**5.2 Hypoxia improves differentiation potential of cells**

Most of the studies use stem cells for regenerative purposes. MSC are multipotent cells that can differentiate into adipocytes, osteoblasts, chondrocytes and neurons under appropriate conditions [68]. The main advantage of stem cell therapy is living biological replacement rather than palliation through drugs. The use of stem cells to replace functional loss of specific tissue is determined by effective differentiation [69].

The results of differentiation are controversial; this variability may be due to:

1.Use of stem cells from different sources [70].

2.Due to heterogenous population of cells with similar morphology [71].

3.Designing of delivery system for successful transplantation of stem cells [72].

Biomaterials can be designed to act as carriers for the local delivery of stem cells, support cells or molecular niche cues [73]. Basal nutrients, cell density, spatial organization, mechanical forces, growth factors and cytokines have a profound influence on hMSC differentiation [71]. The role of hypoxia preconditioning in differentiation of stem cells into other lineages seems controversial due to inconsistent results of various studies [74]. Different studies have used different hypoxia percentages with variable times. Previously, cells were usually insulted with hypoxia using hydrogen peroxide, however, new trigas incubators have been developed recently that creates the precise hypoxic conditions even for longer period of times [59].

It has been shown that placenta derived MSC showed up regulation of osteogenic genes including osteopontin (OPN), osteocalcin (OCN), and alkaline phosphatase (ALP) as well as increased mineralization at 5% oxygen levels [75]. Another study found an increased osteogenic differentiation of human MSCs cultured at 5% oxygen for 5 days [66]. Contrary to this, multilineage differentiation potential including osteogenic differentiation of tendon derived MSc was compromised in hypoxia cultures [76]. Minsheng Yang and colleagues found that 9% hypoxia increased osteogenesis whereas 1% results in decreased osteogenic potentials due to upregulation of Notch 1 expression [77]. Cells cultured throughout in hypoxic culture (5%) showed less osteogenic potential, less mineralization as compared to cells primed with hypoxia (5% for 7 days). These results also emphasis that appropriate time for hypoxia is important to maneuver the different potentials [78].

Gale et al. analyzed the chondrogenic differentiation potential of equine synovial membrane and bone marrow derived MSCs and found no appreciable difference between cells cultured either at 5% oxygen or in normoxic conditions for 28 days. The results of expression of chondrogenic genes SOX9, ACAN, and COL2b were also variable between the groups [79]. Similar to this Li J and Pei M found no significant differences in chondrogenic index between normoxic and 5% hypoxic culturing for 7 days in synovium derived MSCs [80]. On the contrary adipose derived MSC showed better chondrogenesis and upregulation of several chondrogenic specific genes when grown in 2% oxygen cultures [81]. Bae et al. found increased expression of COL2A1, ACAN, and the transcription factor SOX9 in synovium derived MSC cultured at 5% oxygen levels. They also observed increased proteoglycan, glycosaminoglycans and collagen II contents from pellets in hypoxic condition [82]. Henrionnet et al. observed the effects of 5% oxygen conditions on bone marrow derived MSC cultures for their chongrogenic potential and resulted

**143**

normoxia group [95].

**Acknowledgements**

the King Edward Medical University, Lahore.

*Hypoxic Preconditioning as a Strategy to Maintain the Regenerative Potential of Mesenchymal…*

in efficient and strong overexpression of chondrogenic genes COL2A1, ACAN, SOX9, and COMP along with down regulation of osteogenic genes ALP, and

Valorani et al. found that pre-exposure hypoxia of 2% oxygen level results in increased expression of adipogenic genes including peroxisome proliferator activated receptor γ (Pparγ), lipoprotein lipase (Lpl) and fatty acid binding protein 4 (Fabp4) and adipogenesis in MSC derived from murine adipose tissue [84]. Another research found enhanced adipogenesis of human adipose tissue mesenchymal stem-cell (hAT-MSC) exposed to 2% hypoxic conditions for 7 days before shifting to normoxia during differentiation [85]. 2% hypoxia resulted in increased adipogenic differentiation of dental pulp and periodontal ligament derived stem cells [86]. Choi JR et al. found decreased adipogenesis and decreased expression of adipogenic genes including LPL, PPARc and FABP4 under hypoxia (2%) as compared to normoxia cultures [87]. Another research also found a decreased differentiation of

