Immature cells transplanted into mouse brains matured and formed connections with blood vessels.
Scientists have developed the chemically defined conditions necessary to prompt human embryonic stem cells (hESCs) and human pluripotent stem cells (hPSCs) to differentiate into immature astrocytes. The University of Wisconsin-Madison team claims the immature astrocytes readily develop into mature astrocytes when implanted in the mouse brain, by forming connections with blood vessels. Writing in Nature Biotechnology, Su-Chung Zhang, Ph.D., and colleagues, report on their achievement in a paper titled “Specification of transplantable astroglial subtypes from human pluripotent stem cells.”
Astroglial cells are the most abundant cell type in the human brain and spinal cord, and different subtypes have been shown to play essential roles in functions such as the formation and insulation of synapses, and the maintenance of a homeostatic environment, the Wisconsin team reports. Abnormalities in astroglial cells have also been linked with a range of human pathologies including neurodegenerative diseases. However, generating these cell types from human pluripotent stem cells (hPSCs) has to date remained elusive.
Dr Zhang’s team has developed a chemically defined differentiation system for generating immature astrocytes from HPSCs including ESCs and iPSCs. To achieve this the hPSCs were differentiated to neuroepithelial cells, specified to regional progenitors, and then expanded. The researchers claim that in contrast with existing protocols for differentiating astroglial cells from human neural stem cells or fetal tissues, which have limited expansion capacities, the new approach allows the generation of a nearly pure population of astroglial progenitors that can be readily expanded to large quantities. Expansion of astroglial progenitors from the different hESC and iPSC cell lines displayed similar efficiencies.
The resulting cultures contained minimal neurons and no immune cells, and the hPSC-derived astroglial progenitors could be expanded continuously for at least eight months, and survive freeze-thaw cycles. Importantly, the authors report, the hPSC-derived immature astrocytes could be triggered to differentiate into region-specific astroglia using a neuroepithelial cell patterning and differentiation approach similar to that for generating region-specific neuronal cell types from hPSCs.
Evaluation of the hPSC-derived astrocytes confirmed that the cells expressed astroglial-specific marker genes, and demonstrated functional properties such as glutamate uptake and the promotion of synaptogenesis. The researchers calculated that if one hPSC was differentiated to neuroepithelial cells, converted to glial cells, and then expanded in suspension, an estimated 2.8 x 1012 immature astrocytes could be generated in about six months, even when taking cell loss into account.
Interestingly, differentiation of human ESCs into GFAP+ astroglia took at least 12 weeks, which is substantially slower than the two weeks taken to derive cells from mouse ESCs, the authors point out. However, this increased time corresponds to astroglial development in the human brain. Astroglial progenitors or immature astrocytes could be identified by the expression of relevant genes such as S100β and GFAP at four to eight weeks after hPSC differentiation, and more mature astrocytes were evident by 8-12 weeks. hPSC-derived day 210 astroglia expressed high levels of additional cell-specific genes.
To determine whether the hPSC-differentiated cells maintain their identity in vivo, immature astrocytes were transplanted into the brains of experimental neonatal mice. Up to 100 days after engraftment, the human cells were found as clusters adjacent to the implantation site, and were migrating into the corpus callosum. The cells also demonstrated the correct gene-expression markers according to their location in the brain. Moreover, hESC-derived astroglia were capable of maturing and participating in blood-brain barrier structure formation in the mouse brain, and retained unique features characteristic of human astrocytes, even though the cells had been implanted into a different species.
“Our ability to derive and expand an enriched population of astroglial progenitors, as well as differentiating them to immature astrocytes, opens up an avenue for studying the role of human astrocytes in the normal and diseased brain and for the development of transplantation therapy in neurological diseases,” the authors conclude. “In addition, astoglial cells derived from patient-specific iPSCs offer yet another approach for therapeutic discovery.”