Добавил:
Sekretar
kiopkiopkiop18@yandex.ru
t.me/Prokururor I Вовсе не секретарь, но почту проверяю
Опубликованный материал нарушает ваши авторские права? Сообщите нам.
Вуз:
Предмет:
Файл:Ординатура / Хирургия / Библиотека им академика М.И. Перельмана / Книга_896_Библиотеки_им_академика_М_И_Перельмана
.pdf
14 Cell Therapies: New Frontier fortheManagement ofDiabetic Foot Ulceration
https://t.me/med1917
255
studies in which xenogeneic, allogeneic, and autologous
sources of stem cells have been tested and screened to enable
future clinical trials (Table14.1). We will also review human
clinical trials in which allogeneic and autologous sources of
stem cells have been tested for therapy (Table14.2). We will
discuss known therapeutic benets of existing sources of
Table 14.1 In vivo animal studies of diabetic foot ulcer therapies
Cell type Animal model Outcome References
Bone marrow mesenchymal stem cells
BM-MSCs Rats/
BM-MSCs Mice/diabetic Re-epithelialization enhanced by application of BM-MSCs to injury site Javazon etal. [22]
BM-MSCs Mice/diabetic Improved angiogenic response Wu etal. [23]
BM-MSCs +
chemokines
BM-MSCs Mice/
BM-MSCs (xenograft) Rabbit/
BM-MSCs (autograft,
allograft, xenograft)
BM-MSCs + EGF Mice/diabetic Increased blood ow with EGF compared to BM-MSCs alone (70%) Amin etal. [27]
BM-MSCs Mice/diabetic Streptozotocin-induced type I diabetic mouse treated with BM-MSCs resulted
BM-MSCs Rats/
BM-MSCs Rats/diabetic Increased VEGF and improved granulation tissue at the wound site Wan etal. [30]
Endothelial progenitor cells
EPCs Mice/
EPCs Mice/
EPC delivery systems Mice/
Fetal aorta-derived
EPCs
Embryonic-EPCs CM Mice/
EPCs Mice/diabetic Allogenic EPCs from normal diabetic mice accelerated wound closure
EPCs Mice/diabetic EPCs that were applied topically to wounds on the diabetic mice accelerated
EPCs Mice and
Mononuclear stem cells
PB-MNCs Mice/diabetic Human PB-MNCs decreased wound size in diabetic mice Sivan-Loukianova
PBMCs + broblasts Mice/diabetic PBMCs mixed with broblasts accelerated wound healing in mice Ueno etal. [38]
Adipose stem cells
ASCs Mice/diabetic ASCs promoted neovessel formation and better tissue remodeling in treated
ASCs Mice/diabetic ASCs overexpressing SDF-1 promoted the healing of wound in STZ-induced
ASCs Rat/diabetic ASCs enhanced wound healing in STZ-induced diabetic rat model, but did not
non-diabetic
Mice/
non-diabetic
non-diabetic
non-diabetic
Pig/
non-diabetic
non-diabetic
non-diabetic
non-diabetic
non-diabetic
Mice/diabetic Wound healing and angiogenesis in streptozotocin-induced diabetic mice Barcelos etal. [33]
non-diabetic
rabbits/
diabetic
Improved in healing compared to skin-derived broblasts McFarlin etal.
BM-MSCs combined with chemokines are more effective Sasaki etal. [24]
Chemokine release enhanced wound healing Chen etal. 2007
Inhibited scar formation and increased tensile strength of full-thickness
cutaneous wounds
Treatment with autologous, allogeneic, or xenogeneic cells induced wound
healing and regeneration of skin and hair follicles
in increase in tissue regeneration biomarkers and decrease in pro-inammatory
markers
3D collagen allograft with BM-MSCs upregulated MMP-9 promoting repair Kim etal. [29]
Human-isolated EPCs injected systemically into nude mice with ischemia
selectively localized to areas of ischemia and induced new blood vessel growth
EPC injection in mice dermal excisional wound model promoted wound
closure
Bioactive material was the most effective method of EPC delivery for
improving vascularization in ischemic mouse hindlimb
Paracrine factors released from cultured media of human embryonic epithelial
progenitor stimulated wound healing in nude mouse
compared to autologous EPCs
wound closure
Observed inconsistencies at the cellular level among diabetic humans, mice,
and rabbits
mice compared with the control group
diabetic mice
enhance angiogenesis
adult stem cells and implications for future stem cell DFU
treatments using novel sources of stem cells, such as induced
pluripotent stem cells (iPSCs). We will also review potential
application of genetic modication of adult stem cells and
iPSC through gene editing (CRISPR/Cas9) to improve future
treatments for DFUs.
[21]
Stoff etal. [25]
Mansilla etal. [26]
Kuo etal. [28]
Park etal. [31]
Suh etal. [20]
Silva etal. [32]
Lee etal. [34]
Marrotte etal. [35]
Asai etal. [
Tecilazich etal.
[15]
etal. [37]
Kim etal. [29]
Di Rocco etal.
[39]
Maharlooei etal.
