A Manual for Differentiation of Bone Marrow-Derived Stem Cells to Specific Cell Types

Kee Woei Ng, Gilson Khang, Tatsuya Shimizu

• Publisher: WSPC
• Year: 2014
• ISBN:
  9789814578257
• Topic: Biological Sciences
• Subtopic: Cell Biology
• Index: Biological Sciences

This is the first experimental protocol book that covers the differentiation of bone marrow-derived stem cells (BMSCs) into specific cell types, targeted at the undergraduate and graduate student level. The 19 chapters deal with the differentiation methods using small molecules, cytokines and polymeric scaffolds.

BMSCs are pluripotential in that they not only act as myelo-regenerative and supportive cells, but can also differentiate into almost any kind of cells in our body. In addition, when implanted in vivo, they could help repair multiple tissues such as blood vessels, heart, liver and so on.

For the differentiation of BMSCs, many methods have been introduced to adjust their microenvironment (chemical and physical cues), including chemical induction methods using large or small molecules and pellet culture; mechanical stimulation induction methods using cyclic mechano-transduction or ultrasonication; cytokine-released method using scaffolds; and so on.

Contents:

• Introduction (Gilson Khang)
• Chondrogenic Differentiation of Rat BMSCs in Hydrogel (Hazel Y Stevens, David S Reece, Rhima M Coleman and Robert E Guldberg)
• Protocol of Chondrogenesis from BMSCs Using TGF-β Loaded Alginate Bead (Soon Hee Kim and Gilson Khang)
• Protocol of Chondrogenesis from Human BMSCs by Pellet Culture (Jeong Eun Song, Eun Young Kim, Soon Hee Kim and Gilson Khang)
• Protocol of Chondrogenesis from BMSC on a Porcine Chondrocytes-Derived Extracellular Matrix Scaffold (Byoung-Hyun Min)
• Protocol of Chondrogenesis of MSC by Ultrasonication (Byoung-Hyun Min, So Ra Park and Hyun Jung Lee)
• Protocol of Chondrogenesis of BMSC to Chondrocyte Using Chitosan-Modified Poly(L-Lactide- co -ε-Caprolactone) Scaffolds (Zheng Yang, Xiaoyan Tang, Chao Li and Zigang Ge)
• Protocol of Osteogenesis of BMSCs Using Hydroxyapatite/Tricalciumphosphate Scaffold (EunAh Lee, Pamela Gehron Robey and Youngsook Son)
• Protocol of Osteogenesis from BMSC Cultured with Dexamethasone-Loaded Dendrimer Nanoparticles onto Ceramic and Polymeric Scaffolds: In Vivo Studies (Joaquim Miguel Oliveira, João F Mano, Hajime Ohgushi and Rui Luís Reis)
• Protocol for Osteogenesis of BMSC in Calcium Phosphate Ceramics (EuiKyun Park, Hong-In Shin, Shin-Yoon Kim and Jiwon Lim)
• Protocol of Osteoblastic Differentiation of BMSC in Biodegradable Scaffolds Composed of Gelatin and β-Tricalcium Phosphate (Masaya Yamamoto and Yasuhiko Tabata)
• Protocol of Cardiomyogenic Induction of hMSCs on Dendrimer-Immobilized Surfaces Displaying with D-Glucose (Mee-Hae Kim and Masahiro Kino-oka)
• Protocol of Cardiomyocyte Differentiation of BMSC by Small Molecules (Ki-Chul Hwang, Woochul Chang and Byeong-Wook Song)
• Protocol for the Differentiation of BMSCs to a Smooth Muscle Cell for the Application of Engineering Small Diameter Blood Vessels (Hyunhee Ahn, Young Min Ju and Sang Jin Lee)
• Protocol of Schwann Cell Differentiation of BMSC by Direct Co-Culture Method Using Insert System (Jeong Eun Song, Soon Hee Kim, Cho Min Kim and Gilson Khang)
• Protocol of Neurogenesis of BMSC Using β-Mercaptoethanol Released System from β-Mercaptoethanol-Loaded PLGA Film (Jeong Eun Song, Eun Young Kim, Hyeon Yoon, Dongwon Lee and Gilson Khang)
• Protocol of Neural Differentiation from BMSCs Using bFGF and Laminin-Coating Plate (Byung Hyune Choi, Jin-Mo Kim and So Ra Park)
• Protocol of Differentiation of Retinal Pigment Epithelial-Like Cells from BMSC Using Co-Culture Method (Su Ji Kang, Eun Young Kim, Hyeon Yoon, Chun-Ki Joo and Gilson Khang)
• Protocol of Differentiation of Olfactory Ensheathing Cells from BMSCs by Insert and Conditioned Media System (Jeong Eun Song, Yun Mi Lee, Hyeon Yoon, Chun-Ki Joo and Gilson Khang)
• Protocol of the Differentiation of BMSC to Corneal Endothelial Cells by Direct and Indirect Co-Culture (Eun Young Kim, Hyeon Yoon, Jin San Choi, Gilson Khang and Shay Soker)

Readership: Undergraduate and graduate students and researchers in biomedical engineering, tissue engineering, stem cell research, nanotechnology and material science.

