**3.4 Human umbilical cord blood and placental stem cells**

The idea to use human umbilical cord blood arose in the early 1980s, when experiments using cord blood from near-term mice demonstrated hematopoietic reconstitution in lethally irradiated mice [105]. Further, cord blood-derived HSCs were found to have a higher proliferative capacity than those in bone marrow and sufficient doses of HSCs, and hematopoietic progenitor cells were contained in a single collection of cord blood [106]. In 1988 a 5-year-old boy with Fanconi anemia underwent the first cord blood transplantation from his HLA-identical newborn sister [107]. In 1993 the first unrelated cord blood transplantation was performed in a 3-year-old with refractory T-cell acute lymphoblastic leukemia. Since then, related and unrelated cord blood has been increasingly utilized as an alternative source of HSCs [108].

In addition to the HSCs and hematopoietic progenitors, many populations of non-hematopoietic stem cells have been reported in human cord blood, such as MSCs, endothelial progenitor cells [109], embryonic-like stem cells (CBE) [110], very small embryonic-like (VSEL) stem cells [111], multi-lineage progenitor cells (MLPC) [112], and unrestricted somatic stem cells (USSCs) (**Figure 3**) [113]. There have been controversial reports on the identity of VSEL. VSEL stem cells were isolated based on the sorting of CXCR4<sup>+</sup> SSEA-1+ Sca-1+ Lineage<sup>−</sup>CD45<sup>−</sup> cells originally from murine bone marrow cells and subsequently in human CB [111]. Several other groups also reported the isolation of VSEL cells from different adult tissues and implied that VSEL cells were originated during embryonic development and deposited in bone marrow and other organs as dormant precursor cells of adult stem cells (reviewed in [114]). These cells are reported to be very small (3–5 μm), possess large nuclei, express pluripotency markers Oct4 and Nanog, and undergo multi-lineage differentiation. However, the stem cell characteristics of this cell type were challenged by the other group [115].

Among the other cord blood-derived non-hematopoietic stem cells, USSCs are the best characterized stem cell population and represent a novel universal allogenic stem cell source for degenerative diseases. USSCs are generated from full-term CB based on outgrowth of plastic adherent and spindle-shaped colonies in the presence of 30% fetal bovine serum, 10<sup>−</sup><sup>7</sup> M dexamethasone, and 2 mM ultra-glutamine in low glucose DMEM [113]. USSCs possess the ability to differentiate in vitro into the bone, cartilage, adipocytes, hematopoietic cells, and neural cells and in vivo into myocardial cells, Purkinje fibers, and hepatic cells [113]. Although USSCs share cell surface marker with MSCs, they have distinct gene expression, epigenetic signatures, and cytokine profiling [113, 116–120]. USSCs have high proliferation and expansion properties. They can be cultured for more than 20 passages without any spontaneous differentiation or slowing down doubling time [113, 116]. This is a significant advantage of USSCs over MSCs, the doubling time of which dramatically increases after four passages, leading to cell senescence [121]. Significantly, even after 13 passages, the average telomere length of USSCs is 8.6kbp, which is significantly longer than

**111**

**Figure 4.**

of the recipient mice [127].

*Innovations in Human Stem Cell Research: A Holy Grail for Regenerative Medicine*

the 7.27kbp telomere length of MSCs at passage 4 [113]. In preclinical studies, USSCs demonstrated the ability to alleviate myocardial infarction, liver injury, spinal cord injury, and intraventricular hemorrhage [122–124]. Moreover, USSCs also promote wound healing and improve manifestation of an inherited skin blistering disease, i.e., RDEB [120, 125]. Suppression of TGFβ-mediated fibrosis and modulation of extracellular matrix remodeling have been accounted as part of the mechanisms of

*Dual effects of cord blood-derived USSCs on modulating RDEB skin microenvironment. USSCs exert antifibrotic function by suppressing phosphorylation of Smad2/3 in the fibroblasts (Fb) and macrophages (MΦ) of the skin, inhibiting matrix metalloproteinases (MMP)-9 and -13 dermal expression and upregulating antifibrotic TGFβ3 and DCN expression. USSCs also attenuate secretion of MMP-9 and MMP-13, which correlate with epithelial malignant transformation, from keratinocytes (Kc) and cutaneous squamous cell carcinoma (cSCCs) derived from patients with RDEB. This figure was adapted from* Stem Cells *with permission [125].*

