**2.6. Mesenchymal and pluripotent stem cells, cell therapy and tissue engineering aspects**

MSC and other cell types such as the pluripotent stem cells have huge potential for cell therapy and tissue engineering in various diseases. Recently, most clinical trials in cardiovascular field have been performed with MSC or MSC-derivatives [55]. Furthermore, cardiovascular derivatives of pluripotent stem cells are promising tools to differentiate new cardiovascular cells and to build cardiovascular tissue. Latest tissue engineering methods comprise biodegradable matrices combined with cellular building blocks.

MSC and PSC behave and differentiate altered in normal hypoxic or in hyperbaric oxygen conditions PSC studies concluded that altered oxygen levels may mimic in utero conditions

Despite huge promises, the CHART-1 trial failed to reach primary composite endpoint and cell implantation did not improve functional status of the patients [38–40, 60, 61]. The preimplanted cells received a growth factor cocktail, but none-of oxygen level modification was performed during pretreatment [62]. Earlier studies proved that hypoxic preconditioning of MSC increases their secretion of pro-angiogenic, anti-fibrotic, anti-apoptotic secretome, which are known as the paracrine mechanism [63]. Some of these trials comprised temporary anoxia as well [64]. Beside hypoxic pretreatment, other studies aimed hyperbaric pretreatment of regenerative studies [65]. Preconditioning in HBOT circumstances had advantageous effects on neuronal cells as well [65]. *In vitro* part of small animals' trials proved that HBOT can induce hypoxia tolerability of spinal neurons. The mechanisms of actions behind these beneficial effects were metabolic coping, especially altered glucose homeostasis [66]. Thus, these experiments underpinned that HBOT has direct effect on metabolomics and energy homeostasis of cellular compartments, as different oxygen levels await altered metabolic actions [66]. HBOT in MSC resulted in increased proliferative capacity of the cells when compared to those MSC treated in normal oxygen circumstances [11, 29]. In this study, secretome of MSC was evaluated via the ELISA method and levels of BDNF were investigated. This peptide has pivotal role in neurodegenerative diseases but also reported to play a role in salvage mechanisms of the central nervous system after a cardiac arrest [67]. BDNF secretion of MSC significantly increased after HBOT treatment, but was also improved in hypoxia [45]. These results widely clue if normal oxygen levels are suitable to culture and maintain MSC and their derivatives. Further cell therapy trials are needed to standardize cell culture protocols, because recent

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Endothelial progenitor cells [68] are circulating in blood and released from bone marrow. Some studies outline their potential biomarker role for ischemic cardiovascular conditions, as far as their level is increased in acute myocardial infarction and chronic hind-limb ischemia [69]. Endothelial progenitor cells may also have therapeutic effects and phase II/III clinical trials aim boosting them by external infusion of activating factors [70]. An activator drive of these circulating progenitor cells could also be HBOT [71]. Repeated HBOT resulted in significant release of circulating CD34 positive progenitor cells in the peripheral blood. The

If directed differentiation is aimed to be supported, MSC may be cultured in HBOT circumstances. Interestingly, HBOT enhanced osteogenic differentiation of MSC, which *in vitro* description was further proved via *in vivo* proof-of-concept studies as well [11]. Interestingly, metabolic activities, especially calcium influx and exchange, were also modulated by HBOT in MSC. HBOT increased the activity of calcium homeostasis, which is key for osteoblasts via

Pluripotent stem cells are sensitive cell cultures *in vitro*. Their maintenance requires special techniques and expertise in the field. Pluripotent cells form pluripotent cell clusters *in vitro*. They require special maintenance pluripotent stem cell media. Through passaging, spontaneously differentiated cells have to be picked and removed from culture. Recently, enzymatic passage became more advantageous than mechanical breaking of pluripotent colonies.

variations disable direct comparative analyses.

mechanisms were NOS dependent [71].

proliferation and bone formation [11].

**Figure 4.** Effects of HBOT on wound healing and fibroblasts SOD: superoxide dismutase, MMP: matrix-metalloproteinase, ROS: reactive oxygen species.

better and thus may initiate differentiation potency [56]. Latest state-of-the-art molecular biology protocols comprise epigenetic or genetic modifications for example reprogramming and CRISPR/Cas9 genome editing technique [57, 58]. These are often utilized parallel with altered oxygen levels. Signaling steps related to these mechanisms also changed including MAP kinases [44].