[40]
36]
(continued)
256
https://t.me/med1917
Table 14.1 (continued)
Cell type Animal model Outcome References
ASCs Rat/diabetic ASCs improved vasculogenesis and accelerated wound closure in both normal
ASCs Rat/diabetic Autologous ASC transplantation enhanced skin graft survival in diabetic rats,
ASCs Mice/diabetic ASCs harvested from non-diabetic mice had signicantly improved wound
ASCs Mice/
non-diabetic
ASCs Mince/
non-diabetic
ASCs Mice/diabetic Signicantly advanced granulation tissue formation, capillary formation, and
ASCs Mice/
non-diabetic
ASCs cell sheets Mice/diabetic ASC sheets combined with articial skin accelerated wound healing in type 2
ASCs Mice/diabetic Cell sheets that were combined with articial skin accelerated wound healing
and diabetic rat model.
and promoted angiogenesis through the secretion of growth factors such as
VEGF
healing outcomes when compared to autologous diabetic ASCs
Single-layer sheets of ASCs sheet delivery system on wound Lin etal. [44]
Multi-layered ASC sheets promoted a thicker epidermal surface in a mouse
wound than single-layered ASC sheets
epithelialization at wound site treated with ASCs and matrix
Cell sheet delivery limitations due to poor neovascularization Cerqueira etal.
diabetic mice
and more rapid vascularization
S. Shenk et al.
Nie etal. [41]
Zografou etal.
[42]
Cianfarani etal.
[43]
McLaughlin etal.
[45]
Nambu etal. [10]
[46]
Kato etal. [47]
Jiang etal. [48]
Table 14.2
Bone Marrow Mesenchymal Stem Cells
BM-MSCs Topical brin spray system Both diabetic and non-diabetic wounds healed Falanga etal. [49]
BM-MSCs Articial collagen sponge The improved wound healing in 18 of 20 patients Yoshikawa etal. [50]
BM-MSCs Standard wound dressing Improvement in pain-free walking distance and
BM-MSCs vs PBMCs Injection DFU healing with BM-MSCs more effective than
U-MSCs Endovascular infusion and
Endothelial progenitor cells
EPCs Injection and topical application Complete wound healing achieved 4 ulcer patients,
G-CSF stimulated peripheral
blood CD34+ progenitor
cells
Mononuclear stem cells
BM-MNCs Injection Dermal rebuilding and closure of nonhealing
BM-MNCs Subcutaneous injection Reduction of wound size and increased
BM-MNCs Injection Complete wound healing achieved in 1 patient Kirana etal. [55]
BM-MNCs Intra-arterial implantation Wound healing and neovascularization seen in 20
BM-MNCs + platelets +
brin glue + collagen matrix
BM-MNCs alone vs.
combined with BM-MSCs
PB-MNCs Intramuscular injection Increased blood ow and improved angiogenesis
BM-MNCs vs. PB-MNCs Injection BM-MNC and PBMC treatments in patients with
Adipose stem cells
ASCs Intramuscular injection DFU patients with critical limb ischemia showed
ASCs Injection All 3 patients with DFU treatment showed
ASCs + e-PRP Injection ASC in combination with e-PRP increased ulcer
Human/clinical trials of diabetic foot ulcer therapies
injection
Subcutaneous injection Complete DFU closure in all 5 patients Tanka etal. 2014
Injection and topical application Complete wound healing in 3 cases and signicant
Intramuscular injection or
intra-arterial
reduction in DFU size
BM-MNCs
28 DFU patients showed signicant ulcer healing
from U-MSCs treatment following angioplasty
1 of which due to diabetes
chronic wounds were achieved in all 3 patients
vascularization for 1 patient
diabetic patients
improvement in the remaining 5 cases
18 out of 22 patients showed signiicant
improvement
in patients treated with PBMCs
DFUs and critical limb ischemia (CLI) have no
signicant difference in therapeutic outcome
improved ulcer healing rates
improved clinical outcomes
healing rate in 3 of 3 diabetic patients
Dash etal. [51]
Lu etal. [52]
Qin etal. [53]
Badiava etal. [54]
Badiavas and Falanga
[18]
Humpert etal. [54]
Ruiz- Salmeron etal. [56]
Ravari etal. [56]
Kirana etal. [57]
Ozturk etal. [58]
Dubsky etal. [59]
Lee etal. [60]
Marino etal. [61]
Raposio etal. [62]
14 Cell Therapies: New Frontier fortheManagement ofDiabetic Foot Ulceration
https://t.me/med1917
257
Mesenchymal Stem Cells
Mesenchymal stem cells (MSCs) are found in many tissue
types, including bone marrow, umbilical cord blood, adipose
tissue, dermis of skin, and amniotic membrane [63, 64]
(Fig.14.2). Bone marrow-derived mesenchymal stem cells
(BM-MSCs), also known as marrow stromal cells, are currently the most common source of MSCs for clinical use in
DFU treatment [65, 66]. These clinical applications are
based on the well-characterized plasticity of MSCs, as seen
by their capacity to differentiate into mesenchymal cells
such as osteoblasts, adipocytes, and chondroblasts invitro
[67]. This multipotent differentiation potential, their ability
to be grown and expanded efciently in tissue culture, and
their weak immunogenicity and strong immunoregulatory
effects make them a useful source of cells that can stimulate
human tissue regeneration and wound repair [68].
The cellular plasticity of MSCs for DFU therapy has been
demonstrated in pre-clinical animal and clinical human studies. These studies showed accelerated wound closure through
MSC modulation of the inammatory environment, recruitment of inammatory cells, promotion of neovascularization, and regeneration of appendages [69]. BM-MSCs appear
to be critical for wound repair as seen by elevated production
of cytokines known to enhance wound healing. These cytokines include epidermal growth factor (EGF), keratinocyte
growth factor (KGF), insulin-like growth factor 1 (IGF-1),
vascular endothelial growth factor A (VEGF-A), erythropoietin (EPO), stromal cell-derived factor 1(SDF-1), macrophage inammatory protein 1 alpha and beta (MIP-1 α/β),
and transforming growth factor beta (TGF-β). The release of
these cytokines by BM-MSCs has been shown to recruit
additional MSCs to the wound site, leading to their differentiation into a spectrum of healing-competent cell types [19].
