REVIEW

Development of Cell-processing Systems for Human Stem Cells(Neural Stem Cells, Mesenchymal Stem Cells, and iPS Cells)
for Regenerative Medicine
Yonehiro Kanemura

Department of Regenerative Medicine, Institute for Clinical Research,
Osaka National Hospital, National Hospital Organization, Osaka, Japan(Received for publication on October 20, 2009)(Revised for publication on November 27, 2009)(Accepted for publication on December 18, 2009)

Regenerative medicine using human stem cells is one of the newest and most promising fields
for treating various intractable diseases and damaged organs. For clinical applications, choosing which human stem cells to use, i.e. according to tissue of origin and progenitor type, is a
critical issue. Neural stem/progenitor cells (NSPCs) hold promise for treating various neurological diseases. We have shown that the transporter protein ABCB1 is predominantly expressed in immature human fetal NSPCs, and thus could be used as a phenotypic marker to investigate and monitor NSPCs in culture. We describe our proposed model for the in vitro proliferative process of aggregated human NSPCs and show that neurosphere enlargement and
NSPC proliferation are mutually reinforcing. We have established that human neurospheres
contain a heterogeneous cell population, knowledge that will contribute to the development of
human neurospheres with desirable characteristics for clinical applications. Furthermore, decidua-derived mesenchymal cells (DMCs), which we isolated from human placenta, haveunique properties as mesenchymal stem cells. They also generate a pericellular matrix (PCMDM) that supports the growth and pluripotency of human embryonic stem cells and induced
pluripotent stem cells (hiPS) cells. The newly developed re-programming techniques for generating hiPS cells should greatly contribute to cell therapies using human pluripotent stem cells,
including those derived from DMCs. Our DMC-derived hiPS cells are a promising candidate
source of allogeneic hiPS cells for clinical applications. We hope our findings will contribute to
the development of cell-culture systems for generating human allogeneic stem cells for clinicaluse in regenerative medicine. (Keio J Med 59 (2) : 35－45, June 2010)

Keywords: neural stem cells, mesenchymal stem cells, iPS cells, cell processing system, regenerative
medicine

Introduction

aging results,1-3 and the first clinical trial using cells derived from human embryonic stem (hES) cells4 for thetreatment of spinal cord injuries has been launched.
For clinical applications, choosing which human stem
cells to use, i.e. according to tissue of origin and progenitor type, is a critical issue.1-3 Regenerative treatments
using patient-derived autologous somatic stem cells are
the most desirable for reasons of safety and ethics.1- 3

Regenerative medicine, which involves advanced techniques using human stem cells that are processed exvivo, is one of the newest and most promising fields fortreating various intractable diseases and damaged organs. Many preclinical and clinical studies using stem
cells from various somatic tissues have reported encour-

Presented at the 1627th meeting of The Keio Medical Society in Tokyo, June 8, 2009.
Reprint requests to: Yonehiro Kanemura, MD, PhD, Department of Regenerative Medicine, Institute for Clinical Research, Osaka National Hospital, National
Hospital Organization, 2-1-14 Hoenzaka, Chuo-ku, Osaka 540-0006, Japan, E-mail: kanemura@onh.go.jp
35

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Kanemura Y: Cell Processing Systems for Human Stem Cells