MSCs are multipotent stem cells which by definition have the potency to differentiate into cartilage bone muscle tendon ligament and fat tissue. MSC can be characterized via cell surface markers: they widely express CD73, CD90 and CD105 but do not express CD11, CD14, CD19, CD34 and CD45 [57]. They are easy to culture adhere to plastic and most cell culture surfaces and can proliferate in MSC media and others as well. By directed differentiation they can differentiate into chondrogenic osteogenic myogenic and adipogenous linage [57]. It is debated if mature cardiomyocytes can derive from MSC.

Hypoxic preconditioning is currently being investigated also in human clinical trials as a protective mechanism of ischemia-reperfusion injury in the ischemic myocardium [59]. Related to this, ischemic preconditioning is being evaluated in the *in vitro* setting and in clinical trials. Recently, MSC were the most robust players in cardiovascular cell therapy trials. For instance, the CHART-1 clinical trial involved hundreds of patients suffering from chronic ischemic heart failure. Cardiopoietic cells, derived from MSC, were implanted endomyocardially. Despite huge promises, the CHART-1 trial failed to reach primary composite endpoint and cell implantation did not improve functional status of the patients [38–40, 60, 61]. The preimplanted cells received a growth factor cocktail, but none-of oxygen level modification was performed during pretreatment [62]. Earlier studies proved that hypoxic preconditioning of MSC increases their secretion of pro-angiogenic, anti-fibrotic, anti-apoptotic secretome, which are known as the paracrine mechanism [63]. Some of these trials comprised temporary anoxia as well [64]. Beside hypoxic pretreatment, other studies aimed hyperbaric pretreatment of regenerative studies [65]. Preconditioning in HBOT circumstances had advantageous effects on neuronal cells as well [65]. *In vitro* part of small animals' trials proved that HBOT can induce hypoxia tolerability of spinal neurons. The mechanisms of actions behind these beneficial effects were metabolic coping, especially altered glucose homeostasis [66]. Thus, these experiments underpinned that HBOT has direct effect on metabolomics and energy homeostasis of cellular compartments, as different oxygen levels await altered metabolic actions [66].

HBOT in MSC resulted in increased proliferative capacity of the cells when compared to those MSC treated in normal oxygen circumstances [11, 29]. In this study, secretome of MSC was evaluated via the ELISA method and levels of BDNF were investigated. This peptide has pivotal role in neurodegenerative diseases but also reported to play a role in salvage mechanisms of the central nervous system after a cardiac arrest [67]. BDNF secretion of MSC significantly increased after HBOT treatment, but was also improved in hypoxia [45]. These results widely clue if normal oxygen levels are suitable to culture and maintain MSC and their derivatives. Further cell therapy trials are needed to standardize cell culture protocols, because recent variations disable direct comparative analyses.

Endothelial progenitor cells [68] are circulating in blood and released from bone marrow. Some studies outline their potential biomarker role for ischemic cardiovascular conditions, as far as their level is increased in acute myocardial infarction and chronic hind-limb ischemia [69]. Endothelial progenitor cells may also have therapeutic effects and phase II/III clinical trials aim boosting them by external infusion of activating factors [70]. An activator drive of these circulating progenitor cells could also be HBOT [71]. Repeated HBOT resulted in significant release of circulating CD34 positive progenitor cells in the peripheral blood. The mechanisms were NOS dependent [71].

better and thus may initiate differentiation potency [56]. Latest state-of-the-art molecular biology protocols comprise epigenetic or genetic modifications for example reprogramming and CRISPR/Cas9 genome editing technique [57, 58]. These are often utilized parallel with altered oxygen levels. Signaling steps related to these mechanisms also changed including

**Figure 4.** Effects of HBOT on wound healing and fibroblasts SOD: superoxide dismutase, MMP: matrix-metalloproteinase,

Decreased tissue ROS levels

**HBOT Wound healing**

Increased SOD levels

> Increased MMP activity

Decreased ROS levels

MSCs are multipotent stem cells which by definition have the potency to differentiate into cartilage bone muscle tendon ligament and fat tissue. MSC can be characterized via cell surface markers: they widely express CD73, CD90 and CD105 but do not express CD11, CD14, CD19, CD34 and CD45 [57]. They are easy to culture adhere to plastic and most cell culture surfaces and can proliferate in MSC media and others as well. By directed differentiation they can differentiate into chondrogenic osteogenic myogenic and adipogenous linage [57]. It is

Hypoxic preconditioning is currently being investigated also in human clinical trials as a protective mechanism of ischemia-reperfusion injury in the ischemic myocardium [59]. Related to this, ischemic preconditioning is being evaluated in the *in vitro* setting and in clinical trials. Recently, MSC were the most robust players in cardiovascular cell therapy trials. For instance, the CHART-1 clinical trial involved hundreds of patients suffering from chronic ischemic heart failure. Cardiopoietic cells, derived from MSC, were implanted endomyocardially.

debated if mature cardiomyocytes can derive from MSC.