The potential for using MSCs for treatment of DFUs was
rst established using animal models of normal wound repair
[21, 25, 26, 29]. The efcacy of BM-MSCs in healing acute
wounds was shown in non-diabetic rats, where their topical
application signicantly improved repair when compared to
application of mature broblasts. The BM-MSCs increased collagen production, wound strength, and growth factor secretion
[21]. In another study, BM-MSCs, applied as a collagen allograft
to full-thickness wounds on the dorsum of rats, stimulated production of the ECM-degrading enzyme matrix metalloproteinase-9 (MMP-9), which mobilized the angiogenic growth factor
VEGF to accelerate wound healing [29]. Intradermal injection
of human BM-MSCs improved the repair of full-thickness incisional wounds in rabbits [21]. The study also showed that these
cells inhibited scar formation while increasing tensile strength
in the acute, non- diabetic wounds [25]. Similarly, skin regeneration of porcine burn wounds was improved using acellular dermal matrices when combined with autologous, allogeneic, and
xenogeneic BM-MSCs, that resulted in skin and appendage
regeneration with little scarring [26].
Fig. 14.2 Sources of adult stem cells used for treatment of diabetic
foot ulcers. Adult stem cells can be obtained from a variety of sources
including bone marrow, umbilical cord, peripheral blood, fetal aorta,
and adipose tissue. These stem cells have been tested in animal and
human studies and shown to manifest cellular functions that improve
the repair of diabetic foot ulcers. BM-MNC bone marrow-derived
mononuclear cell, BM-MSC bone marrow-derived mesenchymal stem
cell, BM-EPC bone marrow-derived endothelial progenitor cell,
BM-HSC bone marrow-derived hematopoietic stem cell, PBMC peripheral blood mononuclear cell, PB-HSC peripheral blood hematopoietic
stem cell, PB-EPC peripheral blood endothelial progenitor cell,
UC-EPC umbilical cord endothelial progenitor cell, UC-MSC umbilical cord mesenchymal stem cell, UC-HSC umbilical cord hematopoietic stem cell, UC-ENDO umbilical cord endothelial cell, A-EPC aorta
endothelial progenitor cell, ASC adipose stem cell
258
https://t.me/med1917
S. Shenk et al.
These studies in non-diabetic animal models paved the
way for pre-clinical studies in diabetic animals which
demonstrated that BM-MSCs can enhance wound repair
through improved re-epithelialization and granulation tissue
formation, stromal activation, and increased angiogenesis
[22, 23]. The role of BM-MSCs in the stimulation of blood
vessel in- growth was seen in a diabetic mouse model of hindlimb ischemia, where a dramatic increase in blood ow and
increased vessel density was seen in the presence of
BM-MSCs, which was further augmented in the presence of
epidermal growth factor (EGF) [23]. Similar results were
found following the subcutaneous injection of BM-MSCs in
a diabetic rat, which led to enhanced wound healing by promoting angiogenesis, cellular proliferation, and augmented
granulation tissue thickness [30]. Additionally, when
BM-MSCs were topically applied to wounds in streptozotocininduced Type I diabetic mice, they signicantly enhanced
wound healing by increasing angiogenesis and VEGF secretion, decreasing inammation and leukocyte recruitment
which resulted in improved tissue remodeling [28].
These diabetic animal studies provided proof of concept
for the application of BM-MSCs in human clinical trials
(Table 14.2). Human BM-MSCs have been shown to effectively heal DFUs when they were delivered using a spray
delivery system consisting of brinogen and thrombin [49]. A
recent study evaluating feasibility of transplanting BM-MSCs
demonstrated wound healing and improved leg perfusion in
all but two of twelve patients with diabetic foot ulcers [57].
When BM-MSCs were incorporated into an articial dermis
consisting of a collagen sponge and implanted subcutaneously, the DFU patients demonstrated regeneration of subcutaneous tissues and a robust vascular response [50]. Wound
dressings delivering BM-MSCs were found to effectively
treat DFU patients, resulting in reduced pain and ulcer size
following dermal regeneration [51]. Improved angiogenic
responses induced by BM-MSCs were also seen when these
cells were injected into the lower limb of Type II diabetic
patients [52]. Additionally, umbilical cord mesenchymal stem
cells (UC-MSCs) were found to reduce lower limb amputation rates and increase vessel count, suggesting that UC-MSCs
improved angiogenic responses in these patients [53].
Hematopoietic Stem Cells
Hematopoietic stem cells (HSCs) are CD45-positive and
CD34-positive progenitor cells that give rise to all differentiated blood cell types. HSCs are the most abundant cell type
in the bone marrow but can also be isolated from peripheral
or umbilical cord blood (UCB) (Fig.14.2). HSCs have been
shown to enhance DFU healing during both the inammatory and proliferative phases of diabetic wounds [16, 70, 71].
It was shown that UCB-derived HSCs had the capacity to
differentiate into endothelial cells that when combined with
HSCs could heal wounds in diabetic mice when applied in a
topical gel [72]. Recent ndings in diabetic rats indicate
CD93-positive, hematopoietic stem cells can improve diabetic wound healing by increasing activation of vascular
endothelial growth factors (VEGF) protein expression while
simultaneously downregulating the death-associated protein
kinase 1 (DAPK-1) gene [73]. Similar ndings were shown
in human studies when UCB-derived HSCs were combined
with UCB-derived MSCs to augment granulation tissue production and improve DFU healing [74].
