**2. Experimental procedures, materials and methods**

#### **2.1. Cord blood**

CD34+ CBCscan be procured from Riken Bio Resource center (Riken BRC, Ibaraki, Japan) or other commercial suppliers. Alternatively CD34+ CBCscan be obtained from fresh cord blood using a mononuclear cells isolation kit (Lymphoprep TM, Cosmo Bio Co., Japan), and a human CD34 Micro Bead kit (Miltenyi Biotec, 130-046-702) or Auto Macs columns (Miltenyi Biotec, Germany) in accordance with the manufacturer's instruction. CD34+ CBCs were cultured in the density of 1.0 x 105 cells in two mL of hematopoietic culture medium [serum-free X-Vivo10 containing 50 ng/mL IL-6 (Peprotech, London, UK), 50 ng/mL sIL-6R (Peprotech) 50 ng/mL SCF (Peprotech), 10 ng/mL TPO (Peprotech), and 20 ng /mL Flt3/4 ligand (R&D System, Bostone, USA)] for one day prior to viral infection [23].

#### **2.2. Preparation of coated dish for feeder-free generating iPS cells**

PronectinFplus® coated-dish for reprogramming of CBCs is prepared as follows: One mg/mL stock solution PronectinFplus® (hereafter, Pronectin F, Sanyo Chemical Industries, Japan) was prepared by adding one mL of 37 o C deionized water to lyophilized Pronectin F. Ten ug/mL of Pronectin F working solution was prepared by diluting the stock solution with phosphate buffered saline (PBS). The culture dish (BD Life Science, Canada) was covered completely with Pronectin F and left overnight at room temperature. The coating solution was then removed by aspiration., and then dish was rinsed twice with PBS.

To make vitonectin-coated culture dish, the vitronectin-N (VTN-N) (Life Technology,USA) is used for a six-well plate. Dilute thawed VTN-N with 1xPBS (Life Technology,USA). in accordance with the manufacturer's instruction. Keep coated wells in culture medium at 37o C, 5% CO2 during passaging procedure until cells are ready to be re-plated.

#### **2.3. Sendai virus infection and reprogramming**

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

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

CBCscan be procured from Riken Bio Resource center (Riken BRC, Ibaraki, Japan) or

cells in two mL of hematopoietic culture medium [serum-free X-Vivo10

C deionized water to lyophilized Pronectin F. Ten ug/mL

using a mononuclear cells isolation kit (Lymphoprep TM, Cosmo Bio Co., Japan), and a human CD34 Micro Bead kit (Miltenyi Biotec, 130-046-702) or Auto Macs columns (Miltenyi Biotec,

containing 50 ng/mL IL-6 (Peprotech, London, UK), 50 ng/mL sIL-6R (Peprotech) 50 ng/mL SCF (Peprotech), 10 ng/mL TPO (Peprotech), and 20 ng /mL Flt3/4 ligand (R&D System,

PronectinFplus® coated-dish for reprogramming of CBCs is prepared as follows: One mg/mL stock solution PronectinFplus® (hereafter, Pronectin F, Sanyo Chemical Industries, Japan) was

of Pronectin F working solution was prepared by diluting the stock solution with phosphate buffered saline (PBS). The culture dish (BD Life Science, Canada) was covered completely with Pronectin F and left overnight at room temperature. The coating solution was then removed

To make vitonectin-coated culture dish, the vitronectin-N (VTN-N) (Life Technology,USA) is used for a six-well plate. Dilute thawed VTN-N with 1xPBS (Life Technology,USA). in

CBCscan be obtained from fresh cord blood

CBCs were cultured in the

tested vitronectin in two feeder-free ES/iPS mediums. (mTeSR-1 and ReproFF2).

and stable and easy maintenance culturing methods of hESC/iPSC.

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

**2. Experimental procedures, materials and methods**

Germany) in accordance with the manufacturer's instruction. CD34+

**2.2. Preparation of coated dish for feeder-free generating iPS cells**

other commercial suppliers. Alternatively CD34+

Bostone, USA)] for one day prior to viral infection [23].

by aspiration., and then dish was rinsed twice with PBS.