Decreased cellular apoptosis and necrosis

Increased fibroblast proliferation and growth

76 Hyperbaric Oxygen Treatment in Research and Clinical Practice - Mechanisms of Action in Focus

MAP kinases [44].

ROS: reactive oxygen species.

If directed differentiation is aimed to be supported, MSC may be cultured in HBOT circumstances. Interestingly, HBOT enhanced osteogenic differentiation of MSC, which *in vitro* description was further proved via *in vivo* proof-of-concept studies as well [11]. Interestingly, metabolic activities, especially calcium influx and exchange, were also modulated by HBOT in MSC. HBOT increased the activity of calcium homeostasis, which is key for osteoblasts via proliferation and bone formation [11].

Pluripotent stem cells are sensitive cell cultures *in vitro*. Their maintenance requires special techniques and expertise in the field. Pluripotent cells form pluripotent cell clusters *in vitro*. They require special maintenance pluripotent stem cell media. Through passaging, spontaneously differentiated cells have to be picked and removed from culture. Recently, enzymatic passage became more advantageous than mechanical breaking of pluripotent colonies.

Pluripotent stem cell maintenance and differentiation are new and difficult cell culture techniques. These involve monolayer or three-dimensional/cell suspension culture as well. Pluripotent stem cell may be cultured on feeder layer of feeder-free surface on biomatrices. The pluripotent stem cells themselves have excellent viability and proliferative capacity in normal oxygen circumstances [72]. Additionally, they are immortal and can continuously proliferate in pluripotent state. Reasonably, most study protocols emphasize the importance of altered oxygen levels, once differentiation steps are in progress. After differentiation, steps are initiated altered oxygen levels usually increase the yield of developed cells and increase functional activity, for example, insulin secretion of beta cells, derived from pluripotent cells [14].

**Conflict of interest**

**Abbreviations**

**Author details**

Edit Gara

**References**

**33**(5):998-1003

The author declares no conflict of interest.

CO carbon monoxide

NOS nitric oxide synthase

MSC mesenchymal stem cells

PSC pluripotent stem cells

ROS reactive oxygen species

SOD superoxide-dismutase

Semmelweis University, Budapest, Hungary

Brain Res. 2013 Sep 19;**1531**:113-121

HBOT hyperbaric oxygen treatment

MRSA methicillin-resistant *Staphylococcus aureus*

Address all correspondence to: gara.edit@med.semmelweis-univ.hu

England Journal of Medicine. 2002;**347**(14):1057-1067

[1] Cevik NG, Orhan N, Yilmaz CU, Arican N, Ahishali B, Kucuk M, Kaya M, Toklu AS. The effects of hyperbaric airand hyperbaric oxygen on blood-brain barrier integrity in rats.

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[2] Weaver LK et al. Hyperbaric oxygen for acute carbon monoxide poisoning. The New

[3] Löndahl M, Katzman P, Nilsson A, Hammarlund C. Hyperbaric oxygen therapy facilitates healing of chronic foot ulcers in patients with diabetes. Diabetes Care. 2010;

CO2 carbon dioxide

NO nitric oxide

Pluripotent stem cells proliferate in low-oxygen levels in utero. It is also agreed that MSC have low oxygen circumstances *in vivo* in the bone marrow niche. Taking these into consideration, *in vitro* culture of the cells in normal oxygen circumstances is out of their normal niche. Interestingly, all of these cell types improved performance in therapeutic potential when cultured in hypoxic environment [45]. MSC improved angiogenesis-related gene expressions and protein expressions in hypoxia, furthermore implanting them into various *in vivo* models of ischemia resulted in better outcomes [73].

HBOT would have a significant role in tissue engineering and preconditioning the engineered construct *in vitro*, before *in vivo* transplantation. As an example, tissue engineered mucosa were further developed in HBOT. The mucosal cells enhanced expression of angiogenesisrelated factor (e.g., VEGF, FGF and HGF) [74]. Enhanced angiogenesis by mucosal tissue may be beneficial for graft homing and retention.

Wide range of differentiation protocols exist, which aim improving the number of cardiovascular derivatives after the differentiation steps. These are increasing in endothelial cell and cardiovascular cells as well. With endothelial cells, recent protocols reached about 50% differentiation yield. Latest studies aim hypoxia as a diver to mesodermal and then to endothelial lineage specification [75].