Endothelial Progenitor Cells
Endothelial progenitor cells (EPCs) are recruited from the
bone marrow (BM-EPCs) into the peripheral circulation in
response to stimuli, where they acquire the capacity to stimulate angiogenesis and wound repair [15, 75, 76] (Fig.14.2).
These cells display surface markers that are typical of both
endothelial and hematopoietic cells and for vascular structures important for vasculogenesis and angiogenesis [77].
BM-EPCs are found in decreased numbers in diabetic
patients, thus suggesting a link between reduced BM-EPCs
and patient at risk for developing DFUs [15, 16]. Issan etal.
(2012) found that there was attenuation and senescence of
EPCs in high glucose environments which ultimately led to
endothelial dysfunction in coronal arteries [78].
A recent study in diabetic mice demonstrated decreased
numbers of EPCs not only in the peripheral blood, but also in
the wound bed. In addition, numbers of EPCs were increased
after blocking the pro-inammatory receptor CXCR4 with
its antagonist and mice showed increased angiogenesis and
cell proliferation and subsequent wound closure in the presence of increased numbers of EPCs, suggesting enhancement of DFU healing [79]. Human BM-EPCs have been
injected into the circulation of nude mice where they have
been found to localize to areas of tissue ischemia and induce
blood vessel in-growth. This study suggested that transplantation of human BM-EPC may have a potential role in the
regeneration of ischemic tissue in humans [31]. In Type II
diabetic mice, BM-EPCs collected from normal mice accelerated wound closure compared to BM-EPCs collected from
diabetic mice [35]. BM-EPCs that were applied topically to
full-thickness wounds on the dorsum of diabetic mice were
found to accelerate wound closure and increase vascularity
[36] and to accelerate wound closure in ischemic mouse hind
limbs when delivered in a bioactive vehicle [32].
EPCs derived from other sources have also been shown to
improve wound healing. Human fetal aorta-derived EPCs
have been found to stimulate wound healing and promote
angiogenesis in streptozotocin-induced diabetic nude mice
[33] while human cord blood-derived EPCs activated kerati-
14 Cell Therapies: New Frontier fortheManagement ofDiabetic Foot Ulceration
https://t.me/med1917
259
nocyte and broblast proliferation to stimulate wound closure [80]. Injection of peripheral blood-derived EPCs into
mice accelerated wound re-epithelization and improved
macrophage responses when compared to injection with
mature endothelial cells [20]. These therapeutic effects were
also seen using endothelial cell-conditioned media, suggesting that the delivery of paracrine growth factors may be a
viable therapeutic option for DFUs instead of delivery of
cells themselves. In addition, it was found that the secreted
growth factors released into cultured media by EPCs derived
from human embryonic stem cells were able to successfully
close wounds in a nude mouse model [34].
Relatively few human clinical studies have been performed to assess potential benets of EPCs in the treatment
of DFUs. Badivas et al. showed increased blood vessel
growth and wound closure when BM-EPCs were topically
applied to or injected in human DFUs [54]. In addition,
DFUs have shown improved wound closure and vascular
perfusion when peripheral blood-derived EPCs were stimulated with the cytokine granulocyte-colony stimulating factor (G-CSF) in a prospective human clinical trial. This trial
showed that combined therapies of CD34-positive stem cells
and cytokines could offer a safe and efcacious DFU treatment [71].
Mononuclear Stem Cells
Mesenchymal stem cells, hematopoietic stem cells, and
endothelial progenitor cells are all found in the mononuclear
cell fraction derived from bone marrow or peripheral blood
[81–84] (Fig.14.2). This heterogeneous population of mononuclear cells has been used collectively in cell therapy applications for treating diabetic ulcers [85]. Autologous bone
marrow-derived mononuclear cells (BM-MNCs) were found
to stimulate wound vascularization following their intraarterial delivery in DFU patients with peripheral artery disease [56]. An alternative approach was used to improve
healing of DFUs that were refractory to other treatments by
delivering BM-MNCs in a collagen-brin gel [86]. Multiple
case reports have demonstrated a similar efcacy of
BM-MNCs to improving both DFU repair and angiogenic
responses in diabetic patients [55, 87].
As an alternative to BM-MSCs, peripheral blood mononuclear cells (PBMCs) have been found to improve wound
healing. Human-derived PBMCs alone [37] and PBMCs
mixed with broblasts [38] have both been found to accelerate epidermal wound healing in mice [58]. Mildner et al.
(2013) were able to show that supernatant emulsions containing the secretome of PBMCs signicantly accelerated
wound healing in mice and induced wound healingassociated activation in human, primary skin cells.
Importantly, patients with Type II diabetes with DFUs and
limb ischemia showed increased blood ow and improved
angiogenesis when treated with PBMCs [88].
Since both PBMCs and BM-MNCs have shown efcacy
for DFU therapy, it was important to determine if there were
relative benets for each in DFU patients. Dubsky et al.
(2013) compared BM-MNC and PBMC treatments in patients
with DFUs and critical limb ischemia (CLI) and reported no
signicant difference between them in therapeutic outcome
[59], suggesting that their healing potential is similar.
Additionally, Lu etal. (2011) explored differences in both the
safety and efcacy between BM-MNCs and BM-MSCs. In
this study, patients with Type II diabetes exhibiting DFUs and
CLI and treated with BM-MSCs were found to have greater
healing than with BM-MNCs [52]. In contrast, when treatments with BM-MNCs or BM-MSCs were compared using
two different delivery methods (intramuscular injection vs.
intra-arterial infusion), it was found that both types of cells
showed similar healing responses [57].
