Scientists report on the development of new methods for generating large numbers of neural precursor cells from both human embryonic stem cells (hESC) and mouse embryonic fiboblasts using a cocktail of small molecule inhibitors and reprogramming factors in chemically defined growth media.
The protocol for hESCs resulted in the generation of primitive neural stem cells (pNSCs) that were capable of self-renewing, retained high neurogenic potential, and could be triggered to differentiate into a range of neural cell types. The method used to generate NPCs from mouse embryonic fibroblasts critically bypassed the pluripotent cell stage. It triggered the conversion of fibroblasts directly into NPCs in one step.
Sheng Ding, Ph.D., at the University of California Los Angeles’ (UCLA) Gladstone Institute of Cardiovascular Disease, and colleagues claim that compared with other techniques for generating induced neurons (iN) from fibroblasts, their transdifferentiated NPCs have the distinct advantage of being expandable in vitro and retaining the ability to give rise to multiple neuronal subtypes and glial cells.
The researchers claim their achievements will finally allow the production of stable, renewable neural stem cells or downstream products in quantities sufficient for clinical trials and eventually therapeutic applications. Both sets of research by Dr. Ding’s teams are published in the same early online edition of PNAS.
“To our knowledge, this is the fastest and most efficient method so far to produce neural stem cells from hESCs,” the authors claim. “In addition, pNSCs differ from previously reported hESC-derived NSCs in that they represent the primitive prerosette neuroepithelium that has never been long-term expanded in vitro before.”
A major challenge for hESCs dfferentiation is overcoming the current inability to effectively capture and, in the long term, stably expand primitive lineage-specific stem/precursor cells that retain broad differentiation potential and, more importantly, developmental stage-specific differentiation propensity, Dr. Ding and colleagues write in their paper, which is titled “Rapid induction and long-term self-renewal of primitive neural precursors from human embryonic stem cells by small molecule inhibitors.”
In the case of inducing neural precursors from hESCs, in particular, robust, chemically defined conditions for the long-term maintenance of primitive neural epithelial precursor cells has been lacking. The researchers cultured the cells with human leukemia inhibitory factorLIF (hLIF) and two small molecules, CHIR99021 (CHIR) and SB431542 (SB), under chemically defined conditions. CHIR is an inhibitor of glycogen synthase kinase 3 (GSK3) and can activate canonical Wnt signaling, which has been implicated in ESC self-renewal, they note. SB is an inhibitor of transforming growth factor β (TGF-β) and activin receptors and has been implicated in mesenchymal-to-epithelial transition and reprogramming.
Under these conditions the resulting hESC-derived pNSCs were capable of long-term self renew over serial passages with SB, CHIR, and hLIF while maintaining their stable pNSC phenotype, the researchers report. pNSCs cultured on Matrigel exhibited typical epithelial morphology and positive ALP staining. Immunostaining showed that both the early-passage and late-passage pNSCs continued to stably express genes recently identified as rosette-type NSC markers as well as CNS neural stem cell markers, anterior neural markers, and midrain markers, the authors write.
Importantly, the pNSCs could be triggered to differentiate into a range of mature neural cell types. Analysis of differentiated neurons indicated they could fire action potentials and form functional synapses. When the pNSC were transferred into the brains of neonatal mice, they distributed into many brain areas and were found to express differentiated neuronal markers.
The researchers’ parallel work with mouse embryonic fiboblasts (MEF) sought to induce the conversion of somatic cells into lineage-specific stem/ progenitor cells of another germ layer in one step, bypassing the intermediate pluripotent stage. They achieved this through transient induction of the four reprogramming factors Oct4, Sox2, Klf4, and c-Myc. This led to the transdifferentiation of fibroblasts into functional NPCs with appropriate signaling inputs. The results are published in a paper titled, “Direct reprogramming of mouse fibroblasts to neural progenitors.”
The researchers claim their MEF transdifferentiation process is highly specific and efficient, reaching completion within 9–13 days. Importantly, the resulting NPCs are expandable progenitor cells, unlike iN cells generated by other approaches, they stress. Moreover, the NPC technique uses general reprogramming factors rather than lineage-transcription factors.
In fact, the same research team recently triggered the direct reporgramming of MEFs into spontaneously contracting patches of differentiated cardiomyocytes. These results were published in the online edition of Nature Cell Biology in January. “Changing the duration of transgene expression and culture conditions may allow a transient, plastic developmental state established early to effectively serve as a cellular platform for transdifferentiation toward various lineages,” they claim.
The new approach could also lead to better patient-derived cell models of certain neruodegenerative disorders, they add. Currently, iPSCs derived from patients with late-onset neurological diseases do not readily recapitulate disease phenotypes when redifferentiated. One potential reason for this is that complete reprogramming of the cells to a pluripotent state fesibly resets certain epigenetic hallmarks of the disease state, the authors suggest.
“Considering this negative correlation between the degree of reprogramming and the manifestation of a particular disease phenotype, we think that the transdifferentiated NPCs—which are derived with limited reprogramming, thus avoiding the establishment of a pluripotent state—could eventually prove to be a better-suited model system than iPSCs when studying such late-onset diseases.”