**2.1. Cord blood**

density of 1.0 x 105

prepared by adding one mL of 37 o

CD34+

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/* TS15⊿F, SeV18+*GFP/*TS⊿F) were supplied by DNAVEC Corp. 1.0 x 10<sup>4</sup> CD34<sup>+</sup> CBCs were 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 C. The 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 plates, after which 1 x 10<sup>4</sup> infected CBC were seeded on a Pronectin F-coated 6-well dish in primate ES cell medium ReproFF2 supplemented with 5 ng/mL bFGF (ReproCELL Inc, RCHEMD006B, JAPAN) to generate ES cell-like colonies under 20% O2, 37 o C conditions. The amount of SeV constructs in the transfected cells was reduced by incubation cells at 5% CO2, 38 o C for three days.

#### **2.4. Cell culture in naïve state**

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 at 37 o 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; LIF1005, Billerica).

#### **2.5. Gene chip analysis**

Total RNAs from several established iPSCs lines (prime [1st, 2nd] and naïve), khES-1 (Riken BRC) and CD34<sup>+</sup> CBCs (Riken BRC) were purified with an RNeasy Mini kit (QIAGEN), 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 plus 2.0 Array Affymetrix).

#### **2.6. Karyotype analysis**

After the iPS cells have reached the 80% of confluence, it must be harvested and fixed to make a cytogenetic suspension. iPS cells are growth arrested and accumulated in metaphase or prometaphase by inhibiting tubulin polymerization and thus preventing the formation of the mitotic spindle using colcemid (Sigma, #D7385). Following exposure to colcemid, iPS cells are treated with a hypotonic solution to enhance the dispersion of chromosomes and fixed with carnoy fixative (Methanol: Acetic Acid=3:1). Once fixed, the cytogenetic preparation can be stored in cell pellets, under fixative conditions and 20o C for several months. Fixed cells are spread on slides and air-dried, to be finally banded for the correct identification of chromo‐ somes.

structure of fibronectin, was chosen and tested for reprogramming CBCs. Pronectin F was

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


has thirteen RGD motifs and is folded at the RGD sequence. Thus, the RGD motif is effectively exposed at the limbs of the peptide bundle, facilitating its potent binding affinity to the integrin

**Figure 1.** Protocol for generation of iPSCs from CD34+CBCs on Pronectin F-coated dishes with temperature sensitive

Human ES cell-like colonies (first prime state) were picked up at day 24 and cultured on

passage three (P3). Colonies emerged from single cells in Pronectin F-coated 96-well plates under naïve conditions at P4, dome-shaped colonies at P5 under naïve conditions, ES cell-like

The medium was changed every other day for transformed adherent cell stage (day 1-12). However, during day 13-17, primate ES medium was changed every day. The reprogramming process was monitored by checking the morphology of the transfected cells. CD34+cells infected with SeV constructs were cultured in serum-free hematopoietic cell culture, as shown in Figure 2 (day1). Some cells attached to Pronectin F-coated dishes by day four in Figure 2 (day 4). Cobble stone-like cell colonies emerged at day nine and cell clumps with round and small cells emerged inside the colonies at day 13 on Pronectin F-coated dishes (Figure 2, day 9, day 13). Cell clumps within cobble stone-like colonies grew (Figure 2, day 17) and finally human ES cell-like colonies emerged (Figure 2, day 24) on Pronectin F-coated dishes which were then picked up for serial passage. Fifteen to twenty-two dish-shape human ES cell-like colonies were picked out of 10,000 CD34+CBCs seeded on Pronectin F-coated dish in primate ES medium. Colonies were picked approximately three weeks after viral infection. Cells from individual colonies were transferred to a Pronectin F-coated 48-well plate to select passage-

Pronectin F-coated dishes. The colonies were subjected to heat treatment (38o

colonies (second primed) cultured under primed culture conditions at P6,P7.

able ES cell-like colonies capable of passage.

in tandem to produce a-

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

121

C, three days) at


synthesized by fusing two amino acid motifs, RGD and (GAGAGS)9

**3.2. Generation of iPS cells on synthetic peptide (Pronectin F®)**

Protocol for generating iPSC on feeder less condition is shown in Figure 1.


RGD-(GAGAGS)9

SeV vectors. P: passage.

α5/β1 dimer.
