Generation of Induced Pluripotent Stem Cells from Patients with Multiple Myeloma

Abstract

Objective: Patient-specific induced pluripotent stem cells (iPSCs) have potential in human disease modeling and regenerative medicine. The in vitro phenotype of disease-specific iPSC-derived cells can be used to bridge the knowledge gap between clinical phenotype and molecular or cellular pathophysiology and to understand the pathology of diseases, along with further applications, such as creating new strategies for drug screening or developing novel therapeutic agents. The aim of our study was to generate iPSCs from multiple myeloma (MM) patients.

Materials and methods: Mesenchymal stem cells (MSCs) isolated from MM patients were induced for pluripotency via the Sendai virus. Fibroblasts were used as a control. Microscopic analysis was performed daily. For colony selection, live staining was done using alkaline phosphatase staining. Reprogramming experiments were confirmed by flow cytometry, immunofluorescence (IF) staining, and gene expression analyses. To confirm the spontaneous differentiation potential, an in vitro embryonic body (EB) formation assay was performed.

Results: Fibroblasts and MSCs obtained from MM patients were reprogrammed using the Sendai virus, which contains reprogramming vectors with the four Yamanaka factors, Oct3/4, Sox2, Klf4, and c-Myc. Microscopic analysis revealed that the generated iPSCs possessed classical embryonic stem cell-like morphological characteristics. Reprogramming experiments further showed that both cell lines can be reprogrammed up to the pluripotent stage, which was confirmed by flow cytometry, IF staining, and gene expression analyses. Spontaneous differentiation potential was confirmed by in vitro EB formation assays.

Conclusion: iPSCs have been successfully obtained from MM patients for the first time. These cells could clarify the molecular mechanisms behind this disease.

Keywords: Multiple myeloma; Sendai virus; Induced pluripotent stem cells; Mesenchymal stem cells.

Figure 1

Experimental timeline for the reprogramming experiment for MM-MSCs.

Figure 2

Development of the first iPSC colonies produced after Sendai virus transfection was monitored. A) On the 6^th day after transfection, B) 7^th day after transfection, C) 8^th day after transfection, and D) 9^th day after transfection. Scale bars: 50 μm (A, B, C, and D). iPSC: Induced pluripotent stem cell.

Figure 3

Development of new iPSC colonies obtained by mechanical passaging method was observed in culture plates. A, B, C, D, E, F, G, and H) iPSC colonies cultured in the feeder layer were picked up and cultured under feeder-free conditions. I and J) Microscopic views of iPSC colonies in the culture plate, not feeder free, are monitored. A) 2^nd day of P0, B) 14^th day of P0, C) 3^rd day of P1, D)4^th of P1, E) 2^nd day of P2, F) 5^th day of P2, G) 5^th day of P3, H) 7^th day of P3, I) 2^nd day of P0 on Geltrex, J) 5^th day of P0 on Geltrex. Scale bars: 20 μm (A), 100 μm (B, D), 200 μm (C, F, G, H, I, and J), 50 μm (E). iPSC: Induced pluripotent stem cell.

Figure 4

Combined images of light and fluorescent microscopes in which produced iPSC colonies reacting positively with ALP are observed. A and D) Brightfield; B and E) FITC; C and F) overlay. Scale bars: 200 μm. iPSC: Induced pluripotent stem cell; ALP: alkaline phosphatase.

Figure 5

Flow cytometric analysis of pluripotency marker antigens (SSEA-4, Tra-1-81, and Oct 3/4) in normal fibroblast iPSCs and MM-MSCs-iPSCs. iPSC: Induced pluripotent stem cell.

Figure 6

Immunofluorescence staining of pluripotency marker antigens Oct4 (A, B; green), TRA1-60 (C, D; red), Nanog (E, F; green), TRA1-81 (G, H; red) and Sox2 (I, J; red) in fibroblasts and MM-MSCs-iPSCs. All markers were positive for the colonies. Scale bars: 20 μm (A), 100 μm (B, D), 200 μm (C, F, G, H, I, and J), 50 μm (E).

Figure 7

Pluripotent gene expression analysis of colonies formed after viral infection. Gene expressions were monitored for 3 weeks (w1, w2, w3). As a result, it was seen that iPSCs express pluripotent markers.

Figure 8

Measurement of the expression of pluripotent genes by real-time polymerase chain reaction. The HPRT gene was used as the reference gene. Gene expression values are expressed according to fold values relative to the HPRT gene. iPSCs obtained from fibroblasts were used as a control in gene expression analysis.

Figure 9

Embryoid body (EB) formation of iPSCs. A, B) EBs generated from fibroblasts. C, D) EBs generated from MM-MSCs. Scale bars: 200 μm (A and C), 100 μm (B and D).