 min.
Critical: Do not keep the media in the water bath for more than 1 h at 37 • C as continued exposure to 37 • C will reduce the effectiveness of the growth factors.

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Replace the MEF medium with 4 mL of iPSCs complete medium (+LIF).

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Remove frozen mouse iPSCs/ESCs from liquid nitrogen storage. Critical: Avoid seeding mouse iPSCs/ESCs at high density because they tend to aggregate and give rise to cells with mixed morphologies.

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Change the medium the next day to remove the dead cells and daily thereafter until the cells have been cultured for 7 days or the colonies reach 80% confluency ( Figure 2).
• Seed 0.1 × 10 6 of cells onto 6 cm MEF dishes. Critical: Avoid seeding mouse iPSCs/ESCs at high density because they tend to aggregate and give rise to cells with mixed morphologies.
• Change the medium the next day to remove the dead cells and daily thereafter until the cells have been cultured for 7 days or the colonies reach 80% confluency ( Figure 2).

Conversion of Mouse iPSCs/ESCs into CSCs
To initiate the conversion of iPSCs/ESCs into CSCs, we used CM derived from a cancer cell line. We suppose that chronic inflammatory conditions trigger stem cells to develop into CSCs [6]. During this process, cytokines and other soluble mediators in the tumor microenvironment (CM) bound to stem cell surface receptors stimulate the intracellular signaling cascade in order to direct stem cell fates into the CSCs phenotype. We recommend using conversion medium containing CMs and iPSCs complete medium at a 1:1 ratio in order to convert stem cells into CSCs. Monitor the conversion using GFP protein if controlled under a Nanog promoter and photograph the cells every week using Olympus IX81 microscope equipped with fluorescence ( Figure 3).
Inject miPS-PLCcm cells into the liver. V.
• Inject the cells slowly into the organ or subcutaneous until it is completely injected.
Critical: Cells should be infused very slowly.   Critical: Do not remove the needle immediately after infusion to avoid the backflow of cells as well as bleeding. After 1 min, clotting will occur at the site of puncture. • Remove the needle.

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Close the abdominal peritoneum and then skin with the degradable sterile suture separately.

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Keep the mice in a pre-warmed cage directly after surgery for recovery until the mouse shows regular breathing patterns.

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Allow free access to water and food.

Malignant Tumor Detection and Primary Culture
• After 30 days of transplantation, anesthetize the mice with 2% isoflurane in an anesthesia chamber. Check self-renewal potential of new derived primary cultured cells by sphere formation assay.

Sphere Formation Assay
• After maintaining the primary culture from tumor and treating with puromycin, trypsinize the cells and count to perform the sphere formation assay. Critical: The seeded cells should appear as singlets.
• Place the plate in an incubator set at 37 • C with 5% CO 2 . • Wait for 7 days then count the sphere under an optical microscope.

Expected Result
The protocol described here outlines a technique utilized for the induction of CSC using CM derived from different cancer cell lines. The converted cells are highly tumorigenic with malignancy while mouse iPSCs/ESCs form benign teratoma. All the tumors derived from converted cells showed the malignancy, for example, the tumor formed by miPS-PLCcm cells exhibited malignancies such as mitotic figures, nuclear atypia, high nuclear to cytoplasmic ratio and angiogenesis (Figure 4).

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Dissociate the pellet by pipetting in a serum-free medium. • Count the cells and take the required number of live cells.

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Seed the cells in sphere medium, prepare at least 4-6 replicates for each primary culture.
Critical: The seeded cells should appear as singlets.

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Place the plate in an incubator set at 37 °C with 5% CO2. • Wait for 7 days then count the sphere under an optical microscope.

Expected Result
The protocol described here outlines a technique utilized for the induction of CSC using CM derived from different cancer cell lines. The converted cells are highly tumorigenic with malignancy while mouse iPSCs/ESCs form benign teratoma. All the tumors derived from converted cells showed the malignancy, for example, the tumor formed by miPS-PLCcm cells exhibited malignancies such as mitotic figures, nuclear atypia, high nuclear to cytoplasmic ratio and angiogenesis (Figure 4). The malignant tumor derived from miPS-PLCcm cells was assessed for several markers by immunohistochemistry ( Figure 5). GFP staining indicated undifferentiated population derived from The malignant tumor derived from miPS-PLCcm cells was assessed for several markers by immunohistochemistry ( Figure 5). GFP staining indicated undifferentiated population derived from miPS-PLCcm cells. Strong immunoreactivity to Ki67 indicated high proliferation rate in the tumor tissue. Immunoreactivity to CD44, E-cadherin and N-cadherin implied the heterogeneous phenotypes in malignant tumor derived from miPS-PLCcm cells. miPS-PLCcm cells. Strong immunoreactivity to Ki67 indicated high proliferation rate in the tumor tissue. Immunoreactivity to CD44, E-cadherin and N-cadherin implied the heterogeneous phenotypes in malignant tumor derived from miPS-PLCcm cells. The converted cells and the primary from all derived tumors sustained the expression of stemness and CSCs markers. For example, miPS-PLCcm cells and miPS-PLCcmPcells sustained the expression of stemness markers such as Nanog, Klf-4, and c-Myc as well as acquired CSC markers such as CD24, CD44, and CD90 ( Figure 6). These primary cells in an adhesive culture will exhibit two subpopulations: one is a colony, expressing GFP if directed by a Nanog promoter, surrounded by the other, which is epithelial-like cells with suppressed GFP expression by an inactive Nanog promoter. Simultaneously, these cells will also exhibit self-renewal potential in a sphere formation assay (Figure 7). These primary cells in an adhesive culture will exhibit two subpopulations: one is a colony, expressing GFP if directed by a Nanog promoter, surrounded by the other, which is epithelial-like cells with suppressed GFP expression by an inactive Nanog promoter. Simultaneously, these cells will also exhibit self-renewal potential in a sphere formation assay (Figure 7). Methods Protoc. 2019, 2, x FOR PEER REVIEW 13 of 15 Primary cultures cells from different tumors derived from converted cells: miPS-LLCcmP cells, miPS-PK8cm P cells, miPS-T47DcmP cells miPS-PLCcmP cells, and B6G-LLCcmP cells exhibited two subpopulations: one is a colony expressing GFP and epithelial-like cells surrounding it in adhesive condition and with self-renewal potential in a non-adherent culture condition. Scale bars represent 100 μm. Primary cultures cells from different tumors derived from converted cells: miPS-LLCcmP cells, miPS-PK8cm P cells, miPS-T47DcmP cells miPS-PLCcmP cells, and B6G-LLCcmP cells exhibited two subpopulations: one is a colony expressing GFP and epithelial-like cells surrounding it in adhesive condition and with self-renewal potential in a non-adherent culture condition. Scale bars represent 100 µm.