**4. Conclusion**

**Figure 14.** Expression of endogenous pluripotency related genes in iPSC (PFX#9) on VTN-N determined by qRT-PCR.

**Primers Size(bp)**

hOCT3/4-R TCG CTT GCC CTT CTG GCG

hSOX2-R TTG CGT GAG TGT GGA TGG GAT TGG TG

132 Pluripotent Stem Cell Biology - Advances in Mechanisms, Methods and Models

hMYC-R TCT TGG CAG CAG GAT AGT CCT T

hKLF4-R CGA TCG TCT TCC CCT CTT TG

hTERT-R ATG ACG CGC AGG AAA AAT GT

hOCT3/4-F GAA ACC CAC ACT GCA GCA GA undifferentiated

hSOX2-F GGG AAA TGG GAG GGG TGC AAA AGA GG undifferentiated

hMYC-F CGT CTC CAC ACA TCA GCA CAA undifferentiated

hKLF4-F CGC TCC ATT ACC AAG AGC TCA T undifferentiated

hTERT-F CGT ACA GGT TTC ACG CAT GTG undifferentiated

conditions.

Following, the PFX#9 cells cultured with VTN-N was the gene expression of pluripotency markers comparable iPS cells cultured on Matrigel or on SNL in Figure 12. It was found that only a recombinant vitronectin (VTN-N) can be maintained in culture for long-term feeder free

ES cell

ES cell

ES cell

ES cell

ES cell

103

151

68

77

82

In this chapter we have shown the method for generating iPSC from non-cultured CD34+ cord blood cells using feeder-free conditions. The established cell clones were characterized at a single cell level. This robust iPSC generation method will solve some of the safety concerns related to tumorigenicity ariseing from chromosomal integration of exogenous genes and/or infection hazards associated with the use of by xenogeneic biological products in the culture system. These methods will contribute to future application of iPSCs-derived cell therapy.

## **Acknowledgements**

We thank SI Nishikawa for useful discussions, N Fusaki for samples gift, and S Sawada for gene chip analysis. We thank Riken Bio Resource Center (Tukuba, Japan) for fresh CD34+ CBCs. This work was partly supported by the Regulatory Science Study for Safety issue for pluripotent stem cells of JST Tokyo Japan (2010-2014) and Adaptable and Seamless Technology Transfer Program through Target-driven R&D (A-STEP) project of JST Tokyo Japan (2013).
