**2. Trans-differentiation of bone marrow stem cells**

Blood is one of the most highly regenerative tissues in our body with almost one trillion cells arising daily. Over the last decade several investigators have demonstrated that BM stem cells not only contribute to development of blood cells but also to the regeneration of various organs and tissues [5, 6]. MSC isolated from various sources can differentiate into diverse cell types, showing a unique ability to cross lineage borders (i.e. are able to differentiate towards ectoderm-, mesoderm- and endoderm-derived cell types) and do not express the major histocompatibility complex (MHC) class II Human Leukocyte Antigen (HLA-DR) antigens. This, together with their *in vitro* proliferative potential and their immunoregulatory properties, renders them extremely promising for regenerative medicine applications in several diseases [7].

These observations were mainly explained by the hypothesis that the BM stem cells are 'plastic' and thus could dedifferentiate into various cell types of non-hematopoietic organs and tissues [8]. The possibility that HSC/MSC are plastic and able to trans-differentiate raised hope that HSC/MSC isolated from BM, mobilized into the peripheral blood (mPB) or cord blood (CB) could become a universal source of stem cells for tissue/ organ repair. This was supported by several demonstrations of the remarkable regenerative potential of HSC in animal models, for example after heart infarct [5], stroke [9], spinal cord injury [10], and liver damage [11] and of MSC in skeletal regeneration [12], cardiac regeneration [13], diabetes [14] and osteogeneis imperfect [15]. The potential of adult stem cells also resulted in slow growth of research and funding restrictions on ES cells during President Bush regime in USA – based on the argument that destroying embryos to derive human ES cell lines was not essential, when better alternatives including adult stem cells are available for regenerative medicine (http://en.wikipedia.org/wiki/Stem\_cell\_controversy). However the excitement over plasticity of HSC reduced when their role in repair of damaged organs became controversial [16, 17].

70 Blood Cell – An Overview of Studies in Hematology

support HSC transplantation [3].

applications in several diseases [7].

In the 1950s, researchers discovered that the bone marrow contains stem cells i.e. hematopoietic stem cells (HSC) with the ability to self-renew and give rise to cell types in the blood and immune system (Figure1). Multipotent HSCs reside at the apex of hematopoietic hierarchy and they are connected to mature cells by a complex roadmap of progenitor intermediates. The HSC differentiate into two different kinds of progenitors viz. Common Myeloid Progenitors (CMP) and Common Lymphoid Progenitors (CLP), which further differentiate to various blood cells including platelets, granulocytes, lymphocytes and macrophages. As a result, bone marrow transplantation became the standard method of care for most hematopoietic malignancies whereby the HSCs were able to repopulate bone marrow after any kind of hematopoietic failure. A recent review by Doulatov et al [1]

Besides HSC, another stem cell population, the mesenchymal stem cells (MSC) was identified in the bone marrow about 40 years ago [2]. MSCs comprise of the adherent stem cell population with immune-modulatory properties. Besides bone marrow, MSCs can also be extracted from virtually all post-natal as well as extra-embryonic tissues such as amniotic membrane, placenta and umbilical cord. They can differentiate along multiple lineages and exhibit significant expansion capability *in vitro*. Co-transplantation with MSCs improves engraftment of HSCs after autologous intra-bone marrow transplantation [3]. MSCs are also considered useful as vehicles for emerging cell and gene therapies in the field of tissue engineering [4]. Recently it has been postulated that MSC provide the conducive microenvironment for HSCs and thus maintain the stemness and proliferation of HSCs and

Blood is one of the most highly regenerative tissues in our body with almost one trillion cells arising daily. Over the last decade several investigators have demonstrated that BM stem cells not only contribute to development of blood cells but also to the regeneration of various organs and tissues [5, 6]. MSC isolated from various sources can differentiate into diverse cell types, showing a unique ability to cross lineage borders (i.e. are able to differentiate towards ectoderm-, mesoderm- and endoderm-derived cell types) and do not express the major histocompatibility complex (MHC) class II Human Leukocyte Antigen (HLA-DR) antigens. This, together with their *in vitro* proliferative potential and their immunoregulatory properties, renders them extremely promising for regenerative medicine

These observations were mainly explained by the hypothesis that the BM stem cells are 'plastic' and thus could dedifferentiate into various cell types of non-hematopoietic organs and tissues [8]. The possibility that HSC/MSC are plastic and able to trans-differentiate raised hope that HSC/MSC isolated from BM, mobilized into the peripheral blood (mPB) or cord blood (CB) could become a universal source of stem cells for tissue/ organ repair. This was supported by several demonstrations of the remarkable regenerative potential of HSC in animal models, for example after heart infarct [5], stroke [9], spinal cord injury [10], and

describes the knowledge gathered over the years on Hematopoiesis.