Hypoxic preconditioning results in enhanced proliferation and increased colony

I acknowledge the valuable suggestions and critical analysis of my colleagues at

forming units as compared to mesenchymal stem cells cultured in normoxia. Higher oxygen tensions increase oxidative stress to cells and activate apoptosis [55]. Zhang et al. explored the effects of 1 and 5% oxygen culture conditions on rat bone marrow derived MSC and compared them with their counter parts at 18% oxygen level cultures. They found significant increase in proliferation along with upregulation of BCL2 (antiapoptotic gene) and down regulation of BAX (apoptotic gene) [89]. About 5% oxygen tension resulted in greater size, cell number and cell density of MSC colonies [90]. Antebi et al. evaluated the potential of cells at different oxygen concentrations and found that proliferative potentials of porcine MSC was higher at 1, 2, and 5% oxygen tensions as compared to normoxic conditions. They also found that 48 hour hypoxia in their study resulted in more proliferation as compared to proliferative potential of cells when cultured for longer times (10 days) [64]. Elabd et al. suggested that hypoxic preconditioning should be used as a strategy for in vitro expansion of MSC before their clinical use. They cultured human bone marrow MSCs in 5 and 20% oxygen and observed greater effects of hypoxia not on the regenerative potentials but also on the gene expressions of hypoxia exposed MSCs [91]. Notch2-c-Myc signaling cause's proliferation under hypoxia and inhibits apoptosis. Hypoxia can have a great effect on proliferation of MSCs [92]. Hypoxia (1%) increases the proliferation not only in early passages but also in late passages as compared to normoxic cultures and extends the lifespan of MSCs [61]. Significantly higher number of cells as well as increased viability of ADSC occurs in hypoxic conditions [93]. Rat bone marrow MSC cultured at 5% oxygen levels exhibited increased number of colonies and shorter population doubling time as compared to normoxic cultured cells [94]. Asadpoor Dezaki et al. showed increased expansion, population doublings, viability and colony forming unit fibroblasts in a group cultured in 2.5% oxygen tension than

*DOI: http://dx.doi.org/10.5772/intechopen.93217*

stem cells under hypoxia [88].

**5.3 Effect of hypoxia on proliferative potential of cells**

RUNX2 [83].

*Hypoxic Preconditioning as a Strategy to Maintain the Regenerative Potential of Mesenchymal… DOI: http://dx.doi.org/10.5772/intechopen.93217*

in efficient and strong overexpression of chondrogenic genes COL2A1, ACAN, SOX9, and COMP along with down regulation of osteogenic genes ALP, and RUNX2 [83].

Valorani et al. found that pre-exposure hypoxia of 2% oxygen level results in increased expression of adipogenic genes including peroxisome proliferator activated receptor γ (Pparγ), lipoprotein lipase (Lpl) and fatty acid binding protein 4 (Fabp4) and adipogenesis in MSC derived from murine adipose tissue [84]. Another research found enhanced adipogenesis of human adipose tissue mesenchymal stem-cell (hAT-MSC) exposed to 2% hypoxic conditions for 7 days before shifting to normoxia during differentiation [85]. 2% hypoxia resulted in increased adipogenic differentiation of dental pulp and periodontal ligament derived stem cells [86]. Choi JR et al. found decreased adipogenesis and decreased expression of adipogenic genes including LPL, PPARc and FABP4 under hypoxia (2%) as compared to normoxia cultures [87]. Another research also found a decreased differentiation of stem cells under hypoxia [88].

