Kasper C., van Griensven M., P?rtner R. (Eds.) Bioreactor Systems for Tissue Engineering II: Strategies for the Expansion and Directed Differentiation of Stem Cells
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essential to control cell quality. The development of adequate in situ sensors for the
monitoring of the cultures will be of great interest [90]. Furthermore, it has been
shown that UC-MSCs can be frozen and thawed efficiently, which makes them
suitable for their use in clinical cell banking. The therapeutic use of MSCs will
require storage prior to clinical applications. In this regard, it appears worthwhile
that UC-cells isolated at birth, may be safely stored and delivered decades later to a
patient. Nevertheless, additional studies may be necessary to attest the stability of
long term cryopreserved cells.
The clinical potential of MSCs is primary dependent on their differentiation
potential. Like BM stromal cells, UC-derived MSCs were demonstrated to be
multipotent. Interestingly, their differentiation repertoire does not seem to be
restricted to the mesodermal lineages, since the cells could be successfully induced
to neurones, liver, and pancreatic cells. A growing body of evidences suggests,
however, that UC-MSC populations are rather heterogeneous, harboring a subset of
primitive cells. The next generation of studies should focus on the identification and
characterization of these sub-populations. In particular, the question of whether the
differentiation potential of the isolated populations is dependent on their location in
the UC-tissues is of great interest for clinical application. Newly described MSCs
markers may be helpful in this regard.
Additionally, first in vitro and in vivo animal studies evidenced immune-
privileged and immune-modulatory properties of UC-derived MSCs. Low levels
of rejection were observed in all reports of in vivo transplantation experiments and
encouraging results in tissue repairs were obser ved. In particular, supportive func-
tion through paracrine effects seems to be involved. The next generation of studies
and first clinical trials will clarify whether the benefit of UC-derived MSCs after
transplantation experiments relies on supportive effects and/or on differentiation
in vivo.
One of the ambitious aims of regenerative medicine is the engineering of tissue
in vitro. Few but very promising applications of UC-derived MSCs have been
reported in this field. For instance, UC-MSCs are believed to have a high potential
in cardiovascular tissue engineering [91]. They grew very well on bio-degradable
polymer for the elaboration of cardiovascular constructs [33] and could be used for
the construction of human pulmonary conduits [92], for the engineering of biologi-
cally active living heart valve leaflets [ 27], and for the elaboration of living patches
with potential for pediatric cardiovascular tissue engineering [93]. The use of newly
developed scaffolds, mechanical strain approaches, or 3D bioreactors for tissue
generation, which were successfully applied with MSCs from other sources [94],
will also be a highly interesting issue.
Considering the very encouraging results obtained in recent years, it may only be
a question of time until UC-derived MSCs will be routinely used for clinical and
tissue engineering applications.
Acknowledgments The authors are grateful to Martina Weiss for her support with the production
of the figures and to Stefanie Boehm for the critical reading of this work.
Mesenchymal Stromal Cells Derived from Human Umbilical Cord Tissues 47
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DOI: 10.1007/10_2009_24
#
Springer-Verlag Berlin Heidelberg 2010
Published online: 21 January 2010
Isolation, Characterization, Differentiation,
and Application of Adipose-Derived Stem Cells
Jo¨rn W. Kuhbier, Birgit Weyand, Christine Radtke, Peter M. Vogt,
Cornelia Kasper, and Kerstin Reimers
Abstract While bone marrow-derived mesenchymal ste m cells are known and
have been investigated for a long time, mesenchymal stem cells derived from the
adipose tissue were identifi ed as such by Zuk et al. in 2001. However, as subcuta-
neous fat tissue is a rich source which is much mor e easily accessible than bone
marrow and thus can be reached by less invasive procedures, adipose-derived stem
cells have moved into the research spotlight over the last 8 years.
Isolation of stromal cell fractions involves centrifugation, digestion, and filtra-
tion, resulting in an adherent cell population containing mesenchymal stem cells;
these can be subdivided by cell sorting and cultured under common conditions.
They seem to have comparable properties to bone marrow-derived mesenchymal
stem cells in their differentiation abilities as well as a favorable angiogenic and
anti-inflammatory cytokine secretion profile and therefore have become widely
used in tissue engineering and clinical regenerative medicine.
Keywords Adipose-derived stem cells, ASC, Fat harvesting, Isolation protocoll,
Stem cell application
Contents
1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 56
2 Isolation of ASC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
2.1 Influence of Donor Site and Age . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 58
2.2 Techniques of Harvesting Fat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 60
2.3 Isolation of ASC from Fat Grafts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
J.W. Kuhbier (*)
Department of Plastic, Hand and Reconstructive Surgery, Podbielskistrasse 380, 30659 Hannover,
Germany
e-mail: joernkuhbier@gmx.de
B. Weyand, C. Radtke, P.M. Vogt, C. Kasper, and K. Reimers
Department of Plastic, Hand and Reconstructive Surgery, Medical School Hannover, Podbielski-
strasse 380, 30659 Hannover, Germany
3 Characterization of ASC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3.1 Characterization via Surface Marker and Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . 65
4 Differentiation of ASC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 67
4.1 Adipogenic Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
4.2 Osteogenic Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 72
4.3 Chondrogenic Differentiation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
5 In Vivo Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
5.1 Cytokine Profile Secreted by ASC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
5.2 Angiogenesis and Functional Improvement of Ischemic Muscle Tissue . . . . . . . . . . . . . 76
5.3 Neurological and Skeletal Application of ASC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80
5.4 ASC for Enhancement and Acceleration of Wound Healing . . . . . . . . . . . . . . . . . . . . . . . . 83
5.5 Immunomodulatory Effect of ASC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86
5.6 Other Purposes in Current In Vivo Application . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
6 Tissue Engineering Applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6.1 Adipose Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 91
6.2 Bone Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
6.3 Cartilage Tissue . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
7 Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 94
1 Introduction
Despite bone marrow being the primary and best stud ied source of stem cell
populations, multilineage progenitor cells with the potential to differentiate into
multiple cell lines have been found in the heart [1, 2], muscle [3, 4], lung [5, 6],
intestine [7], kidney [8], liver [9], pancreas [10], skin [11], and even in the brain
[12, 13]. These tissue-resident stem cells seem to posses certain repair functions in
their native tissue, but also have potential for and might contribute to tissue
regeneration processes in regions other than their origin [14, 15].
Due to their many advantages, several recent studies favor adipose-tissue
derived mesenchymal stem cells for their study purposes. A multilineage stem
cell population that was derived from the stroma of adipose tissue was first
described by Zuk et al. in 2001 [16], who used the term “processed lipoaspirate”,
as they isolated the cells from an aspirate of a liposuction. Since the lipoaspirate is
composed of several cell types, it requires processing by stepwise centrifugation in
order to isolate the stem cell fraction.
Fat tissue is composed mainly of mature adipocytes which are aligned in lobules
and surrounded by connective tissue. Within the connective tissue, a vascular network
allows nutrition and also transportation of endocrine mediators produced by adipose
tissue, especially leptin and adiponectin, an insulin-sensitizing hormone [17, 18]. In
specific anatomical locations, such as the abdominal wall, the fat layer is divided by a
thin facia, so-called Scarpa’s facia, into a superficial and a deeper layer of fat tissue.
After centrifugation of the lipoaspirate, the most voluminous fraction contains
adipocytes, which may burst and die during processing. Alongside, another
56 J.W. Kuhbier et al.