**2. Trans-differentiation of bone marrow stem cells** 

Several alternative mechanisms were proposed to explain the trans-differentiation of bone marrow stem cells [18] including (i) epigenetic changes i.e. factors present in the environment of damaged organs may induce epigenetic changes in the genes that regulate pluripotency of HSCs (ii) cell fusion during which infused HSCs may fuse with cells in damaged tissues and form heterokaryons which express markers of both donor and recipient cells (iii) paracrine effect i.e. HSCs are source of different trophic and angiopoietic factors that may promote tissue/organ repair (iv) microvesicles- dependent transfer of molecules like receptors, proteins and mRNA between HSC and damaged cells and (iv) **presence of pluripotent stem cell population in the bone marrow in addition to HSC & MSC that may contribute to regeneration**. Presence of other stem cells in the BM may also explain the loss of contribution of BM cells to organ regeneration with the use of highly purified population of HSC [16]. Of these various possibilities (i) and (ii) are extremely rare and most likely the fact that BM houses heterogeneous and perhaps pluripotent stem cells may explain transdifferentiation potential of bone marrow. It has been demonstrated that there are heterogeneous stem cell populations in adult bone marrow compartment. Under appropriate experimental conditions, a certain type of bone marrow stem cells appears to differentiate (or transdifferentiate) into a variety of non-haemopoietic cells of ectodermal, mesodermal and endodermal origins (such as myocytes, neural cells and hepatocytes) [67].Various investigators have reported pluripotent stem cells in the bone marrow by using varied approaches to demonstrate their presence and are listed in Table 1.

The potential relationship of the BM-derived pluripotent stem cells reported by various investigators and compiled in Table1 is not clear. It is possible that these are overlapping populations of cells identified by slightly different isolation/ expansion strategies and likely that all of these versatile BM-derived Oct-4+ non-hematopoietic stem cells, which were given different names, are in fact very closely related to the same type of BM-residing Pluripotent Stem Cells (PSC). This overlap was elegantly described earlier by Ratajczak and his group [25] that various investigators are looking from different "keyholes" at the same population of stem cells that are hiding in a "darkroom" of the bone marrow environment. They further suggested that a 'founder cell' may exist in the bone marrow which is responsible for multi-lineage differentiation. Table 2 is a compilation of various markers reported on these differently described PSCs in the bone marrow responsible for their mobility (CXCR4), pluripotency (Oct-4, Nanog, Rex, Tert), non-hematopoietic lineage (CD45), immune status (MHC-1) and their developmental migration similarity to PGCs (SSEA1).


VSELs in Bone Marrow and Cord Blood 73

Small Small Small

No data available <sup>+</sup>

Do not form teratoma Do not form teratoma

Characteristics

Shape and size

Quiescent by nature

Teratoma formation

Markers MAPC MIAMI MACS RS VSEL

Form small colonies in culture

CXCR4 + + + + + CD 133 ND ND - - + Sca 1 ND ND ND ND + CD 45 - - - - - OCT-4 + + + ND + REX-1 ND + + ND + Nanog + + + + + TERT + + ND + + SSEA 1 + ND ND ND + MHC-1 - ND + ND -

> No data available

Do not form teratoma

Besides these pluripotent stem cells, BM also houses Tissue Committed Stem Cells (TCSCs) including Epithelial Progenitor Cells (EPCs). Available literature suggests that postnatal neovascularization does not rely on formation of new blood vessels from pre-existing ones (angiogenesis) rather on EPCs migrating from the BM to induce neovascularization. EPCs and HSCs share certain markers like Flk-1, Tic2, Sca-1, and CD34. As a result it has been

Interestingly the trans-differentiation ability of adult BM cells into various TCSCs like hepatocytes, cardiomyocytes, vascular endothelial cells, neuronal cells etc. occurs only when there is a need i.e. into hepatocytes when damage is inflicted on the liver by radiation or chemical damage [27], into cardiomyocytes when myocardial infarction is induced [28], into endothelial cells on inducing ischemia [29] and into neural stem cells on inducing stroke [30]. In the same manner, the BM stem cells have also been shown to trans-differentiate into germ cells when gonadal function is compromised e.g. by treating with busulphan in female [31] and male [32] mice. Freshly prepared BM may also exhibit early tissue-specific markers

but are up-regulated several folds when the function of organ is compromised [33].

Pluripotent stem cells are expected to be more primitive to TCSCs based on their developmental hierarchy (totipotent - pluripotent - multipotent– unipotent stem cells). This

No data available

Do not form teratoma

Form small colonies in culture

No data available

ND-experiment not done; + positive; - negative

Do not form teratoma

**Table 2.** Compilation of Various Markers on BM Pluripotent Stem Cells

suggested that they both may arise from a common precursor [26].

is also supported by various observations shown below.

**Table 1.** Pluripotent Stem Cells Reported in the Bone Marrow


ND-experiment not done; + positive; - negative

72 Blood Cell – An Overview of Studies in Hematology

**MAPCs**  Multipotent Adult Progenitor Cells

**MIAMI**  Marrow Isolated Adult Lineage Inducible Cells

**RS cells**  Recycling Stem

Cells

**MACS**  Multipotent Adult Progenitor Cells

**MPCs**  Mesodermal Progenitor Stem Cells

**VSELs**  Very Small Embryonic-like Stem Cells

**Stem Cell Functional attributes (in brief)** 

Ability to transdifferentiate Do not form teratomas

MHC I–, MHC II–m SSEA1+, OCT-4+

Pluripotent properties even after 50 doublings

Positive for OCT-4, REX-1 and telomerase

Many characteristics like ES cells MAPCs maintain telomere length

Pluripotent by nature

Small in size Proliferate rapidly

CD45-

CD45-

Rex1[22]

High telomerase activity

Wide range of differentiation potential.