### **5.3 Effect of hypoxia on proliferative potential of cells**

Hypoxic preconditioning results in enhanced proliferation and increased colony forming units as compared to mesenchymal stem cells cultured in normoxia. Higher oxygen tensions increase oxidative stress to cells and activate apoptosis [55]. Zhang et al. explored the effects of 1 and 5% oxygen culture conditions on rat bone marrow derived MSC and compared them with their counter parts at 18% oxygen level cultures. They found significant increase in proliferation along with upregulation of BCL2 (antiapoptotic gene) and down regulation of BAX (apoptotic gene) [89]. About 5% oxygen tension resulted in greater size, cell number and cell density of MSC colonies [90]. Antebi et al. evaluated the potential of cells at different oxygen concentrations and found that proliferative potentials of porcine MSC was higher at 1, 2, and 5% oxygen tensions as compared to normoxic conditions. They also found that 48 hour hypoxia in their study resulted in more proliferation as compared to proliferative potential of cells when cultured for longer times (10 days) [64]. Elabd et al. suggested that hypoxic preconditioning should be used as a strategy for in vitro expansion of MSC before their clinical use. They cultured human bone marrow MSCs in 5 and 20% oxygen and observed greater effects of hypoxia not on the regenerative potentials but also on the gene expressions of hypoxia exposed MSCs [91]. Notch2-c-Myc signaling cause's proliferation under hypoxia and inhibits apoptosis. Hypoxia can have a great effect on proliferation of MSCs [92]. Hypoxia (1%) increases the proliferation not only in early passages but also in late passages as compared to normoxic cultures and extends the lifespan of MSCs [61]. Significantly higher number of cells as well as increased viability of ADSC occurs in hypoxic conditions [93]. Rat bone marrow MSC cultured at 5% oxygen levels exhibited increased number of colonies and shorter population doubling time as compared to normoxic cultured cells [94]. Asadpoor Dezaki et al. showed increased expansion, population doublings, viability and colony forming unit fibroblasts in a group cultured in 2.5% oxygen tension than normoxia group [95].

### **Acknowledgements**

I acknowledge the valuable suggestions and critical analysis of my colleagues at the King Edward Medical University, Lahore.

*Regenerative Medicine*

times [59].

effective differentiation [69].

**5.2 Hypoxia improves differentiation potential of cells**

1.Use of stem cells from different sources [70].

Most of the studies use stem cells for regenerative purposes. MSC are multipotent cells that can differentiate into adipocytes, osteoblasts, chondrocytes and neurons under appropriate conditions [68]. The main advantage of stem cell therapy is living biological replacement rather than palliation through drugs. The use of stem cells to replace functional loss of specific tissue is determined by

The results of differentiation are controversial; this variability may be due to:

3.Designing of delivery system for successful transplantation of stem cells [72].

It has been shown that placenta derived MSC showed up regulation of osteogenic genes including osteopontin (OPN), osteocalcin (OCN), and alkaline phosphatase (ALP) as well as increased mineralization at 5% oxygen levels [75]. Another study found an increased osteogenic differentiation of human MSCs cultured at 5% oxygen for 5 days [66]. Contrary to this, multilineage differentiation potential including osteogenic differentiation of tendon derived MSc was compromised in hypoxia cultures [76]. Minsheng Yang and colleagues found that 9% hypoxia increased osteogenesis whereas 1% results in decreased osteogenic potentials due to upregulation of Notch 1 expression [77]. Cells cultured throughout in hypoxic culture (5%) showed less osteogenic potential, less mineralization as compared to cells primed with hypoxia (5% for 7 days). These results also emphasis that appropriate time for

hypoxia is important to maneuver the different potentials [78].

Gale et al. analyzed the chondrogenic differentiation potential of equine synovial membrane and bone marrow derived MSCs and found no appreciable difference between cells cultured either at 5% oxygen or in normoxic conditions for 28 days. The results of expression of chondrogenic genes SOX9, ACAN, and COL2b were also variable between the groups [79]. Similar to this Li J and Pei M found no significant differences in chondrogenic index between normoxic and 5% hypoxic culturing for 7 days in synovium derived MSCs [80]. On the contrary adipose derived MSC showed better chondrogenesis and upregulation of several chondrogenic specific genes when grown in 2% oxygen cultures [81]. Bae et al. found increased expression of COL2A1, ACAN, and the transcription factor SOX9 in synovium derived MSC cultured at 5% oxygen levels. They also observed increased proteoglycan, glycosaminoglycans and collagen II contents from pellets in hypoxic condition [82]. Henrionnet et al. observed the effects of 5% oxygen conditions on bone marrow derived MSC cultures for their chongrogenic potential and resulted

Biomaterials can be designed to act as carriers for the local delivery of stem cells, support cells or molecular niche cues [73]. Basal nutrients, cell density, spatial organization, mechanical forces, growth factors and cytokines have a profound influence on hMSC differentiation [71]. The role of hypoxia preconditioning in differentiation of stem cells into other lineages seems controversial due to inconsistent results of various studies [74]. Different studies have used different hypoxia percentages with variable times. Previously, cells were usually insulted with hypoxia using hydrogen peroxide, however, new trigas incubators have been developed recently that creates the precise hypoxic conditions even for longer period of

2.Due to heterogenous population of cells with similar morphology [71].

**142**