Adipose Tissue-Derived Stromal Cells
Adipose tissue-derived stromal cells (ASCs) are mesenchymal cells found in adipose tissues that are able to differentiate into a spectrum of cell types that may be important for
wound healing [89] (Fig. 14.2). When ASCs were transplanted into non-diabetic animal wounds, they were shown
to promote re-epithelialization and angiogenesis [90–95].
Responses to human-derived ASCs have been studied in diabetic mouse and rat models, where stimulation of angiogenesis and improved tissue remodeling were seen [39].
Genetically modied ASCs, designed to overexpress the
chemokine stromal-derived factor-1 (SDF-1), were found to
promote healing in STZ-induced diabetic mice [40, 96].
ASCs used to treat STZ-induced diabetic rats accelerated
wound healing that was linked to a decrease in the density of
broblasts, suggesting that improved diabetic wound healing
was likely to be controlled by mechanisms other than accumulation of collagen produced by broblasts [41]. This study
demonstrated that autologous ASC treatment in diabetic rats
could close wounds and was linked to the elevated expression of vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), and broblast growth factor 2
(FGF2). This efcacy of ASCs was further supported when
autologous ASCs combined with full-thickness skin grafts
were used in a diabetic rat model. ASCs enhanced skin graft
survival and resulted in greater collagen density, increased
levels of VEGF, and improved angiogenesis [42]. Another
diabetic rat model study found that ASCs that secreted exosomes promoted proliferation and angiopoiesis in EPCs in a
high glucose environment. As a result, wounds in diabetic
rats had a signicantly reduced ulcerated area when treated
with these exosomes. Increased granulation tissue formation,
260
https://t.me/med1917
S. Shenk et al.
angiogenesis, and levels of growth factor expression, as well
as reduced levels of inammation and oxidative stressrelated proteins, were detected in the wound beds [97].
Together these studies support previous ndings that ASCs
promote new vessel formation and granulation tissue deposition to accelerate wound healing. When the wound healing
efcacy of ASCs from diabetic and non-diabetic mice were
compared, it was found that ASCs harvested from nondiabetic mice had signicantly improved wound healing outcomes when compared to autologous, diabetic ASCs. ASCs
from non-diabetic mice stimulated signicantly greater
amounts of granulation tissue, collagen deposition, and vessel density in diabetic wounds [43].
Novel approaches have been used for the delivery of
ASCs to wound sites. Lin etal. formed single-layer sheets of
ASCs by culturing ASCs in a monolayer on a temperaturesensitive N-isopropylacrylamide (PIPAAm) that allowed
formation of ASC sheets which were easily detached from
the cell culture surface and layered on top of each other.
These multi-layered cell sheets generated greater collagen
density in the wounds of athymic nude mice than singlelayered sheets [44]. Similarly, McLaughlin etal. found that
such multi-layered ASC sheets promoted a thicker epidermal
surface in a mouse wound than single-layered ASC sheets
and ASCs generated on standard cell culture surfaces [45].
Other studies have examined seeding ASCs in an articial
dermal substitute and placing it directly on a wound created
on diabetic mice. The treatment signicantly enhanced granulation tissue formation, capillary formation, and epithelialization [10]. However, skin substitutes for stem cell delivery
have been limited by the poor vascularization of these scaffolds [46]. To overcome this obstacle of cell delivery, Kato
etal. treated skin defects in Type II diabetic rats using allogeneic ASCs that were incorporated into cell sheets that were
combined with articial skin, resulting in accelerated wound
healing and more rapid vascularization than with either treatment alone [47]. Similarly, Jiang etal. used a diabetic, porcine wound model to demonstrate that the combination of
collagen scaffolds and autologous ASC sheets resulted in
higher vascularization and expression of VEGF when compared to ASC sheets alone or topically applied ASCs [48,
97]. Thus, the combination of three-dimensional scaffolds
with ASC sheets offers a novel delivery modality to accelerate wound closure and enhance angiogenesis, cell migration,
and proliferation.
DFU patients with critical limb ischemia and injected
intramuscularly with cells differentiated from ASCs showed
clinical improvement as evidenced by decreased claudication, healed amputation sites, lengthened walking distances,
increased ulcer healing rates, and formation of more numerous vascular collateral networks [60]. Similarly, Marino
et al. showed improved healing in patients with diabetic
ulcers and peripheral arterial disease after intradermal injec-
tion of ASCs harvested from non-diabetic patient donors
[60]. The treatment of non-healing chronic ulcers, including
DFUs, was also enhanced when ASCs were used with or
without enhanced platelet-rich plasma (e-PRP) [61].
In summary, bone marrow-derived mesenchymal stem
cells, hematopoietic stem cells, endothelial progenitor cells,
bone marrow-derived and peripheral blood mononuclear
cells, and adipose tissue-derived stem cells have all been
shown to improve wound healing in pre-clinical animal models and human clinical trials. While great strides have been
made in using such stem cell therapy for treating diabetic
foot ulcers with these cell types, more detailed analysis of
the therapeutic mode of action of these cells remains to be
elucidated. Importantly, a limitation of these sources is that
they may not always supply an unlimited quantity of autologous cells for therapy.
Future Directions andNew Technologies
A new replenishing source of multiple stem cell types that
have been shown to be important for wound healing is
induced pluripotent stem cells (iPSCs). In addition, gene
editing strategies, such as CRISPR/Cas9, are emerging as
promising technologies to modify specic genes, thus altering the production of proteins that may improve wound healing in patients with DFUs. We review the principles of these
two novel technologies and discuss their potential impact on
DFU therapy.