Recently developed re-programming techniques to generate human induced pluripotent stem (hiPS) cells may
make the ideal cell therapy, the use of autologous pluripotent stem cells, a reality.5 On the other hand, the transplantation of allogeneic human fetus-derived stem
cells1, 2 or hES cells4 is also a hopeful strategy, and theestablishment of cell banking systems will increase thefeasibility of using these cell sources.
In addition, the preparation and storing of human stem
cells for clinical use requires time-consuming and difficult techniques. Such cells are strictly prepared using
aseptic processing procedures to prevent contamination
with microbiological and hazardous materials. Methods
are still being developed for cell-processing and cellbanking systems to generate and maintain the various
human stem cells intended for clinical use.
Neural Stem Cells (NSCs)
NSCs are characterized as self-renewing and multipotent progenitors of neurons, astrocytes, and oligodendrocytes.1, 2, 6, 7 During development, NSCs inhabit the ventricular zone and subventricular zone (SVZ) of thebrain.1, 2, 6, 7 Postnatally, they reside in only particular regions of the brain, including the SVZ of the lateral ventricles, the dentate gyrus of hippocampus, and the olfactory bulb, and they can be found in all of these places
throughout life in rodents.1, 2, 6, 7 In vitro, NSCs of rodent
or human origin can be selectively expanded in serumfree culture medium supplemented with fibroblast
growth factor 2 (FGF-2) and/or epidermal growth factor(EGF) as free-floating aggregates termed ‘neurospheres,’which contain a population of immature neural progenitor cells (neural stem/progenitor cells; NSPCs). 8 - 1 0
Many preclinical studies indicate that the transplantationof cultured NSPCs holds promise as a treatment for various neurological diseases that could aid in functional recovery.11-1 4 Furthermore, the first clinical trial using human NSPCs, defined by their expression of CD133,1 5 totreat neuronal ceroid lipofuscinosis (NCL, often called
Batten disease) has begun. 1 6 Although several studies
have already described some of the biological properties
of in-vitro-expanded human NSPCs and the molecules
involved in the maintenance, proliferation, and/or differentiation of human NSPCs,9, 1 7 many others remain to beinvestigated.
The transporter protein ABCB1 is predominantly expressed in human NSPCs
Neurospheres are very heterogeneous cell aggregatesconsisting of stem cells, progenitor cells, and more-differentiated cells.1 7-1 9 Because of their heterogeneity andthe potential for tumorigenesis or other unexpected effects of transplantation, it is very important to establish
strict standards for determining and controlling the qual-

ity of the human NSPCs used for investigations and therapies.
ABCB1 (MDR1/PGY1) is a member of the ATP-binding cassette (ABC) transporter superfamily and was
originally characterized by its ability to confer a multidrug resistance (MDR) phenotype on cancer cells.2 0-2 2
In the mammalian central nervous system, ABCB1 is expressed in cells at the blood-brain barrier, where it presumably helps to transport compounds that cannot be removed by diffusion out of the brain.2 1, 2 3 It is highly expressed in hematopoietic stem cells, which ABCB1 mayprotect from toxins. ABCB1 is thought to play importantroles in various immature stem cells, and it is recognized
as a functional marker molecule for some stem cells.2 2, 2 4
We previously reported that functional ABCB1 is expressed in human NPSCs in vitro, and not in differentiated cells.2 2, 2 5 Recently we performed a detailed examination of ABCB1’s expression on human NSPCs in vitro
and in human fetal brain tissues, and analyzed the cellular properties of the human NSPCs expressing
ABCB1.26
In our analysis, we used four different phenotypicmarkers (ABCB1, CD133,1 5 Nestin,2 7, 2 8 and CD241 5)
and showed that most cells (over 90%) in human neurospheres express Nestin, suggesting that the neurosphereforming cells are immature. About 68.6%(34.8/34.8+15.9) of the ABCB1-positive NSPCs co-expressed CD133 on their cell surface (Fig. 1).2 6 In the opposite analysis, about 73.1% (34.8/34.8+12.8) of theCD133-positive NSPCs also co-expressed ABCB1 on
their cell surface (Fig. 1).2 6 This analysis revealed that
two thirds of the cells expressing ABCB1 also express
CD133 and vice versa, and the remaining one-third expresses either ABCB1 or CD133. Although about 24.5%(14.5/14.5+45) of the ABCB1-positive NSPCs co-expressed CD24 on their cell surface, its level was relatively low compared to the ABCB1 non-expressing NSPCs
and the expression of CD24 in human NSPCs was incontrast to that of ABCB1 (Fig. 1). 2 6 This finding
showed that cells expressing both ABCB1 and CD24
make up a minor population in neurospheres. Thus, thefour phenotypic markers used in our analysis are applicable to the quantitative analysis of heterogeneity in human neurospheres (Fig. 1). Furthermore, the predominant expression of ABCB1 in immature human fetal
NSPCs in vitro indicates that ABCB1 might serve as agood functional marker for human NSPCs.
Effect of cell aggregation on the growth of human
NSPCs
Neurospheres can form from the clonal growth of asingle NSC, under suitable culture conditions (Fig. 2A),and it has been long assumed that neurospheres typicallyarise as such clones.8, 9 Furthermore, in many studies, theself-replication and neurosphere-forming potential of

