Combinatorial programming of human neuronal progenitors using magnetically-guided stoichiometric mRNA delivery

Abstract

Identification of optimal transcription factor expression patterns to direct cellular differentiation along a desired pathway presents significant challenges. We demonstrate massively combinatorial screening of temporally-varying mRNA transcription factors to direct differentiation of neural progenitor cells using a dynamically-reconfigurable magnetically-guided spotting technology for localizing mRNA, enabling experiments on millimetre size spots. In addition, we present a time-interleaved delivery method that dramatically reduces fluctuations in the delivered transcription factor copy numbers per cell. We screened combinatorial and temporal delivery of a pool of midbrain-specific transcription factors to augment the generation of dopaminergic neurons. We show that the combinatorial delivery of LMX1A, FOXA2 and PITX3 is highly effective in generating dopaminergic neurons from midbrain progenitors. We show that LMX1A significantly increases TH-expression levels when delivered to neural progenitor cells either during proliferation or after induction of neural differentiation, while FOXA2 and PITX3 increase expression only when delivered prior to induction, demonstrating temporal dependence of factor addition.

Roles of Transcription Factors in the Development and Reprogramming of the Dopaminergic Neurons

“…As previously stated, Lmx1a is more important in developing mdDA, and Lmx1b acts as a compensatory factor for Lmx1a. It was reported that Lmx1a, Foxa2 and Pitx3 could increase Th expression when delivered to NPCs during neural proliferation, but only Lmx1a increases expression after induction, which is more efficacious [ 15 ]. When embryoid body cells (EBCs) transplanted into the adult intact SNpc, they differentiate into NPCs without acquiring DA phenotypes.…”

“…After decades of research, it was found that stem cell therapy in PD can selectively slow the disease progression, and, more importantly, it may potentially resolve the root problems in the future [ 14 ]. Thus, tremendous effort and research have been focused on the vital step for stem cell therapy of generating functional mdDA neurons by cell reprogramming from stem cells or other non-neural cells [ 15 , 16 , 17 ]. As a result, a serial of transcription factors (TFs) has been characterized, which are essential elements in mdDA neuronal development, differentiation, specification, and transmitter synthesis.…”

“…Ascl1, Ngn2, and NeuroD1 are neural progenitor cell markers, whereas Pitx3 and Nurr1 are mdDA neuron-specific markers. These TFs have long been known to involve the differentiation, maturation, and maintenance of mdDA [ 15 , 16 , 17 ]. Additionally, some neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and glial-cell-derived neurotrophic factor (GDNF), also play an essential role in this process.…”

Transcription Factor-Based Fate Specification and Forward Programming for Neural Regeneration

“…Other midbrain-specific TFs that were tested for their potency to instruct dopaminergic fates include (i) Foxa2, which, when overexpressed in mouse primary midbrain-derived and ESC-derived NPCs, was found to boost the derivation of TH-positive neurons (Kittappa et al, 2007), (ii) Lmx1a, which efficiently specifies murine Shh-and Fgf8-treated (Andersson et al, 2006;Panman et al, 2011) as well as human ESC-derived NPCs toward a dopaminergic fate (Friling et al, 2009), and in combination with Foxa2 and Barhl1 drives dopaminergic differentiation from Fgf8-and CHIR99021-exposed murine ESCderived NPCs (Kee et al, 2017), (iii) En1 and (iv) Otx2, which were reported to drive dopaminergic differentiation of murine NPCs alone, each in combination with Lmx1a, or as a 3 TF cocktail (Panman et al, 2011), (v) Dmrt5, which does not increase overall neuronal yield after being overexpressed in mouse ESC-derived dopaminergic NPCs but specifically induces an increase of certain midbrain dopaminergic markers on RNA level (Gennet et al, 2011), and (vi) PBX1, which appears to cooperate with NURR1 promoting dopaminergic specification from human PSC-derived NPCs (Villaescusa et al, 2016). Another good example is the recently published study by Azimi et al (2018), who used magnetically guided mRNA spot delivery to screen single TFs and TF combinations for their capacity to commit human ESC-derived NPCs toward a dopaminergic fate, and revealed that transfection of FOXA2, LMX1A, and PITX3 mRNA results in an increased yield of TH-positive neurons. Combinatorial delivery of these 3 TFs at their respective most effective stage results in almost 68% of TH-and MAP2-doublepositive cells (Azimi et al, 2018).…”

“…Another good example is the recently published study by Azimi et al (2018), who used magnetically guided mRNA spot delivery to screen single TFs and TF combinations for their capacity to commit human ESC-derived NPCs toward a dopaminergic fate, and revealed that transfection of FOXA2, LMX1A, and PITX3 mRNA results in an increased yield of TH-positive neurons. Combinatorial delivery of these 3 TFs at their respective most effective stage results in almost 68% of TH-and MAP2-doublepositive cells (Azimi et al, 2018).…”