Induced Pluripotent Stem Cells
James Thomson’s landmark study in 1998 discovered a
method for isolating human embryonic stem cells (hESCs)
[62]. These pluripotent stem cells were shown to give rise to
cells from all three embryonic germ layers and possibly primordial germ cells (PGCs) [98]. hESCs have the potential to
be used as a source of cells for regenerative medicine; however, due to ethical and legal issues regarding research using
human embryos, progress on the clinical application of these
cells has been limited. An alternative source of pluripotent
stem cells was discovered when [99] showed that mouse
embryonic broblasts (MEFs) could be reprogrammed to a
state that was similar to mouse embryonic stem cells by
using only four transcription factors (OCT-4, KLF4, SOX2,
c-MYC (OKSM)) [100]. In 2007, Yamanaka etal. demonstrated an approach that successfully reprogrammed human
broblasts to a pluripotent state. These cells, known as
“induced pluripotent stem cells” (iPSCs), show many of the
same self-renewal and differentiation capabilities as hESCs.
This same reprogramming approach has now been applied to
a wide variety of somatic cell sources [101–108].
14 Cell Therapies: New Frontier fortheManagement ofDiabetic Foot Ulceration
https://t.me/med1917
261
The ability to reprogram many types of somatic cells into
iPSCs shows promise for diabetic patient-specic cell derivation. iPSCs have been generated from skin broblasts of
patients with maturity-onset diabetes of the young (MODY)
[109, 110], patients with Type I and Type II diabetes [111–
113], Juvenile-onset Type I diabetes [114], and directly from
DFU-derived broblasts in patients with Type II diabetes
[115]. Gerami-Naini et al. successfully reprogrammed primary broblast cell lines derived from DFUs to iPSCs and
compared them to iPSCs derived from non-ulcerated foot
skin from diabetic patients and from healthy foot skin from
non-diabetic patients [115]. These studies have established
that primary, DFU-derived broblasts can be reprogrammed
with efciencies similar to non-diabetic control broblasts,
thus holding promise for future diabetic patient-specic
regenerative therapy of DFUs.
In order for iPSCs to serve as an improved source of cells
for both autologous and allogeneic cell therapies, it will be
necessary to differentiate them into the multiple cell types
needed for the treatment of DFUs. This is a feasible goal, as
pluripotent stem cells have previously been differentiated
into endothelial cells [116–119], smooth muscle cells [117,
120], adipocytes [121, 122], broblasts [123–125], keratino-
cytes [126–131], motor and sensory neurons [132–135], and
mesenchymal stem cells [123, 136–140]. All of these are relevant for improving wound healing in patients with diabetic
foot ulcers. However, to date, iPSC-derived cells that were
initially reprogrammed from DFU-derived broblasts have
only been used to generate cells in a broblast lineage [115]
(Fig.14.3).
Improvements in reprogramming methods for iPSC generation will be required to ensure the safe and effective use of
iPSCs for DFU treatment in clinical trials. One barrier to the
safe clinical translation of iPSCs [141] is the use of lentiviral- and retroviral-based vectors to express the OKSM reprogramming factors in a somatic cell of interest. These systems
are both accompanied by transgene integration into the
genome, leading to an increased risk of acquisition of harm-
Fig. 14.3 Cells differentiated from iPSCs for use as a cell source for
DFU treatment. iPSCs reprogrammed from many cell types and directly
from DFUs can be differentiated into multiple cell types needed for
DFU treatment, including keratinocytes, endothelial cells, adipocytes,
mesenchymal stem cells, smooth muscle cells, and neurons
262
https://t.me/med1917
S. Shenk et al.
ful changes in gene expression. To address this problem,
non-integrative viral approaches, such as adenovirus and
Sendai virus, have been developed. However, the efciency
of iPSCs with adenovirus generation is much lower compared to lentivirus and retrovirus [142]. During the last
decade, several non-viral reprogramming methods have also
been established. These include delivering a plasmid vector
[143], using episomal plasmids as “minicircle vectors” [103,
144], expressing OKSM mRNA [145] directly delivering
OKSM proteins [146] or inducing expression of OKSM factors with microRNA [147] or with small molecules [148–
150]. When these varied reprogramming methods have been
compared, Sendai virus has been shown to be the most efcient means of reprogramming somatic cells to a pluripotent
state without integration [151].
The risk of tumor formation from iPSC-derived cells is
another challenge associated with their safe use for DFU
treatment [152, 153]. This risk arises because formation of
teratomas upon implantation into mice is the sine qua non for
establishing the pluripotency of fully reprogrammed iPSCs
[154, 155]. Thus, reliable in vitro differentiation protocols
must be coupled with screening assays that will ensure the
absence of residual pluripotent cells before use in DFU therapy [156]. Virus-free, transgene-free methods of reprogramming such as miRNA, episomal plasmid, non-integrating
Sendai virus, and recombinant protein methods will reduce
the risk of tumorigenicity that can result from viral- mediated,
insertional mutagenesis. In addition, it is likely that iPSCderived cells will be extensively manipulated exvivo by the
time that are ready for clinical use. This is known to lead to
chromosomal aberrations and altered cell phenotypes [157,
158]. In light of this, cell therapies for DFUs will require
extensive pre-clinical testing to ensure that cells differentiated from iPSCs do not contain genetic alterations, or harbor
residual virus or residual pluripotent cells that might generate harmful tumors.