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Fig. 1 Expression of ABCB1 on the cell surface of human NSPCs from neurospheres.
(Left) Flow cytometry analysis of cells immunolabeled for ABCB1 and Nestin, CD133, or CD24 expression. Single-cell suspensions
were prepared from neurospheres using enzymatic dissociation and stained with an anti-ABCB1 antibody (Ab) (MRK16, Kyowa,
Tokyo, Japan) and phycoerythrin (PE)-conjugated CD133/1 (AC133) Ab (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany),
PE-conjugated CD24 Ab (BD Biosciences), or anti-human Nestin Ab (Nakamura, et al., 2003). After several washes, the samples
were incubated with AlexaFluor 488-conjugated goat anti-mouse IgG Ab (Molecular Probes, Eugene, OR) and/or PE-Cy5 (R-phyco
erythrin-cyanine) -conjugated goat anti-rabbit IgG Ab (Santa Cruz Biotechnology Inc., Santa Cruz, CA) as appropriate. The stained
samples were analyzed on a FACS Calibur cytometer (BD Biosciences). All the results show the mean ± S.D. from three independent
experiments (Reproduced with revision from Yamamoto, et al., J Neurosci Res 2009; 87: 2615–2623). (Right) Venn diagram showing
the relative proportions of the cells in neurospheres labeled by each marker, indicating the heterogeneity of human neurospheres. Ourfindings do not prove the presence of some cells that co-express several markers, and their proportions in neurospheres are uncertain;these hypothetical populations are shown in gray in the diagram (Reproduced with revision from Yamamoto, et al. J Neurosci Res
2009; 87: 2615–2623).

dissociated cells have been regarded as indicators for thepresence of NSCs, and the number of neurospheres generated in a primary heterogeneous culture has been accepted as representing the number of NSCs plated. 9
However, it is inefficient and time-consuming to use single human NSCs to form neurospheres. For example,
Uchida, et al. established neurospheres from human
NSCs plated as single cells, but only after an 8-week
culture period.1 5 In our hands, in contrast, large humanneurospheres form in cultures of dissociated cells in only
a few days, even though the proliferation of human
NSPCs is very slow compared with rodent cells.1 0 Thesefindings call into question whether the cells in a neurosphere are indeed typically clones of a single NSC.
Recently, several groups including ours reported thatneurospheres can form not only from the clonal progenyof a single NSC, but also from aggregated cells or neuro-

spheres (Fig. 2B).1 7, 2 9-3 1 Therefore, we have proposed a
new model for the proliferative process of human
NSPCs that includes in vitro aggregation (Fig. 2B).1 7 In
this model, human NSPCs in vitro first increase their cellnumber by clonal growth. Meanwhile, cell-cell interactions initiated between human NSPCs in small aggregates (i.e. small neurospheres), signal the cells to divide.
Subsequently, each neurosphere becomes larger boththrough cell division and agglomeration with other neurospheres (Fig. 2B).
Interestingly, each human NSPC in an aggregate contributes to its own proliferation through the formation ofneurospheres.1 7 To examine the human neurosphere formation process, we conducted an in vitro culture of human NSPCs focusing on the cell proliferation and themolecular mechanisms involved, using a combination ofcell biological assays and morphological image analysis.

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Kanemura Y: Cell Processing Systems for Human Stem Cells

Fig. 2 Proposed models of the in vitro proliferative process of human NSPCs during neurosphere formation.
(A) Original concept of neurosphere formation and growth. All neurospheres are clones derived from a single neural stem cell (NSC),
which divides to produce NSCs (gray circles) and/or other progenitor cells (black circles). As the NSCs divide (curved arrows), thesize of the neurosphere increases with the proliferation of the clonally related NSCs. (B) Proposed model. Neurospheres can form not
only from the clonal progeny of a single NSC (gray circles) (shown at right), but also by the aggregation of cells into neurospheres(left and middle). In this model, several human NSCs divide (curved arrows), increasing their cell number by clonal growth (right).
Meanwhile, other human NSCs and progenitor cells (black) begin to divide in response to a signal derived from cell-cell interactions
that is initiated by the aggregation of NSPCs (open arrows) to form small neurospheres. Subsequently, each neurosphere becomeslarger, either through cell division (right) or through the combination of cell division and agglomeration with other neurospheres (leftand middle).

