**3. Oxygen levels vary in tissues**

*Regenerative Medicine*

transdifferentiation into respective tissues and/or through their paracrine effects by

The potential of adult stem cells such as MSCs is severely compromised in vitro by culture conditions and by number of passages of the cells [5]. In addition, "disease conditions" and "age" of the donor also reduces regenerative functionality of MSCs and their clinical use for repair and regeneration of damaged and lost tissues [6]. It is pertinent to note that elderly population is the main portion of population for potential stem cell-based regenerative therapies. However, autologous use of cells from aged individuals seems not to provide the expected benefits of stem cell-based therapies due to age depleted function of stem cells from such patients [7]. It is therefore required to enhance the potential of stem cells before clinical use. Different strategies have been employed for this purpose such as growth factors preconditioning [8], mild

Different pretreatment strategies have been employed to enhance the regenerative potential of stem cells, however; hypoxic preconditioning seems more effective for enhancing stem cell function because relatively low oxygen concentrations prevail in stem cells niches as compared to normoxic conditions. Hypoxia can be an effective strategy for enhancing the cells function because it can make the cells adapt external microenvironment, reduce oxidative stress, shift metabolism towards glycolysis, enhance proliferation, differentiation and maintain stemness, and improve their motility to tolerate the hypoxic preconditioning after transplantation [11].

**2. Regenerative potential of stem cells is compromised with age**

Autologous stem cell-based therapies seem promising for several diseases. As humans get older, stem cell function deteriorate like other cells of the body. Diseases, especially degenerative diseases generally affect elderly people and therefore using autologous cells for such patients may have practical concerns [12]. With aging, superoxide dismutase activity (SOD) declines [13]. Studies indicate that differentiation potential of stem cells is negatively related with age of donor [12, 14–16], and therefore cell potential to form osteoblasts [12, 15–17], cartilage [12, 14–16] and other cell types is compromised. Another important aspect for cell-based therapies, that is, proliferation is also adversely effected with increasing donor age. Choudhery et al. [15] indicated that the number of population doublings decreased while the time of population doublings increased for cells obtained from aged donors as compared to cells from the young donors. Similarly, number of the colony forming units, size of colonies and plating efficiency of aged cells decreases in vitro [13–17]. Overall, growth kinetics and differentiation potential of the cells

Cell-based therapies require large number of cells to get the favorable results in patients. For this purpose cells are expanded in vitro before use in most of clinical applications [12]. Leonard Hayflick in 1960 described that after a limited number of cell divisions the cells stop dividing. Cell morphology changes and they become enlarged and irregular in shape. The cells undergo a replicative senescence and this limited life span of cells is called as Hayflick's limit [19]. In this way replicative senescence limits the therapeutic potential of stem cells. The differentiation

**2.1 Effect of donor age on regenerative potential of stem cells**

are inversely proportion to the donor age [15–18].

**2.2 Effect of in vitro passaging**

releasing different cytokines and growth factors [4].

heat shock [9], and glucose depletion [10].

**138**

The structural and functional microenvironment in tissues where stem cells reside is known as stem cell niche described for the first time by Schofield [24]. A cell niche maintains the identity and functional characteristics of resident cells [25]. Important identified stem cell niches are in bone marrow [26], vascular vessels [27], liver [28] adult kidneys [29] intestine [30] endometrium [31], oral tissue [32], skin [33] and adipose tissue [34]. **Table 1** shows variable oxygen concentrations in some important stem cell niches.


#### **Table 1.**

*Oxygen levels in different tissue in-vivo.*

It indicates that oxygen levels significantly vary in various tissues of the body and are significantly low as compared to normoxic oxygen concentration. Initially, oxygen level of 21% were adopted for in vitro culturing the cells based on normal oxygen conditions in environment, however, latter, it was realized that the cells grow better in vitro when cultured in those oxygen conditions which are representative of their respective niche.

Similarly, when cells are transplanted in the body, they face hypoxic in vivo environmental conditions [49, 50]. A large number of grafted cells die due to harsh in vivo environmental conditions (such as hypoxia) at transplanted site. The cell death due to hypoxic microenvironment is especially considerable for tissues that are not vascularized and or already injured or wounded [51].