The efcacy and safety of several iPSC-derived cells are currently being tested in a small number of clinical trials. Previous
trials have tested iPSC-derived cells as a treatment for acute graft
versus host disease (AGVH), wet age-related macular degeneration (AMD), dry AMD, and Stargardt’s macular dystrophy (clin-
icaltrials.gov ID NCT02923375, NCT02464956, NCT01691261,
NCT01344993, NCT02463344, NCT02286089, NCT02755428,
NCT01345006, NCT01469832, NCT02941991, NCT02445612,
NCT02749734). In 2023, there are 7 clinical trials testing the
efcacy of iPSC-derived interventions; multiple studies are
underway using iPSC-derived cardiomyocytes as treatment for
heart failure and cardiovascular diseases (clinicaltrials.gov ID
NCT05647213, NCT05566600, NCT04696328, NCT04982081,
NCT04396899). Additionally, there are numerous studies testing
the use of iPSC cells for treatment of macular degeneration and
related retinal diseases (clinicaltrials.gov ID NCT04339764,
NCT05445063, NCT04339764). This increases the promise of
iPSC therapies being applied to diabetic wounds in the future.
Gene Editing ofDFU-Derived Cells forStem
Cell Therapy
Genome editing technologies, such as zinc nger nucleases
[159], TALENs [160], and CRISPR/Cas9 [161, 162], can
now be used to modify specic genes in primary DFUderived cells or iPSCs to improve their wound healing potential. In recent years, genome editing has advanced
dramatically with the discovery of Clustered Regularly
Interspaced Short Palindromic Repeats (CRISPR), an important bacterial immune defense system [163–165] that has
been harnessed to specically target and modify genes with
high efciency. CRISPR technology relies on CRISPRassociated protein 9 (Cas9), which is an RNA-guided DNA
endonuclease [48, 166], and a “guide RNA” (gRNA) consisting of CRISPR RNA (crRNA) fused to transactivating
RNA.A crRNA is necessary for the precise targeting of the
CRISPR/Cas9 complex to a specic sequence in the genome.
Gene editing occurs as transactivating RNA binds Cas9 protein [161, 162, 167], making it possible for Cas9 to introduce
a double strand break, disrupting the expression of the gene
of interest. Alternatively, CRISPR/Cas9 can also correct a
specic mutation, by inserting a sequence provided on the
donor template.
At this point, a relatively small number of mutations associated with poor wound healing in diabetic patients have
been identied, thus limiting the number of genes that could
be targeted by gene editing. While the role of genetic mutations in the pathogenesis of DFUs remains poorly understood, numerous genome-wide association studies (GWAS)
have identied single nucleotide polymorphisms (SNPs)
associated with Type I and Type II diabetes [168–171], that
may prove to be useful gene editing targets in the future. For
example, SNPs in IL-6, TNF-α, and SDF-1, which are genes
known to be associated with DFUs [172], may be useful
genomic targets that could be CRISPR modied in order to
improve DFU healing. The potential use of CRISPR/Cas9 to
treat DFUs will also be complicated by the multifactorial
nature of chronic wound healing, which will likely necessitate modication of multiple genes in individual cells.
Currently, up to seventeen different genomic loci have been
simultaneously targeted in individual cells using CRISPR/
Cas9 [161, 173–180]. While modifying multiple gene targets
in the same cell may be feasible, the number of targets sufcient to improve wound healing in diabetic patients remains
to be determined. Clinical applications of CRISPR/Cas9
14 Cell Therapies: New Frontier fortheManagement ofDiabetic Foot Ulceration
https://t.me/med1917
263
technology will also require consideration of possible offtarget effects, which can lead to unintended changes in
expression of genes, requiring further optimization and
renement of Cas9 delivery [181–186]. Until 2018, CRISPR/
Cas9 technology for clinical trials was only approved in
China for the treatment of cancer (ClinicalTrials.gov ID
NCT02793856, NCT02867345, NCT02863913,
NCT02867332). In 2019, the rst clinical trials using
CRISPR/Cas9 technology in the United States began for the
treatment of cancer, beta thalassemia, and sickle cell disease
(ClinicalTrials.gov ID NCT03399448, NCT03745287,
NCT03655678). The results in clinical trials have been
promising. In one study, researchers isolated blood stem
cells from bone marrow of seven sickle-cell disease patients
and utilized CRISPR to modify the gene responsible for production of usable hemoglobin. Upon re-infusion of the
CRISPR-modied cells, at the 26-month follow-up, all seven
patients were free of vaso-occlusive crisis. This indicates a
promising new frontier of the use of CRISPR technologies
for DFU patients, with potential to utilize genome modication to improve wound healing and vascular phenotypes. In
2023 there were 19 clinical trials using CRISPR/Cas9 technology, targeting hematopoietic cancer and diseases of the
urinary tract and eyes.
As an alternative approach, the Cas9 protein can be repurposed to regulate transcription of genes that are dysregulated
in diabetic patients. For example, Cas9 protein with mutations in the nuclease domain cannot introduce double strand
breaks but can still be targeted to a specic locus using
gRNA [187, 188]. This mutated Cas9 can be fused to
enzymes which will then epigenetically regulate gene
expression at that locus. Alternatively, a reversible CRISPR
interference technique has been employed by targeting
mutated Cas9 to the transcriptional start site, thus blocking
gene transcription [189]. As genetic mutations may not be
sufcient targets for editing in DFUs, it is likely that transcriptional modication using CRISPR/Cas9 will be useful
in the future.