We cultured human NSPCs under two different conditions: in flat-bottomed 96-well plates (the control) and inplates with U-bottomed wells designed to promote cellaggregation (Fig. 3A).17
In the flat-bottomed wells, the NSPCs formed small
neurospheres (less than 50 μm) that consisted of a few
cells, possibly resulting from clonal cell division and/or
spontaneous sphere aggregation at a low frequency.
These neurospheres grew gradually, reaching a maximum diameter of less than 100 μm during the 144-hour
culture (Fig. 3B).1 7 In contrast, more and larger neurospheres formed in the U-bottomed wells, and theyreached a diameter of approximately 250 μm after only
24 hours in culture (Fig. 3A, B).1 7 The maximum diameter of the neurospheres in the U-bottomed wells was
greater than in the control cultures throughout the experiment and reached more than 300 μm by 144 hours (Fig.
3B).1 7 A comparison of the proliferation profiles in both
culture systems showed that human NSPC proliferationin the U-bottomed wells was significantly better than in
the flat-bottomed ones. That is, at 144 hours the numberof viable cells in the U-bottomed wells was double thatin the flat-bottomed ones. Furthermore, the population
doubling time in the U-bottomed wells was approximately 60 hours, which was two-thirds of the value inthe flat-bottomed wells.
Finally, this effect of aggregation in the U-bottomed
wells involved cell-cell signaling molecules of the

Notch1 pathway, as shown by the higher levels of Hes1
and Hes5, target genes of the Notch signal, express by
the cells in the U-bottomed wells compared with those in
the control flat-bottomed wells.1 7 These results suggestthat the three-dimensional architecture of NSPC aggregates creates a microenvironment that promotes the proliferation of human NSPCs, and that as neurospheres
grow larger, the proliferative signals increase.
Many researchers report the usefulness of sphere formation in vitro for the acquisition and maintenance ofthe physiological properties and functions of various somatic cells. Examples include the neural differentiationof ES cells,3 2 the promotion of albumin production fromhepatocytes,3 3 and the expression of insulin and glucagon by pancreatic islet cells. 3 4 In the case of neurospheres, our findings suggest that the structure of theneurosphere itself may contribute to the proliferation ofhuman NSPCs and the maintenance of their multipotency. We also speculate that cell motility affects neurosphere growth and/or maintenance. A recent study
showed that β1-integrin mediates the migration, differentiation, and proliferation of NSPCs.3 5, 3 6 Moreover, wehave detected several phenomena that suggest cell migration occurs in neurospheres.37
Considering all these observations, we speculate that aneurosphere is not simply a static cell cluster resulting
from the clonal proliferation of a single NSC, but a dynamic three-dimensional biological structure that canKeio J Med 2010; 59 (2): 35－45

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Fig. 3 Effect of cell aggregation on the growth of human NSPCs.
(A) Human NSPCs were cultured as aggregates either in plates with U-bottomed wells (spheroid culture plate, Sumitomo Bakelite
Co. Ltd., Tokyo, Japan), to promote aggregation, or in plates with flat-bottomed wells (NUNC, Roskilde, Denmark), as thecontrol. The NSPCs were seeded as a single-cell suspension of 2.5×104 cells/ml and cultured for 0, 24, 48, 96, or 144 hours in the
DMEM/F-12(1:1)-based defined medium supplemented with EGF (20 ng/ml), FGF2 (20 ng/ml), B27 (Invitrogen), and leukemia
inhibitory factor (LIF) (10 ng/ml; Chemicon, Temecula, CA), as described previously. 10 Typical images of neurospheres taken at 24
and 144 hours. Scale bar: 100 μm (Reproduced with revision from Mori, et al. J Neurosci Res 2006; 84:1682–1691). (B) Human
NSPC proliferation is up-regulated by aggregation. Time-courses showing numbers of viable cells and maximum neurospherediameters (DAmax). The data represent the mean value ± S.E.M. from six independent cultures for each culture condition (Reproducedwith revision from Mori, et al. J Neurosci Res 2006; 84:1682–1691).

