Differentiation of CD133+ stem cells from amyotrophic lateral sclerosis patients into preneuron cells

Abstract

Improvements in quality of life and life expectancy have been observed in amyotrophic lateral sclerosis (ALS) patients transplanted with CD133(+) stem cells into their frontal motor cortices. However, questions have emerged about the capacity of cells from these patients to engraft and differentiate into neurons. The objective of this work was to evaluate the in vitro capacity of CD133(+) stem cells from 13 ALS patients to differentiate into neuron lineage. Stem cells were obtained through leukapheresis and cultured in a control medium or a neuroinduction medium for 2-48 hours. Expression of neuronal genes was analyzed by reverse transcription polymerase chain reaction (RT-PCR) and immunohistochemical techniques. Fluorescence microscopy demonstrated that CD133(+) stem cells from ALS patients incubated for 48 hours in a neuroinduction medium increased the detection of neuronal proteins such as nestin, β-tubulin III, neuronal-specific enolase, and glial fibrillary acidic protein. RT-PCR assays demonstrated an increase in the expression of β-tubulin III, nestin, Olig2, Islet-1, Hb9, and Nkx6.1. No correlation was found between age, sex, or ALS functional scale and the CD133(+) stem cell response to the neuroinduction medium. We conclude that CD133(+) stem cells from ALS patients, like the stem cells of healthy subjects, are capable of differentiating into preneuron cells.

Figure 1.

Representative images of immunofluorescence detection of β-tubulin III in CD133⁺ stem cells from an amyotrophic lateral sclerosis patient. (A): Positive staining for β-tubulin III was detected around the nucleus (green) after 2 hours of incubation in control medium. After 2 h of incubation in NIM, distribution was detected across the cytoplasm. The nucleus was labeled with 4′,6-diamidino-2-phenylindole (blue). Scale bar = 20 μm. (B): Graphic representation of the average expression of β-tubulin III in patient cells detected by reverse transcription-polymerase chain reaction (RT-PCR) after 2–48 hours. Bars represent the SD of patient values. (C): Representative images of agarose electrophoresis of RT-PCR products for β-tubulin III. Lanes show molecular weight markers (M), cells grown in Dulbecco's modified Eagle's medium (DMEM-F12) (C1), cells grown in DMEM-F12 supplemented with 5% fetal bovine serum (FBS) (C2), and cells incubated for 2–48 hours in DMEM-F12 supplemented with 5% FBS and inducers. Abbreviations: C1, control medium 1; C2, control medium 2; Ctrl, control medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, molecular weight markers; NIM, neuroinduction medium.

Figure 2.

Representative images of immunofluorescence detection of nestin in CD133⁺ stem cells from an amyotrophic lateral sclerosis patient. (A): Positive staining for nestin was detected around the nucleus (green) after 2 hours of incubation in control medium. After 2 hours of incubation in NIM, distribution was detected across the cytoplasm. The nucleus was labeled with 4′,6-diamidino-2-phenylindole (blue). Scale bars = 20 μm. (B): Graphic representation of the average expression of nestin in patient cells detected by reverse transcription-polymerase chain reaction (RT-PCR) after 2–48 hours. Bars represent the SD of patient values. Under the graph are representative images of agarose electrophoresis of RT-PCR products for nestin. Lanes show molecular weight markers (M), cells grown in Dulbecco's modified Eagle's medium (DMEM-F12) (C1), cells grown in DMEM-F12 supplemented with 5% fetal bovine serum (FBS) (C2), and cells incubated for 2–48 hours in DMEM-F12 supplemented with 5% FBS and inducers. Abbreviations: C1, control medium 1; C2, control medium 2; Ctrl, control medium; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, molecular weight markers; NIM, neuroinduction medium.

Figure 3.

Representative images of immunofluorescence detection of neuronal-specific enolase (NSE) and glial fibrillary acidic protein (GFAP) (green) in CD133⁺ stem cells from an amyotrophic lateral sclerosis patient after 2 hours of cultivation in control medium and neuroinduction medium (NIM). (A): NSE was not detected in cells incubated in control medium. (B): Moderate staining for NSE was observed around the nucleus in cells incubated in NIM; nucleus was labeled with 4′6-diamidino-2-phenylindole (DAPI) (blue). (C): Cells incubated with control medium showed positive staining against GFAP around the nucleus. (D): Cells incubated in NIM showed a cytoplasmic distribution of GFAP; nucleus was labeled with DAPI (blue). Scale bars = 20 μm.

Figure 4.

Reverse transcription-polymerase chain reaction (RT-PCR) for Olig2 expression in induced CD133⁺ stem cells from amyotrophic lateral sclerosis patients. (A): Graphic representation of the band intensity from electrophoresis of RT-PCR products for Olig2 after 12–48 hours of incubation with neuroinduction medium (NIM). (B): Agarose gel imaging of gene expression after 2–48 hours of cell incubation in control medium and NIM. Positive expression was detected after 12 hours and was increased after 48 hours. (C): Graphic representation of the band intensity after electrophoresis of RT-PCR products for Olig2 expression in CD133⁺ stem cells from 13 patients after 48 hours of incubation in NIM. (D): Agarose gel imaging of gene expression after 48 hours of cell incubation in NIM in cells from 11 patients. Abbreviations: C1, control medium 1; C2, control medium 2; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; M, molecular weight markers; P, patient.

Figure 5.

Reverse transcription-polymerase chain reaction for Islet-1, Nkx6.1, and Hb9 expression in induced CD133⁺ stem cells from an amyotrophic lateral sclerosis patient. Shown is agarose gel imaging of gene expression after 48 hours of cell incubation in control medium and NIM. Abbreviations: C, control medium; NIM, neuroinduction medium.