In summary, CRISPR/Cas9 technology holds future
promise to modify specic diabetic gene signatures either by
correcting disease-causing mutations or through epigenetic
modication of expression of diabetes-associated genes that
may lead to an improved cellular wound healing phenotype.
Additionally, regulation of diabetes induced-gene expression
may be achieved without CRISPR editing, simply by reprogramming primary DFU-derived cells to iPSCs [190], as it is
known that cells undergo extensive transcriptional and epigenetic remodeling during reprogramming [191]. In conclusion, iPSC technology combined with CRISPR/Cas9 genome
engineering will be a powerful approach to obtain cells with
improved wound repair features that can be valuable for
novel cell therapies needed to improve diabetic wound
healing.
3D Tissue Models forPre-Clinical Drug Testing
ofDFU Therapies
The spatially and temporally controlled events that occur
during tissue morphogenesis and repair need to be studied in
biological systems in which a high degree of tissue complexity can be achieved to recreate an invivo-like tissue microenvironment. Biologically meaningful signaling pathways,
including those that direct the proliferation and differentiation of epidermal stem cells, function optimally when cells
are spatially organized in 3D tissues rather than in rudimentary 2D, monolayer culture systems. In this light, the development and application of 3D tissue models that mimic
healthy human skin and chronic wounds will play an important role in moving discovery of new treatments for DFU into
a pre-clinical, drug screening platform. Bioengineered 3D
tissues can provide experimental systems that are characterized by growth factor-directed cell-cell crosstalk and the
presence of ECM-mediated cues that can recreate the complexity of conditions like DFUs. Existing pre-clinical therapeutic testing involves comparing the effects of stem cells on
wound healing in both normal and diabetic animals. However,
it is now clear that safety and efcacy testing of stem cell
therapies for DFU treatment can be streamlined using 3D
tissues that are developed with DFU patient-derived cells.
equivalents (HSEs) (Fig.14.4). These are tissues fabricated
by assembling a layer of primary broblasts in a collagen gel
that serves as the “stroma” for the fully differentiated keratinocytes that are grown above them at an air-liquid interface.
Functional broblasts in the stromal layer consisting of Type
I Collagen and secreted extracellular matrix proteins support
the proliferation and differentiation of primary keratinocytes
to form this fully differentiated, stratied epithelium [192–
194]. These HSEs have been used to successfully model
behaviors of normal skin and disease-specic tissues [194]
and that primary, DFU-derived broblasts can be incorporated into skin-like tissues that mimic DFUs [195]. Maione
etal. compared the phenotype of 3D tissues harboring either
cells from the foot of healthy, diabetic, or DFU patients and
compared differences in the phenotype of patient broblasts
derived from these sources. The proliferation of surface keratinocytes was found to be elevated in the presence of DFU
broblasts when compared to those from healthy donors,
which simulates ndings from DFUs in patients [195].
One example of such a tissue model system is human skin
264
https://t.me/med1917
Fig. 14.4 Skin equivalent
tissue model for testing cells
for DFU therapy. Cells
differentiated from primary
and iPSC-derived cells can be
used to construct 3D HSE
tissues with many features of
human skin. These tissues can
incorporate patient-derived
cells, as well as adult or
iPSC-derived stem cells and
for use in pre-clinical drug
testing, disease modeling, and
characterization of cell
functions
S. Shenk et al.
In addition, a 3D model of wound healing using HSEs has
been developed by creating a full thickness wound in HSEs
and monitoring re-epithelialization of surface keratinocytes
from the wound edge toward the wound center (Fig.14.5)
[192]. Maione et al. demonstrated that in this 3D wound
healing model, DFU-derived broblasts showed delayed
wound re-epithelialization of surface keratinocytes grown at
an air-liquid interface [195]. This wound healing model
using diabetic patient-specic HSEs can now be used to
effectively test the efcacy and safety of potential therapeutics designed to accelerate wound repair before clinical trials. Recently, three-dimensional tissue models have been
used to show that extracellular matrix derived from iPSCderived broblasts that were initially generated from diabetic
patients generated scaffolds that can be applied to wound
healing [196] and for diabetic wound healing [197].
Additionally, HSEs can be integrated into 3D microuidic
devices in which multiple tissue types are grown, known as
organs-on-chips, which can better mimic interactions
between different tissue types to serve as a more predictive
model for pre-clinical testing of cell therapeutics for DFUs
[198, 199]. While tissue models are currently being tested
with pancreatic beta cells to mimic diabetes, it is hoped that
they will be applied to diabetic wounds in the future.
While these existing 3D tissue models of skin and other
stratied epithelial tissues provide many benets for
mimicking human disease states, it would be advantageous to develop additional complexity in these tissues.
To accomplish this, it will be important to incorporate
cells related to wound healing and immune response, such
as macrophages and T cells. Smith et al. demonstrated
that incorporation of patient-derived macrophages could
be incorporated into this tissue model and demonstrated
production of inammatory cytokines important for DFU
pathogenesis and healing [200]. A future step in the development of 3D skin-like tissues will be the ability to generate tissues that are personalized tissue “surrogates” of
their in vivo counterparts, by incorporating multiple
autologous cell types from DFU- derived patients. Beyond
this, 3D tissue models enable development of well-characterized benchmarks to conrm that cells differentiated
from iPSCs have acquired cell phenotypes and functions
that will best mimic features of skin. This can be accomplished by developing reproducible techniques for the
derivation and characterization of iPSC- derived broblasts and other cell types that can assemble endogenous
3D ECM or provide soluble factors essential for optimal
skin fabrication.
Соседние файлы в папке Библиотека им академика М.И. Перельмана