Once considered as a medical waste similar to CB, the human placenta has also been demonstrated to provide a novel stem cell source for cellular therapy. Hematopoietic stem and progenitor cells were identified throughout gestation from human placental blood, vessel perfusate, and cells from digested placenta tissue, and stromal cells generated from placenta possessed pericyte characteristic and may be a supportive microenvironment for hematopoiesis [126]. Recently Celgene Cellular Therapeutics, Inc. manufactured human placental-derived stem cells (HPDSCs) from full-term donor placentas following saline perfusion, red blood cell depletion, and volume reduction. The overall cell types as determined by flow cytometry analysis are similar between HPDSCs and CB [127]. However, HPDSCs contain a significantly higher level of both hematopoietic and non-hematopoietic stem and progenitor cells than CB. In addition, HPDSCs have a lower percentage of T cells than CB and are largely negative for MHC class II molecules, indicative of their potential use as both autologous and allogeneic cells. A pilot clinical study demonstrated that adding HPDSCs as universal donor cells with CB transplantation in patients with malignant and nonmalignant diseases had no adverse effects and may reduce the incidence of aGvHD [128]. Administration of HPDSCs in a mouse model of RDEB, in the absence of any conditioning regimen, also resulted in significant improvement on the survival and disease manifestation

action of USSCs in the treatment of RDEB (**Figure 4**) [125].

*DOI: http://dx.doi.org/10.5772/intechopen.88790*

*Innovations in Human Stem Cell Research: A Holy Grail for Regenerative Medicine DOI: http://dx.doi.org/10.5772/intechopen.88790*

### **Figure 4.**

*Innovations in Cell Research and Therapy*

differentiation [104].

HSCs [108].

self-renewal and bone formation capacity of old MSCs were significantly compromised as compared to the young MSCs [103]. Moreover, similar to the finding in the satellite cells, exposure of the aged MSCs to a young extracellular matrix rejuvenated these functions of aged MSCs [103]. Recent studies also demonstrated that with age as well as treatment of antidiabetic drugs, MSCs favored differentiation into adipocytes resulting in an increased number of adipocytes and a decreased number of osteoblasts, which may be related to osteoporosis. Downregulation of a transcription factor c-Maf has been identified as the age-related switch in MSC

The idea to use human umbilical cord blood arose in the early 1980s, when experiments using cord blood from near-term mice demonstrated hematopoietic reconstitution in lethally irradiated mice [105]. Further, cord blood-derived HSCs were found to have a higher proliferative capacity than those in bone marrow and sufficient doses of HSCs, and hematopoietic progenitor cells were contained in a single collection of cord blood [106]. In 1988 a 5-year-old boy with Fanconi anemia underwent the first cord blood transplantation from his HLA-identical newborn sister [107]. In 1993 the first unrelated cord blood transplantation was performed in a 3-year-old with refractory T-cell acute lymphoblastic leukemia. Since then, related and unrelated cord blood has been increasingly utilized as an alternative source of

In addition to the HSCs and hematopoietic progenitors, many populations of non-hematopoietic stem cells have been reported in human cord blood, such as MSCs, endothelial progenitor cells [109], embryonic-like stem cells (CBE) [110], very small embryonic-like (VSEL) stem cells [111], multi-lineage progenitor cells (MLPC) [112], and unrestricted somatic stem cells (USSCs) (**Figure 3**) [113]. There have been controversial reports on the identity of VSEL. VSEL stem cells

originally from murine bone marrow cells and subsequently in human CB [111]. Several other groups also reported the isolation of VSEL cells from different adult tissues and implied that VSEL cells were originated during embryonic development and deposited in bone marrow and other organs as dormant precursor cells of adult stem cells (reviewed in [114]). These cells are reported to be very small (3–5 μm), possess large nuclei, express pluripotency markers Oct4 and Nanog, and undergo multi-lineage differentiation. However, the stem cell characteristics of this cell type