List of Figures

A

Fig. 1	Cartoon of process of injecting cells and hydrogel into the custom-designed mold.
Fig. 2	Collagen type II (red) and aggrecan (green) immunolocalization in hydrogels at 21 days in culture. Cells were treated with FGF-2 during monolayer expansion and ITS, Dex or TGF-β1+Dex during 3D culture.
Fig. 3	Chondrogenic differentiation of hAFS cells in pellet culture. (A) Size and sGAG (stains purple with toluidine blue) amount is growth factor dependent. (B) Collagen type II immunolocalization detected by red coloration. Scale bar is 100 μm and applies to all images.
Fig. 4	rBMSCs expanded to P2 ± FGF-2 and encapsulated in alginate or agarose and cultured for 14 and 21 days. sGAG normalized to total DNA.
Fig. 5	Live–dead staining of alginate and agarose gels after 21 days of culture. Original magnification was ×10. Live and dead cells fluoresce green and red, respectively.
Fig. 6	Pellet culture. DNA content (A) and sGAG normalized to DNA content for hAFS cells and hBMSCs grown first in expansion media, then later in pellet culture with supplements listed (B). sGAG staining for hBMSC vs. smaller hAFS cell pellets (C).

B

Fig. 1	BMSC morphology from primary culture to passage 1 (P: passage, scale bar: 250 μm in magnification ×100, 100 μm in magnification ×200).
Fig. 2	Confirmation of mesenchymal stem cell marker in cultured BMSCs.
Fig. 3	Schematic representation of process of preparing alginate bead. The concentration of TGF-β1: 0.5 μg/mL, alginate solution: 1.2 w/v%, CaCl2: 102 mM.
Fig. 4	(A, B) The observation of empty beads by inverted microscope and SEM. (C) Fluorescence microscope of alginate beads including BMSCs and TGF-β1 (scale bar: 500 μm in magnification ×40).
Fig. 5	Gross morphology after extracting subcutaneous implanted alginate bead.
Fig. 6	Photomicrographs from H&E histological sections of alginate beads implanted for 4 weeks (scale bar: 100 μm in magnification ×200, 50 μm in magnification ×400).
Fig. 7	Alcian Blue staining, Safranin-O staining, and IHC and immunofluorescence (IF) for type II collagen in beads including BMSCs and TGF-β1 (scale bar: 250 μm for magnification ×100 and 100 μm for magnification ×200).

C

Fig. 1	Bone marrow aspiration from human iliac crest.
Fig. 2	Percoll gradient centrifugation of human BMSC (RBCs: red blood cells).
Fig. 3	Primary culture of human BMSCs for 5 days. (A) Cell morphology using reverse phase microscope (magnification ×50), (B) cell morphology using red cell tracer (magnification ×100).
Fig. 4	Differentiation of human BMSCs by pellet culture system. Cell pellet was maintained in differentiation medium.
Fig. 5	Confirmation of pellet morphology by hi-scope.
Fig. 6	SEM picture of cell pellet (magnification ×1000).
Fig. 7	Safranin-O staining of human BMSCs cultured as aggregates. (magnification ×200)

D

Fig. 1	(A) The gross morphology of consolidated membrane; (B) exterior and inside structure of ECM scaffold. ECM scaffold is a sponge type with uniformly distributed pores and white in color.
Fig. 2	(A) The gross images of specimens. (B) Expression levels of type II collagen, sox-9 and GAPDH by RT-PCR analysis. The implanted specimens were retrieved at 1, 2, 4 and 6 weeks after implantation.
Fig. 3	Histology for chon...

Table of contents

• Cover
• Halftitle Page
• Frontmatter
• Title Page
• Copyright Page
• Preface
• Contents
• List of Figures
• List of Contributors
• Introduction
• A Chondrogenic Differentiation of Rat BMSCs in Hydrogel
• B Protocol of Chondrogenesis from BMSCs using TGF-β Loaded Alginate Bead
• C Protocol of Chondrogenesis from Human BMSCs by Pellet Culture
• D Protocol of Chondrogenesis from BMSC on a Porcine chondrocytes-Derived Extracellular Matrix Scaffold
• E Protocol of Chondrogenesis of MSC by Ultrasonication
• F Protocol of Chondrogenesis of BMSC to Chondrocyte Using Chitosan-Modified Poly(l-Lactide-co-ε-Caprolactone) Scaffolds
• G Protocol of Osteogenesis of BMSCs using Hydroxyapatite/Tricalciumphos-phate Scaffold
• H Protocol of Osteogenesis from BMSC cultured with Dexamethasone-Loaded Dendrimer Nanoparticles onto Ceramic and Polymeric Scaffolds: In Vivo Studies
• I Protocol for Osteogenesis of BMSC in Calcium Phosphate Ceramics
• J Protocol of Osteoblastic Differentiation of BMSC in Biodegradable Scaffolds Composed of Gelatin and β-Tricalcium Phosphate
• K Protocol of Cardiomyogenic Induction of hMSCs on Dendrimer-immobilized Surfaces displaying with D-Glucose
• L Protocol of Cardiomyocyte Differentiation of BMSC by Small Molecules
• M Protocol for the Differentiation of BMSCs to a Smooth Muscle Cell for the Application of Engineering Small Diameter Blood Vessels
• N Protocol of Schwann Cell Differentiation of BMSC by Direct Co-culture Method using Insert System
• O Protocol of Neurogenesis of BMSC Using β-Mercaptoe-thanol Released System from β-Mercaptoe-thanol-Loaded PLGA Film
• P Protocol of Neural Differentiation from BMSCs using bFGF and Laminin-Coating Plate
• Q Protocol of Differentiation of Retinal Pigment Epithelial-like Cells from BMSC using Co-culture Method
• R Protocol of Differentiation of Olfactory Ensheathing Cells from BMSCs by Insert and Conditioned Media System
• S Protocol of the Differentiation of BMSC to Corneal Endothelial Cells by Direct and Indirect Co-culture
• Index