function like a physiological micro-incubator for human
NSPCs. The findings presented here describe some fundamental biological properties of neurosphere formationthat will contribute to establishing new strategies for culturing human NSPCs intended for clinical applications.
The regional distribution of cells in human neurospheres
As the size of a neurosphere increases, so do the number of cell-cell interactions and the proliferation of
NSPCs through Notch signaling.1 7 To inspect the cellular heterogeneity of neurospheres, we developed an algorithm for image cytometry to quantify the area of immunolabeling in living neurospheres cultured from human NSPCs. We also investigated the localization of
specific neural cell types and the regional fluctuation ofcell density in neurospheres.17, 19
To carry out this quantification, we captured images ofneurospheres and measured the diameter of each. To obtain the total number of cells in each sphere, we used an
ATP assay.1 0, 1 7 The viable cell number (N) determinedby the ATP assay correlated fairly well with the diameter(D) of the spheres, and we derived the following equation to describe this relationship: N=1.05×104 D3.18 (r =0.997) (Fig. 4A).1 7 Thus, we were able to estimate thetotal number of viable cells in human neurospheres fromthe sphere diameter alone. Next, neurospheres with different diameters were sectioned, labeled with 1 μM TOPRO-3 iodide (Invitrogen, Carlsbad, CA), and the distribution and density of the nuclei were measured. 1 9 The
radial distance from the periphery of each neurospheresection (d) was adopted as the variable of regional dimension, and the regional cell density (X(d)) was recorded as the total number of cells (TO-PRO-3 labeled nuclei) per unit volume.1 9 As shown in Figure 4B, a similar tendency in the regional variation of total cell density
was observed in terms of the examined d values. The
X(d) values were approximately 1.0×106 cell/mm3 near
the periphery of neurospheres, which decreased gradually to d = 50 μm. Beyond this point, i.e. d=50-180 μm,
the value was constant at approximately 6.0×10 5 cell/
mm3 (Fig. 4B).19

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Kanemura Y: Cell Processing Systems for Human Stem Cells

Fig. 4 Analysis of the regional distribution of cells inside human neurospheres.
(A) Relationship between viable cell number and neurosphere diameter (D A). Images of neurospheres were captured and the
projected area (AA) of each neurosphere and the number of neurospheres were analyzed. The size of each neurosphere was defined
as an equivalent circle diameter, DA = 2(AA/π)1/2. The number of viable human NSPCs was determined indirectly by an ATP assay(Reproduced with revision from Mori, et al. J Neurosci Res 2006; 84:1682-1691). (B) Regional distribution of total cell density (X(d))
in neurospheres of varied sizes. Symbols indicate neurosphere diameter (D): triangle, D=100-200 μm; square, D=200-300 μm; circle,
D=300-400 μm (Reproduced with revision from Mori, et al. J Biosci Bioeng 2007; 103: 384–387). (C) Immunofluorescent imagesof human neurosphere sections. Sections were immunostained with a proliferation marker (anti-BrdU: green) and anti-glial fibrillary
acidic protein (GFAP) (red) or a neuronal cell marker (anti-βIII-tubulin: red), and counterstained with TO-PRO-3 (blue). Scale bar:
100 μm (Reproduced with revision from Mori, et al. J Biosci Bioeng 2007; 103: 384–387). (D) Regional changes in the densities
of proliferating and differentiated cells in neurospheres. Blue: BrdU-positive cell. Red: BrdU-negative/GFAP-positive cell. Green:
BrdU-negative/βIII-tubulin-positive cell (Reproduced with revision from Mori, et al. J Biosci Bioeng 2007; 103: 384–387).

