Neural Stem Cell Properties of an Astrocyte Subpopulation Sorted by Sedimentation Field-Flow Fractionation

Abstract

Astrocytes encompass a heterogeneous cell population. Using sedimentation field-flow fractionation (SdFFF) method, different, almost pure, astrocyte subpopulations were isolated. Cells were collected from cortex of newborn rats and sorted by SdFFF to obtain different fractions, which were subjected to protein analysis and characterized by immunocytofluorescence. The behavior of the cells was analyzed in vitro, under culture conditions used for neural stem cells. These culture conditions were also applied to cells derived from an adult cortical tissue after traumatic brain injury (TBI). Finally, the astrocytic neural stem-like cells were transplanted in damaged sciatic nerve. Protein analysis indicated a high expression of glial fibrillary acidic protein (GFAP) and vimentin in fraction F3-derived cells. These cells formed neurospheres when cultured with epidermal growth factor and large colonies in a collagen-containing semi-solid matrix. Neurospheres expressed GFAP and nestin and were able in addition to generate neurons expressing MAP2 and oligodendrocytes expressing Olig2. When transplanted in a damaged nerve, cells of F3-derived neurospheres colonized the damaged area. Finally, after TBI in adult rats, cells able to form neurospheres containing a subpopulation of astrocytes expressing vimentin were obtained. Using the SdFFF method, an astrocyte subpopulation presenting stem cell properties was isolated from a newborn rat cortex and from an injured adult rat cortex. The specific activation of this astrocyte subpopulation may provide a potential therapeutic approach to restore lost neuronal function in injured or diseased brain.

Isolation of Astrocytes Displaying Myofibroblast Properties and Present in Multiple Sclerosis Lesions

“…1). It has been previously shown that F3-derived astrocytes also express vimentin [16]. In F1, among the total population of astrocytes (96%), a subpopulation of astrocytes (60%) expressed both the astrocytic marker (GFAP) and α-SM actin (F1-derived astrocytes did not express vimentin) (Figs.…”

“…After cell sorting using SdFFF, two astrocyte subpopulations were rapidly obtained: one expressing GFAP and vimentin (F3 fraction) [16] and the other one expressing both GFAP and α-SM actin (F1 fraction). This co-expression of GFAP and of α-SM actin had already been observed in the literature, particularly in primary cultures of astrocytes in embryonic [17] and postnatal [17][18][19] mice.…”

“…In conclusion, using SdFFF, beside the characterization of an astrocyte population presenting stem cell properties [16], a subpopulation able to express α-SM actin in vitro has been isolated underlining the heterogeneity of the astrocyte populations present in the CNS. In vivo, astrocytes coexpressing GFAP and α-SM actin was found in MS lesions that display significant inflammation with macrophages presenting intimal relationships with α-SM actin expressing reactive astrocytes.…”

Field-Flow Fractionation in Molecular Biology and Biotechnology

“…SdFFF shows wider applications, ranging from stem cells for cell model systems and cancer stem cells [234][235][236]. Recently, SdFFF was applied to the study of astrocyte progenitors and human-induced pluripotent stem cells (hiPSc) for the preparation of increasingly complex models for the development of new therapeutic tools in neural and brain pathologies [236][237][238][239]. Promising results were also presented for the study of cancer stem cells for the development of personalized therapies [218,[240][241][242].…”

New Method for Sorting Endothelial and Neural Progenitors from Human Induced Pluripotent Stem Cells by Sedimentation Field Flow Fractionation

“…Among FFF techniques, Sedimentation FFF (SdFFF) is a gentle, noninvasive, tagless cell sorting method. , These advantages are based on substantially limiting cell-solid phase interactions by the use of 1) an empty ribbon-like channel without a stationary phase and 2) the “hyperlayer” elution mode, a size/density driven separation mechanism. − The principle of cell separation is based on physical criteria such as size, density, shape, and rigidity − , and depends on the differential elution of species submitted to the combined action of 1) a parabolic profile generated by flowing a mobile phase through the channel and 2) an external field applied perpendicularly to the flow direction. − , In SdFFF, a multigravitational external field is generated by rotation of the separation channel in a rotor basket, constituting one of the most complex devices used in FFF separation . Since our pioneering report concerning mouse embryonic stem cells, we have demonstrated the utility of SdFFF in preparing enriched and sterile populations of normal and cancer stem cells. − …”