**1. Introduction**

Cord blood (CB) cells are commonly used for the treatment of leukemia and inherited metabolic diseases. To date, more than 20,000 bone marrow transplants have been performed on children and adults with cord blood cells, and There are more than 450,000 HLA-defined CB collections stored frozen cryoperserved form in more than 50 units public CB banks and more than 2,000 CB transplants are being performed world-wide per year. CB cells are the youngest somatic cells and in theory have no post natal DNA damage such as caused by UV or chemical irritant exposure. Therefore, our previous study thought that use to the ability to cryopreserve CB HSC long-term in bank, which conferring a unique advantage to CB cell as a suitable material for generating induced pluripotent stem (iPSC) cells for future clinical use.[1]

iPSC should be generated with methods that do not require integration of exogenous DNA, thereby reducing the chance of tumorigenicity caused by random chromosomal insertion of exogenous genes. Several non-integrating reprogramming methods using EBNA basedplasmids vector [2, 3, 4, 5], piggy-back transposons [6, 7], human artificial chromosome vectors [8], small peptides [9, 10], mRNA [11] and proteins [12] have been reported. Among the vectors employed for these experiments, the Sendai virus (SeV) vector (that lacks a DNA phase) is recognized as a potent reagent for reprogramming of somatic cells [13-15]. However, complete elimination of the SeV construct carrying reprogramming factors is a key issue to assure three germ layer differentiation of individual cells. The presence of residual reprogramming factors in transfected cells could impede differentiation and contribute to formation of tumors after implantation. Therefore, the possible presence of the SeV construct should be checked at a

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single cell level (not at a cell clump level) utilizing single cell cloning techniques in the naïve state [16-18]. Recently, feeder-free culture systems utilizing Laminin 511, LM-E8s or Matrigel have been reported for the maintenance of established iPSCs or ES cells [19-23]. The generation of iPSCs from fibroblasts on vitronectin-coated dishes and maintenance of iPSCs in chemically defined medium on vitronectin-coated dishes has been reported [23]. These studies were to characterize as substrates that support hESCs in a sustainable undifferentiated state under a xeno-free and chemical defined culture condition [20, 23]. On the other hands, multiple matrix proteins, such as laminin, vitronectin fibronectin and synthetic polymer surfaces support hESC/iPSC growth and maintenance. Most of these materials are too expensive for large-scale usage. Because, recombinants vitronectin is relatively easy to over-express and purify, we tested vitronectin in two feeder-free ES/iPS mediums. (mTeSR-1 and ReproFF2).

accordance with the manufacturer's instruction. Keep coated wells in culture medium at 37o

Generation and Maintenance of iPSCs From CD34+Cord Blood Cells on Artificial Cell Attachment Substrate

Temperature-sensitive Sendai virus vector constructs inserting four reprogramming factors (SeV18+HS-*OCT3/4/*TS⊿F, SeV18+HS-*SOX2/*TS⊿F, SeV18+HS-*KLF4/*TS⊿F, SeV(*HNL)c-MYC/*

transferred to one well of a 96-well plate in 180 µL of hematopoietic cell culture medium with 20 µL of viral supernatant containing 20 M.O.I. each of SeV constructs at 5% CO2, 37 o

medium was changed to fresh medium in the following days (15-18 hours after infection). Infected cells were cultured another three days in hematopoietic culture medium in 96-well

primate ES cell medium ReproFF2 supplemented with 5 ng/mL bFGF (ReproCELL Inc,

amount of SeV constructs in the transfected cells was reduced by incubation cells at 5% CO2,

After heat treatment, three hundred cells were resuspened in 100ml of naïve cell culture medium (see below). The cells were seeded in ten well of 96-well plate (100µl/well) pre-coated with Pronectin F. Approximately single cell in every three wells was seed in a 96-well plate. The presence of a single cell per individual well was verified by microscopic observation (phase contrast Olympus CKX31) in the same manner as single cell cloning. These cells were cultured

C in 5% O2, 5% CO2 condition in naïve cell culture medium to form dome-shape colonies. 50 mL of naïve ES/iPS cell culture medium was prepared by mixing 24 mL DMEM/F-12 medium (Invitrogen, 11320, Osaka), 24 mL Neurobasal medium (Invitrogen, 21103), 0.5 mL x100 nonessential amino acids (Invitrogen, 11140), 1 mL B27 supplement (Invitrogen; 17504044), and 0.5 mL N2 supplement (Invitrogen; 17502048). The medium also contained final concentrations of 0.5 mg/mL BSA Fraction V (Sigma, A8412, Nebraska), penicillin-streptomy‐ cin (final x 1, Nacalai, Kyoto), 1 mM glutamine (Nacalai), 0.1 mM β-mercaptoethanol (Invi‐ trogen 21985), 1 µM PD0325901 (Stemgent, 04-0006, Cambridge), 3 µM CHIR99021 (Stemgent, 04-0004), 10 µM Forskolin (Sigma, F6886) and 20 ng/mL of recombinant human LIF (Millipore;

Total RNAs from several established iPSCs lines (prime [1st, 2nd] and naïve), khES-1 (Riken

amplified Ovation Pico WTA System (Takara cat#3300–12), labeled with an Encore Biotin Module (Takara catalog number 4200–12) and then hybridized with a human Gene Chip (U133

CBCs (Riken BRC) were purified with an RNeasy Mini kit (QIAGEN),

infected CBC were seeded on a Pronectin F-coated 6-well dish in

TS15⊿F, SeV18+*GFP/*TS⊿F) were supplied by DNAVEC Corp. 1.0 x 10<sup>4</sup> CD34<sup>+</sup>

RCHEMD006B, JAPAN) to generate ES cell-like colonies under 20% O2, 37 o

5% CO2 during passaging procedure until cells are ready to be re-plated.

**2.3. Sendai virus infection and reprogramming**

plates, after which 1 x 10<sup>4</sup>

C for three days.

**2.4. Cell culture in naïve state**

38 o

at 37 o

LIF1005, Billerica).

BRC) and CD34<sup>+</sup>

**2.5. Gene chip analysis**

plus 2.0 Array Affymetrix).

C,

119

CBCs were

http://dx.doi.org/10.5772/58591

C conditions. The

C. The

In this chapter, we describe the generation of iPSC clones from cord blood cells (CBCs) in feeder-free thought naïve conditions using temperature sensitive SeV vector. Additional, human naïve iPSC culturing methods using feeder-free systems and we introduce to low-cost and stable and easy maintenance culturing methods of hESC/iPSC.
