Despite the general belief that recapitulating astrocyte lineage heterogeneity is necessary for stem cell-based disease modelling and cell transplantation, the extent of astrocyte heterogeneity in different brain regions, their anatomical origins, and associated molecular signatures remain largely elusive. This knowledge gap limits the endpoint characterization of stem cell-derived astrocytes; hence, reliance on the dominant regional characteristics of the initial neural progenitor populations, with the assumption that the lineage representation of the progenitors is preserved in the derived astrocytes. By harnessing an LMX1A-based lineage tracing human iPSC line, we discovered unexpected negative selection against derivatives of LMX1A+ midbrain-patterned progenitors during astrocyte induction and progenitor expansion, highlighting the need for careful characterization of PSC-derived astrocytes and reinforce the need for a deeper understanding of the molecular landscape of astrocytes in different regions of the human brain.
Most neural progenitors used for astrocyte differentiation in this study coexpressed LMX1A and FOXA2. In the ventral midbrain, the combinatorial expression of these transcription factors defines the dopaminergic neural progenitors (Failli et al., 2002; Andersson et al., 2006). We found that astrocytes derived from LMX1A+ progenitors could only be obtained if LMX1A+ cells were purified prior to astrocyte differentiation. In contrast, astrocytes derived from bulk midbrain-patterned progenitors exhibit transcriptomic profiles of the lateral-dorsal midbrain despite LMX1A+ progenitors being the predominant starting population. Our findings demonstrate that the lineage composition of parent progenitors was not preserved during astrocyte induction or progenitor expansion. FGF is the most used inductive molecule for astrocyte differentiation from stem cells (Chandrasekaran et al., 2016). However, it is evident that FGF-expanded neural progenitors, originating either from the brain or neutralized PSCs, exhibit restricted regional competence and positional gene expression. For example, bulk-expanded human ventral midbrain neural progenitors (Jain et al., 2003), fetal forebrain or spinal cord-derived neural stem (NS) cells only give rise to GABAergic neurons (Sun et al., 2008), and lt-NES cells display an anterior hindbrain-like positional profile (Falk et al., 2012), while their antecedents, PSC-derived neural rosettes and early passage derivatives, express anterior forebrain markers (Koch et al., 2009). It is unclear whether this is due to the deregulation of the original patterning at the level of gene expression or the loss of the associated cell population (Gabay et al., 2003). In this study, because BFP+ astrocytes can be generated under the same culture conditions as purified LMX1A+ progenitors, we reasoned that the loss of their derivatives in unsorted cultures was possibly due to their differential growth capacity.
Our study highlights the need for a careful assessment of the positional identity of in vitro-derived astrocytes. A common practice in this regard is to confirm the regional identity of founder progenitors following fate-directed neural induction, with the assumption that the dominant positional features are maintained by astrocyte progeny (Krencik et al., 2011). This strategy is, at least partly, dictated by our limited knowledge of the gene expression signatures of regional and/or lineage-specific astrocytes. Hence, endpoint evaluation of PSC-derived astrocytes often relies on region-specific markers defined in the brain during the neurogenic period. For example, LMX1A and FOXA2 expression has been used as criteria for midbrain astrocytes in previous studies (Barbuti et al., 2020; Crompton et al., 2023). However, scRNA-seq of the human fetal ventral midbrain and adult substantia nigra has revealed negligible expression of these transcripts in astrocytes (La Manno et al., 2016; Agarwal et al., 2020; Kamath et al., 2022). Consistent with these findings, we did not detect LMX1A or FOXA2 in BFP+ or BFP − astrocytes. However, our analysis identified new positive and negative markers that can be used to validate astrocytes derived from the LMX1A+ lineage of ventral midbrain progenitors. Future work will benefit from in vivo validation of these putative lineage-specific markers, as a mouse analogous tracer line is available, whereas lineage tracking is not possible in humans.
In addition to the distinct transcriptomic profiles, BFP+ and BFP astrocytes may also be functionally different. Astrocytes generated from progenitors broadly patterned to the dorsal forebrain, ventral forebrain, and spinal cord have been shown to exhibit different GO enrichment profiles as well as different physiological and functional properties (Bradley et al., 2019). In comparison to BFP- and non-patterned astrocytes, the current study revealed that GO terms enriched in BFP+ astrocytes, which originated from the same progenitor giving rise to midbrain dopaminergic neurons, were closely related to various biological processes disrupted in astrocytes carrying familial Parkinson’s disease mutations (di Domenico et al., 2019; Barbuti et al., 2020; Sonninen et al., 2020). Such a distinct enrichment profile suggests that BFP+ astrocytes may be functionally adapted to support midbrain dopaminergic neurons compared with BFP− and non-patterned astrocytes. Indeed, astrocytes isolated from the ventral midbrain have been reported to exhibit stronger neurotrophic effects and the ability to reduce α-synuclein aggregation in the midbrain than cortical astrocytes in cellular and mouse models of PD (Kostuk et al., 2019; Yang et al., 2022), highlighting the importance of understanding astrocyte heterogeneity in iPSC disease modelling.
In conclusion, this study provides further evidence for the regional diversity of astrocytes and identifies a set of midbrain-enriched genes. Crucially, the transcriptomic fingerprint of human astrocytes derived from LMX1A-expressing midbrain progenitors reported here offers a much-needed resource for assessing the authenticity of stem cell-derived astrocytes in studies of Parkinson’s disease.