When neurospheres were cultivated in medium containing BrdU (5-bromo-2’-deoxyuridine; 10 mM) for 72
hours to label proliferating cells, cells with three different molecular profiles were identifiable: BrdU-positive(BrdU ＋), BrdU-negative and βIII-tubulin-positive(BrdU- βIII tub＋), and BrdU-negative and GFAP-positive (BrdU- GFAP＋) (Fig. 4C).1 9 The X(d) values for
BrdU＋ (Fig. 4D, blue) and BrdU- βIII tub＋ cells (Fig.
4D, green) showed a significant decrease from the periphery to d = 28 μm and were nearly constant thereafter.
On the other hand, the X(d) value for BrdU - GFAP ＋
cells (Fig. 4D, red) did not show a significant difference
from its value in the peripheral region.19

These findings revealed that there were differences inthe distributions of proliferating and differentiated cells
within human neurospheres, which should help characterize the nature of the human NSPCs they contain.
Mesenchymal Stem Cells (MSCs)
MSCs comprise a heterogeneous subset of stromal
stem cells that can differentiate into cells of the mesodermal linage, such as osteoblasts, chondrocytes, and adipocytes.3, 3 8 MSCs are typically isolated from the nonhematopietic mononuclear cells of the bone marrow(bone marrow derived-mesenchymal stem cells; BM-

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Fig. 5 In vitro differentiation of bone marrow derived-mesenchymal stem cells (BM-MSCs) and decidua-derived mesenchymal cells(DMCs).
To induce osteogenic differentiation, cells were cultured with dexamethasone (Dex; 0.2 μM), β-glycerol phosphate (10 mM), ascorbic
acid 2-phosphate (100 mM), and 10% fetal bovine serum (FBS) for 28 days. To evaluate the mineralized matrix, cells were stainedwith 0.1% Alizarin red S. To induce adipogenic differentiation, cells were cultured with Dex (1 mM), 3-isobutyl-1-methylxanthine (IBMX) (500 mM), indomethacin (200 mM), recombinant human insulin (20 mg/ml), and 10% FBS for 28 days. To evaluate thepresence of cytoplasmic inclusions of neutral lipids, cells were stained with Oil red O and counterstained with Mayer hematoxylin.
To induce chondrogenic differentiation, cells were pelleted in a polypropylene tube and then cultured with Dex (0.1 μM), ascorbic
acid 2-phosphate (170 μM), sodium pyruvate (1 mM), proline (350 μM), recombinant human transforming growth factor-β3 (TGF-β3) (10 ng/ml), and ITS+Premix (BD Biosciences) for 28 days. To evaluate chondrogenic differentiation, sections of the chondrogenic
cell pellet were incubated with anti-collagen type-II Ab and signals were detected by the avidin-biotin complex (ABC) method with ametal-enhanced DAB Kit (Pierce, Rockford, IL). Scale bar: 100 μm.

MSCs) or they can be also isolated from various human
fetal and adult tissues.3, 38
Isolation of decidua-derived mesenchymal cells (DMCs)
The fetal adnexa is composed of the placenta, fetal
membrane (FM), and umbilical cord. These are ephemeral organs that connect the developing fetal tissues tothe uterine wall, supplying the fetus with maternal nutrients, allowing fetal waste to be disposed of via the maternal kidneys, and contributing to fetal-maternal immune tolerance. Many recent studies have shown that fibroblast-like adherent cells isolated from various placental components are phenotypically similar to BMMSCs.3 9-4 2 Therefore, the fetal adnexa may prove to bea useful source of human MSCs.
We isolated decidua-derived mesenchymal cells(DMCs) from human FMs at term4 3 and examined how
their cellular properties compared with those of BMMSCs. The DMCs adhered to plastic dishes and exhibited a typical fibroblast-like morphology. They showed
excellent potential for in vitro expansion, because they
maintained their morphology for more than 30 population doublings (Fig. 5). The DMCs had an F-actin cytoskeleton that extended throughout the entire cytoplasm,
and they strongly expressed a mesenchymal cell marker,

