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The Controlled Generation of Functional Basal Forebrain Cholinergic Neurons from Human Embryonic Stem Cells



From the May 2011 Issue of Stem Cells

Paper Commentary by Stuart P. Atkinson

Dementia, and specifically Alzheimer's disease (AD), may be among the most costly diseases for society in Europe and the United States, and with the continual increase in the aged population promises only to get worse, with 1 in 85 persons worldwide of all ages predicted to suffer by the year 2050 (Brookmeyer et al). Therefore, treatment for this type of disease, in particular cell replacement therapy, is highly sought after. A constant feature of AD is the loss of basal forebrain cholinergic neurons (BFCNs) and is associated with problems in spatial learning and memory, and therefore a source of these cells for possible replacement therapy would be of great advantage. Using data known about BFCNs arising from studies of the mouse median ganglionic eminence (MGE), the laboratory of John A. Kessler at the Northwestern University's Feinberg School of Medicine, Chicago, Illinois, USA set out to determine a suitable source of cells for cell replacement therapy. This study (Bissonnette et al) is published in the May 2011 edition of Stem Cells.

Previously published in vitro protocols (Lee et al) were utilised to first generate neural progenitors from hESC (hNPCs), which were then treated with diffusible ligands known to be expressed in the developing mouse MGE in an attempt to attain BFCNs. hNPCs were first pre-treated with SHH and FGF8, which are necessary for the patterning of the developing neural tube and specification of the primordial forebrain (Lee et al), which led to an increase in FORSE1, a forebrain progenitor marker, after which cells were dissociated and treated with BMP9 (GDF2). BMP9 has previously been shown to be vitally important for the formation of BFCNs (Lopez-Coviella et al). At day 16 of culture, Q-PCR analyses showed a significant increase in the expression of markers for the BFCN lineage (ChAT, p75, TrkA and AChE), while markers of other cholinergic neurons remained low (SST and NOS). Further, HB9 (MNX1) was lacking from these cells suggesting that these cholinergic neurons were not motor neurons, while markers for astroglial (GFAP) and oligodendroglial (MBP) lineages were also absent. Overall, this shows that precise temporal treatment of hNPCs is sufficient to generate a population of BCNF-like cells.

The research then moved on to study whether the specific overexpression of genes could be used to generate BFCNs, utilising LHX8 and GBX1, both of which are downstream effectors of BMP9 signalling. FGF8/SHH-pre-treated hNPCs were targeted with vectors containing LHX8, GBX1 and eGFP to allow for efficient FACS-based purification of nucleofected cells. Subsequent analysis, after 19 days of culture, showed that nearly all cells became ChAT positive neurons, while around 19% of these cells were dual ChAT and p75 positive suggesting their similarity to the mitogen-differentiated putative BCNF-populations. Subsequent analysis showed that LHX8 RNAi undertaken at the same time as BMP9 treatment on FGF8/SHH treated cells led to a greatly reduced level of BFCN marker expression at day 16 of differentiation, suggesting that the two differentiation techniques alter the same pathway.

Functionality of these cells was then demonstrated using mouse ex vivo entorhinal-hippocampal cortical slice cultures. Putative BFCN cells were cultured with cortical slices for 7 – 19 days, which revealed their capacity to migrate, extend axonal projections within the cortical slice and, importantly, to form functional synapses with explanted cortical tissue. BFCN cells expressed markers of mature neurons and the authors also demonstrate their ability to receive synaptic transmission. Most importantly, these cells were shown to be able to generate cholinergic synapses and displayed appropriate electrophysiological function.

Overall this suggests that utilising the protocols set out within this study, the derivation of a predominantly pure and functional BFCN population for the treatment of neurological disease, such as AD, from human pluripotent cells is possible. Also, the production of these cells and the delineation of the pathways involved in this differentiation process may be useful for in vitro screening of agents which may lead to the discovery of survival factors for the maintenance of BFCNs in vivo. Assessment of the capability of donor BFCN cells to integrate in a functional manner in animal models of dementia will be key in determining their true restorative potential for treatment of this debilitating group of diseases in humans.



Forecasting the global burden of Alzheimer's disease".
Brookmeyer, R; Johnson, E; Ziegler-Graham, K; Arrighi, HM
Alzheimer's and Dementia. 2007 July;3 (3): 186–91.

The controlled generation of functional Basal forebrain cholinergic neurons from human embryonic stem cells.
Bissonnette CJ, Lyass L, Bhattacharyya BJ, Belmadani A, Miller RJ, Kessler JA.
Stem Cells. 2011 May;29(5):802-11.

Stem cells act through multiple mechanisms to benefit mice with neurodegenerative metabolic disease.
Lee JP, Jeyakumar M, Gonzalez R, Takahashi H, Lee PJ, Baek RC, Clark D, Rose H, Fu G, Clarke J, McKercher S, Meerloo J, Muller FJ, Park KI, Butters TD, Dwek RA, Schwartz P, Tong G, Wenger D, Lipton SA, Seyfried TN, Platt FM, Snyder EY.
Nat Med. 2007 Apr;13(4):439-47. Epub 2007 Mar 11.

Bone morphogenetic protein 9 induces the transcriptome of basal forebrain cholinergic neurons.
Lopez-Coviella I, Follettie MT, Mellott TJ, Kovacheva VP, Slack BE, Diesl V, Berse B, Thies RS, Blusztajn JK.
Proc Natl Acad Sci U S A. 2005 May 10;102(19):6984-9.