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iPSCs - Quick Stepping Towards the Clinic?

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Review of “A Facile Method to Establish Human Induced Pluripotent Stem Cells From Adult Blood Cells Under Feeder-Free and Xeno-Free Culture Conditions: A Clinically Compliant Approach” from Stem Cells Translation Medicine by Stuart P. Atkinson

Induced pluripotent stem cells (iPSCs) represent one of the most promising therapeutic tools which we possess today. However, several technical problems still exist which impede their clinical application and must be overcome before this great potential can be translated into a firm reality. A recent paper in Stem Cells Translational Medicine from the group of Linzhao Cheng (Johns Hopkins) has attempted to solve some of these problems through enhancing the strategy used to generate human iPSCs from the adult peripheral blood mononuclear cell (PB-MNC) which represents an easy to expand abundant cell source. They report on their work using EBNA1/ OriP episomal vectors and new culture systems to generate iPSCs in a constant and reliable manner under clinically compliant conditions [1].

The vectors used represented a modified pCEP4 plasmid backbone containing the EBNA1/OriP replicon for non-integrating vectors containing the spleen focus-forming virus (SFFV) long terminal repeat promoter/enhancer, which functions better in hematopoietic cells [2]. This controlled the expression a single transcript of two linked cDNAs, to produce two proteins linked by a 2A self-cleavage peptide. The researchers used three distinct modified (M) episomal vectors for one time nucleofections; MOS (OCT4 and SOX2), MMK (c-MYC and KLF4), and either MBX (BCL-Xl alone) or GBX (GFP and BCL-Xl) as shown in the accompanying figure. EBNA1 expression from the plasmid backbone [3, 4] and BCL-XL expression from the third vector [5] are both able to significantly boost PB-MNC reprogramming. The researchers then compared these newly engineered vectors (the pEV combination) to the pEB combination, a combination consisting of the pEB-C5 episomal vector (expressing Oct4, Sox2, Klf4, c-Myc, and Lin28 genes) and the pEB-Tg episomal vector (expressing SV40 large T antigen to enhance reprogramming), previously successfully used to reprogram adult PB-MNCs. Encouragingly, they found that the pEV combination reprogrammed adult PB-MNC samples at a significantly higher efficiency than the pEB combination when using MEF feeders and MEF conditioned medium (CM). Application of this reprogramming methodology also allowed the generation of iPSCs from the previously refractory PB-MNCs of patients with paroxysmal nocturnal hemoglobinuria (PNH). Replacement of the MBX vector with the GBX vector aided in the selection of fully reprogrammed iPSC colonies that silenced transgene expression (GFP-negative) and so enhanced iPSC production from human patient samples.

To further enhance the suitability of resultant iPSCs to clinical applications, the group successfully replaced MEFs and CM with the simplified and xeno-free E8 medium and vitronectin substrate throughout. Indeed this change actually further enhanced the efficiency of reprogramming. Furthermore, the replacement of bovine serum albumin and fetal bovine serum used in the human erythroblast culture by human albumin present in FDA-approved Plasbumin-25 and BMP-4 still permitted high levels or reprogramming.

Altogether, the application of the new vectors and the new culture conditions allowed the generation of iPSCs from 200 donor samples, which all formed teratomas with three germ layer structures and maintained a normal karyotype when examined at passage 15.

Overall, this represents an easy and efficient method to produce patient specific iPSCs from an abundant and easy to harvest cell population under feeder-free and xeno-free conditions towards their clinical application. The authors hope to confirm whether this method may function efficiently for other cell types (e.g. commonly used fibroblasts and keratinocytes) and to adapt the nucleofection technique to allow for the use of a smaller starting cell number. Furthermore, enhanced analysis of the abilities of the resultant iPSCs to differentiate to specific lineages and their mutational load may also prove that this technique represents a safe and attractive method of generating the large amounts of somatic cells required for cell replacement therapies.

References

  1. Chou BK, Gu H, Gao Y, et al. A facile method to establish human induced pluripotent stem cells from adult blood cells under feeder-free and xeno-free culture conditions: a clinically compliant approach. Stem Cells Translational Medicine 2015;4:320-332.
  2. Meng X, Neises A, Su RJ, et al. Efficient reprogramming of human cord blood CD34+ cells into induced pluripotent stem cells with OCT4 and SOX2 alone. Molecular therapy : the journal of the American Society of Gene Therapy 2012;20:408-416.
  3. Chen G, Gulbranson DR, Hou Z, et al. Chemically defined conditions for human iPSC derivation and culture. Nature methods 2011;8:424-429.
  4. Okita K, Yamakawa T, Matsumura Y, et al. An efficient nonviral method to generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells. Stem Cells 2013;31:458-466.
  5. Su RJ, Baylink DJ, Neises A, et al. Efficient generation of integration-free ips cells from human adult peripheral blood using BCL-XL together with Yamanaka factors. PLoS One 2013;8:e64496.