vimentin, but not cytokeratin 19 or HLA-G. 4 3 In vitro
differentiation assays showed that the DMCs differentiated well into chondrocytes, and moderately into adipocytes, but hardly at all into osteoblasts, as compared with
BM-MSCs (Fig. 5). Although DMCs are not superior to
BM-MSCs in multipotency, their greater proliferative
ability requires less maintenance in cell preparation, andtheir derivation from the maternal portion of humanFMs, which are otherwise discarded, would resolve
many ethical concerns. These unique properties of
DMCs have several advantages for clinical use andmake these cells an attractive alternative to allogeneic
MSCs for use in regenerative medicine.
Pericellular matrix of DMCs (PCM-DM) is a potent human-derived substrate for the maintenance culture ofhuman pluripotent stem cells
Unlike mouse ES cells, hES and hiPS cells are maintained on either feeder cells or special culture substrates
such as Matrigel.4 4 However, to expand hES/hiPS cells
for clinical applications, it is desirable to minimize animal-derived materials in the culture for safety reasons.
We therefore tested the pericellular matrix prepared fromhuman DMCs (PCM-DM) for its ability to support hES
cell growth and pluripotency. DMCs were cultured on

42Kanemura Y: Cell Processing Systems for Human Stem Cells

Fig. 6 Pericellular matrix of decidua-derived mesenchymal cells (PCM-DMs) is a potent human-derived substrate for the maintenanceculture of human pluripotent stem cells.
Undifferentiated hES cells (KhES-1) and hiPS cells (253G4) were maintained on a feeder layer of mouse embryonic fibroblasts(MEFs) in DMEM/F12 supplemented with 20% Knockout Serum Replacement (KSR), glutamine (2 mM), nonessential amino acids(0.1 mM), recombinant human FGF-2 (5 ng/ml), and 2-mercaptoethanol (0.1 mM). MEF-conditioned medium (MEF-CM) was
prepared by incubating the maintenance culture medium with confluent MEFs for 24 hours. (A) Staining of alkaline phosphatasein hES cells cultured on PCM-DM in MEF-CM. Scale bar: 100 μm. (B) Propagation efficiency of hES cells cultured in MEF-CM.
PCM-DM supported propagation better than Matrigel. (C) Alkaline phosphatase activity of hES cells cultured in StemPro on a 4-well
PCM-DM plate. Scale bar: 100 μm. (D) Propagation efficiency of hES cells. StemPro supported the propagation of hES cells. (E) hiPScolonies plated on a PCM-DM 4-well plate showing alkaline phosphatase activity. (F) hiPS cells cultured under various conditions.
StemPro medium and PCM-DM supported the efficient growth of hiPS cells. (Reproduced with revision from Nagase, et al. Dev Dyn
2009; 238: 1118–1130).

gelatin-coated plastic culture dishes and lysed by deoxycholate to prepare the PCM-DM. 4 3 The PCM-DM wasthen used for hES cell culture in mouse embryonic fibroblast (MEF)-conditioned medium (MEF-CM), and its
maintenance-supporting activity was compared with thatof two conventional matrices, fibronectin and Matrigel.44 During multiple passages, PCM-DM supported thegrowth of human ES cells with an efficiency similar to,or slightly better than, that of Matrigel, an animal-derived substrate, in MEF-CM (Fig. 6A, B).43
Since MEF-CM itself contains mouse-derived materials, we next examined whether PCM-DM-based hES
cell culture was compatible with unconditioned serumfree medium. Human ES cell clumps were seeded on
PCM-DM in StemPro hESC SFM (Invitrogen), which
supports hES cell maintenance on Matrigel-type matrices. We found that the PCM-DM-based culture wascompatible with non-conditioned commercial defined
medium, and with the maintenance of dissociated hES
cells (Fig. 6C, D).4 3 We next tested whether hES cells
maintained on PCM-DM in StemPro retained their pluripotency after multiple passages, and found that they
did.4 3 Notably, the maintenance-supporting activity of
PCM-DM was stable and could be preserved for at least
eight months after preparation, by keeping the plate in
the refrigerator under semi-dry conditions.4 3 Moreover,
we tested whether PCM-DM was applicable to the maintenance culture of hiPS cells. Cells from the hiPS line
253G44 5 were cultured as cell clumps on PCM-DM or
Matrigel in MEF-CM or StemPro. In both media, the
PCM-DM supported hiPS cell growth as efficiently as
Matrigel (Fig. 6E, F).43
Since DMCs can be prepared and expanded in large
quantity, PCM-DM is a practical human-derived substi-