Among the other cord blood-derived non-hematopoietic stem cells, USSCs are the best characterized stem cell population and represent a novel universal allogenic stem cell source for degenerative diseases. USSCs are generated from full-term CB based on outgrowth of plastic adherent and spindle-shaped colonies in the presence

low glucose DMEM [113]. USSCs possess the ability to differentiate in vitro into the bone, cartilage, adipocytes, hematopoietic cells, and neural cells and in vivo into myocardial cells, Purkinje fibers, and hepatic cells [113]. Although USSCs share cell surface marker with MSCs, they have distinct gene expression, epigenetic signatures, and cytokine profiling [113, 116–120]. USSCs have high proliferation and expansion properties. They can be cultured for more than 20 passages without any spontaneous differentiation or slowing down doubling time [113, 116]. This is a significant advantage of USSCs over MSCs, the doubling time of which dramatically increases after four passages, leading to cell senescence [121]. Significantly, even after 13 passages, the average telomere length of USSCs is 8.6kbp, which is significantly longer than

SSEA-1+

Sca-1+

M dexamethasone, and 2 mM ultra-glutamine in

Lineage<sup>−</sup>CD45<sup>−</sup> cells

**3.4 Human umbilical cord blood and placental stem cells**

were isolated based on the sorting of CXCR4<sup>+</sup>

were challenged by the other group [115].

of 30% fetal bovine serum, 10<sup>−</sup><sup>7</sup>

**110**

*Dual effects of cord blood-derived USSCs on modulating RDEB skin microenvironment. USSCs exert antifibrotic function by suppressing phosphorylation of Smad2/3 in the fibroblasts (Fb) and macrophages (MΦ) of the skin, inhibiting matrix metalloproteinases (MMP)-9 and -13 dermal expression and upregulating antifibrotic TGFβ3 and DCN expression. USSCs also attenuate secretion of MMP-9 and MMP-13, which correlate with epithelial malignant transformation, from keratinocytes (Kc) and cutaneous squamous cell carcinoma (cSCCs) derived from patients with RDEB. This figure was adapted from* Stem Cells *with permission [125].*

the 7.27kbp telomere length of MSCs at passage 4 [113]. In preclinical studies, USSCs demonstrated the ability to alleviate myocardial infarction, liver injury, spinal cord injury, and intraventricular hemorrhage [122–124]. Moreover, USSCs also promote wound healing and improve manifestation of an inherited skin blistering disease, i.e., RDEB [120, 125]. Suppression of TGFβ-mediated fibrosis and modulation of extracellular matrix remodeling have been accounted as part of the mechanisms of action of USSCs in the treatment of RDEB (**Figure 4**) [125].

Once considered as a medical waste similar to CB, the human placenta has also been demonstrated to provide a novel stem cell source for cellular therapy. Hematopoietic stem and progenitor cells were identified throughout gestation from human placental blood, vessel perfusate, and cells from digested placenta tissue, and stromal cells generated from placenta possessed pericyte characteristic and may be a supportive microenvironment for hematopoiesis [126]. Recently Celgene Cellular Therapeutics, Inc. manufactured human placental-derived stem cells (HPDSCs) from full-term donor placentas following saline perfusion, red blood cell depletion, and volume reduction. The overall cell types as determined by flow cytometry analysis are similar between HPDSCs and CB [127]. However, HPDSCs contain a significantly higher level of both hematopoietic and non-hematopoietic stem and progenitor cells than CB. In addition, HPDSCs have a lower percentage of T cells than CB and are largely negative for MHC class II molecules, indicative of their potential use as both autologous and allogeneic cells. A pilot clinical study demonstrated that adding HPDSCs as universal donor cells with CB transplantation in patients with malignant and nonmalignant diseases had no adverse effects and may reduce the incidence of aGvHD [128]. Administration of HPDSCs in a mouse model of RDEB, in the absence of any conditioning regimen, also resulted in significant improvement on the survival and disease manifestation of the recipient mice [127].

As will be mentioned below, CB hematopoietic progenitor cells are among the few stem cell products that are approved by the FDA. Over 40,000 CB transplantations have been performed worldwide in both adults and children for the treatment of around 80 different disorders [129]. The advantage of using CB and cord blood CB-derived stem cells compared to other adult stem cell sources is the fast availability and ease in collection without causing any discomfort or risk to the donors. Moreover, being early in development, CB stem cells have not been exposed to immunological challenge and are less likely to carry somatic mutations than other adult cells. Any age- or stress-related transcriptional remodeling that might have impacted the stem cell function of adult stem cells, as discussed above, would not be of an issue in CB stem cells.
