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Understanding Gene Expression after Engraftment of iPSC-Derived Cells

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Review of “Gene profiling of human iPSC-derived astrocyte progenitors following spinal cord engraftment” from Stem Cell Translational Medicine by Stuart P. Atkinson.

Human induced pluripotent stem cells (hiPSCs) have become a popular cell of choice for the future treatment of neurological disorders [1], with one important study showing the ability of hiPSC-derived oligodendrocyte progenitors to extensively remyelinate and rescue a mouse model of congenital hypomyelination [2]. Studies of certain disease states (amyotrophic lateral sclerosis (ALS), Rett syndrome, and Huntington’s disease [3]) have underlined the importance of another neural cell type – the astrocyte, which acts as a support cell for neurons. A team from Johns Hopkins University School of Medicine, Maryland, USA led by Nicholas J. Maragakis now has studied the transplantation of hiPSC-derived astrocytes into the rat spinal cord to evaluate their therapeutic use and their use in disease modelling. They report their findings in Stem Cells Translational Medicine on the engraftment, survival, maturation, and expression profile [4].

 

The researchers differentiated hiPSCs towards astrocytes according to a recently published protocol [5]; neuralization using BMP4 and activin/TGF-b antagonists, followed by caudalization and ventralization using retinoic acid and sonic hedgehog before culture in glial differentiation media at day 30. The study found CD44+ CD184+ astrocyte progenitors between day 50-60 while S100b, and Nestin expression by day 100. At this stage, cultures were mainly astrocyte progenitors and GFAP+ astrocytes, with no NG2 + or Olig2 + oligodendrocytes and rare Tuj1 + neurons. At this point, the researchers transplanted progenitor cells bilaterally into the ventral horn of the cervical spinal cord of adult wild-type rats. Cells demonstrated limited rostral-caudal migration (1mm) at 2 and 7 weeks with some cells (0.5%) surviving up to 12 weeks, and as apoptosis was not observed after 2 weeks, this suggests that most cells die or do not engraft during this time point.

 

Of the cells which did engraft, 80% were astrocytes (GFAP+) at 2 weeks, while at 7 and 12 weeks, this rose to 90% of grafted cells. Additionally, cells expressed the mature astrocyte markers Aquaporin 4, while the researchers only observed a few Olig2 + and no Tuj1 + cells, although LCN2, a recently identified marker for reactive astrocytes [6], was not observed. As many markers did not have human specific antibodies for protein detection, further analyses used NanoString technology for detection of small amounts of human-specific RNAs [7], using cells taken from various regions in the cervical spinal cord near the injection site. This allowed for the detection of a wide array of astrocyte lineage genes expressed (GFPA, Aquaporin4, Connexin 43, MLC1 and EAAT1) by the human cells in the spinal cord; being highest at the transplant site and decreased rostrally and caudally. NESTIN and SOX2 (progenitor cell markers) were also found, suggesting that some of the transplanted cells remain in an immature form in vivo. The study made further comparisons between cells just before (in vitro) and just after (in vivo) to understand if and how they differentiate and mature in vivo. This analysis found that while in vitro the astrocyte progenitors expressed high levels of progenitor/stem cell markers, after transplantation in vivo the cells increased expression of astrocyte-specific gene expression [8].

 

The authors have shown that astrocyte progenitors can engraft into the spinal column and effectively demonstrate the differentiation of these cells almost entirely towards astrocytes, taking cues from their transplanted niche, using a novel gene-profiling technology. Furthermore, the cells exhibit excellent long-term survival in vivo. This suggests that these cells could have great utility in cell transplant therapy as well as in the modelling of neurodegenerative disease. However, further work is required to enhance the initial engraftment/survival of cells, which could improve the therapeutic relevance of these cells.

References

  1. Lukovic D, Moreno Manzano V, Stojkovic M, et al. Concise review: human pluripotent stem cells in the treatment of spinal cord injury. Stem Cells 2012;30:1787-1792.
  2. Wang S, Bates J, Li X, et al. Human iPSC-derived oligodendrocyte progenitor cells can myelinate and rescue a mouse model of congenital hypomyelination. Cell Stem Cell 2013;12:252-264.
  3. Molofsky AV, Krencik R, Ullian EM, et al. Astrocytes and disease: a neurodevelopmental perspective. Genes Dev 2012;26:891-907.
  4. Haidet-Phillips AM, Roybon L, Gross SK, et al. Gene Profiling of Human Induced Pluripotent Stem Cell-Derived Astrocyte Progenitors Following Spinal Cord Engraftment. Stem Cells Transl Med 2014;
  5. Roybon L, Lamas NJ, Garcia-Diaz A, et al. Human stem cell-derived spinal cord astrocytes with defined mature or reactive phenotypes. Cell Rep 2013;4:1035-1048.
  6. Bi F, Huang C, Tong J, et al. Reactive astrocytes secrete lcn2 to promote neuron death. Proc Natl Acad Sci U S A 2013;110:4069-4074.
  7. Geiss GK, Bumgarner RE, Birditt B, et al. Direct multiplexed measurement of gene expression with color-coded probe pairs. Nat Biotechnol 2008;26:317-325.
  8. Cahoy JD, Emery B, Kaushal A, et al. A transcriptome database for astrocytes, neurons, and oligodendrocytes: a new resource for understanding brain development and function. J Neurosci 2008;28:264-278.