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Fig. 7 Generation of Human iPS cells.
(A) The reprogramming of human somatic cells to generate human iPS cells has been achieved by the forced expression of exogenous
Oct4, Sox2, Klf4, and c-Myc. (B) Human iPS cells prepared from decidua-derived mesenchymal cell (DMCs). Upper: Phase-contrast
view of DMC-derived human iPS cell colonies. Lower: Alkaline phosphatase activity. Scale bar: 100 μm.

tute for the animal-derived substrates currently in use forthe clinical-grade culture of hES/hiPS cells.
Human iPS Cells
The reprogramming of human somatic cells into iPS
cells has been achieved by the forced transduction of
Oct4, Sox2, Klf4, and c-Myc.5 These iPS cells closely
resemble ES cells in their gene expression pattern andepigenetic profile, and the transduced transcription factors restore the pluripotency of the iPS cells in vitro andin vivo (Fig. 7A).5 Regenerative treatments that use patient-derived autologous somatic stem cells or iPS cells
are the most desirable for reasons of safety and ethics.
Human iPS cells will greatly contribute to making theclinical use of autologous pluripotent stem cells a future
reality.
On the other hand, the transplantation of allogeneic
human somatic stem cells derived from hES cells or
hiPS cells is also a promising approach, especially forthe treatment of acute diseases and injures (e.g. stroke,
spinal cord injury) in which cell transplantation must be
performed in the acute to sub-acute stage.11, 1 3 We havesuccessfully derived hiPS cells from DMCs and are examining their biological properties (Fig. 7B). We think
DMC-derived hiPS cells are likely to have properties
that are virtually equivalent to those of allogeneic hiPS

cells. The establishment of hiPS cell-banking systems
will increase the feasibility of using allogeneic stem
cells for clinical applications.
Conclusions
We found that ABCB1 is predominantly expressed inimmature human fetal NSPCs, and thus could be used asa phenotypic marker to investigate and monitor NSPCs
in culture. We have proposed a new model for the in vitro proliferation of human NSPCs in which cell aggregation increases cell-cell interactions that activate Notch
and promote proliferation. Therefore, the proliferation of
new NSPCs and the increase in neurosphere size are mutually reinforcing. We also found that neurospheres contain a heterogeneous, regionally biased cell population,
which may help improve our understanding of how toisolate neurospheres with desired properties.
To improve cell culture processes and increase thenumber of cell sources available, we compared DMCs
isolated from term human FMs with BM-MSCs andfound that the two stem-cell populations had nearly
identical properties. We also found that the pericellularmatrix made by the DCMs, PCM-DM, supported thegrowth and pluripotency of hES/hiPS cells. These
unique properties of DMCs suggest that they may beparticularly appropriate for clinical use, and we expect

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Kanemura Y: Cell Processing Systems for Human Stem Cells

that future studies will show them to be an attractive alternative to allogeneic MSCs in regenerative medicine.
Recent advances in the re-programming techniques
used for generating iPS cells should also greatly contribute to the development of cell therapies using humanpluripotent stem cells. We have successfully derived
hiPS cells from DMCs, and these hiPS cells may be an
ideal candidate cell type for allogeneic hiPS cells in clinical applications. We hope these findings will promote
the development of cell-processing systems to generate
human allogeneic stem cells for clinical use in regenerative medicine.
Acknowledgements
I am very grateful to the following collaborators: Prof.
Hideyuki Okano (Keio University), Dr. Yoshiki Sasai(Center for Developmental Biology, RIKEN), Prof. Masahiro Kino-oka and Prof. Masahito Taya (Osaka University), and Dr. Hideki Mori (Osaka Prefectural University). I am also grateful to Dr. Chiaki Ban and Dr. Mami
Yamasaki at Osaka National Hospital and to all themembers of our laboratory. These studies were supported by the Project for the Realization of Regenerative
Medicine, the research field for the Development ofStem Cell Therapy, from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan,
the Cooperative Link of Unique Science and Technology
for Economy Revitalization (CLUSTER) project from
MEXT, Japan.
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