Detected in bone marrow and cord blood [23] Exist as a sub-population in MSC culture Fail to divide in culture thus quiescent Multi- to pluripotent by nature

Express SSEA-4, OCT-4, Nanog by IF and RT-PCR

Large nucleus surrounded by a narrow rim of cytoplasm

cells identified in murine BM [24]

Small size (~3.5 μm in diameter)

Do not form teratoma Quiescent population of cells

**Table 1.** Pluripotent Stem Cells Reported in the Bone Marrow

Open-type chromatin (euchromatin) Differentiate into three lineages

Described first by Verfailles and her group [19] Extracted from bone marrow in mouse, rat and human Plastic in nature and give rise to multiple cell types

Single MAPC in early mouse embryo can contribute to all body tissues

Can reconstitute bone marrow and also give rise to HSCs

Capable of differentiating into cells from all three germ layers

Express markers typically associated with embryonic stem cells

Are a sub-population of cells present amongst the MSCs [21]

>50 population doublings with no sign of senescence

SSEA-1+, CD13+, Flk-1low, Thy-1low, CD34–, CD44–, CD45–, CD117(c-kit)–,

Bone marrow derived adult stem cells isolated in humans aged 3- 72 years [20]

Express pluripotent-state-specific transcription factors (OCT-4, Nanog and

Homogenous population of rare (~0.01% of BM mononuclear cells) Sca-1+ Lin–

Express SSEA-1, OCT- 4, Nanog and Rex-1 & Rif-1 telomerase protein

Cloned from human liver, heart, and BM-isolated mononuclear cells

**Table 2.** Compilation of Various Markers on BM Pluripotent Stem Cells

Besides these pluripotent stem cells, BM also houses Tissue Committed Stem Cells (TCSCs) including Epithelial Progenitor Cells (EPCs). Available literature suggests that postnatal neovascularization does not rely on formation of new blood vessels from pre-existing ones (angiogenesis) rather on EPCs migrating from the BM to induce neovascularization. EPCs and HSCs share certain markers like Flk-1, Tic2, Sca-1, and CD34. As a result it has been suggested that they both may arise from a common precursor [26].

Interestingly the trans-differentiation ability of adult BM cells into various TCSCs like hepatocytes, cardiomyocytes, vascular endothelial cells, neuronal cells etc. occurs only when there is a need i.e. into hepatocytes when damage is inflicted on the liver by radiation or chemical damage [27], into cardiomyocytes when myocardial infarction is induced [28], into endothelial cells on inducing ischemia [29] and into neural stem cells on inducing stroke [30]. In the same manner, the BM stem cells have also been shown to trans-differentiate into germ cells when gonadal function is compromised e.g. by treating with busulphan in female [31] and male [32] mice. Freshly prepared BM may also exhibit early tissue-specific markers but are up-regulated several folds when the function of organ is compromised [33].

Pluripotent stem cells are expected to be more primitive to TCSCs based on their developmental hierarchy (totipotent - pluripotent - multipotent– unipotent stem cells). This is also supported by various observations shown below.


Thus we propose following developmental hierarchy of stem cells in bone marrow (Figure:2) as opposed to the existing notion that HSC sit at the apex of hematopoietic system [1].

VSELs in Bone Marrow and Cord Blood 75

In mammals the first primitive HSC are found in the yolk sac and first definitive HSC a few days later in the aorta-gonadmesonephros (AGM) region [35]. From the yolk sac and/or AGM region HSC migrate to the fetal liver (FL), which during the second trimester of gestation becomes the major mammalian hematopoietic organ. By the end of the second trimester of gestation, HSC leave the fetal liver and colonize BM tissue. Signals for the translocation of HSC from the fetal liver to BM are provided by the alpha chemokine – SDF-1 that is secreted by osteoblasts lining the developing marrow cavities, marrow fibroblasts and endothelial cells. In response to SDF-1, HSC that expresses, SDF-1 receptor-a seven transmembrane-spanning G protein coupled CXCR4 receptor, leave the fetal liver and begin

It is very likely that at this point BM is also colonized by several other nonhematopoietic stem cells that may circulate during organogenesis and rapid foetal growth/expansion. In support of this stem cells for different tissues express CXCR4 on their surface and follow an SDF-1 gradient. Thus the SDF-1-CXCR4 axis alone or in combination with other chemoattractants plays a crucial role in the accumulation of non-hematopoietic stem cells in developing BM [36, 37]. These cells find a permissive environment to survive in BM, and may play an underappreciated role as a reserve pool of stem cells for organ/tissue

The presence of these various populations of stem cells in the BM (Table 1) is a result of the 'developmental migration' of stem cells during ontogenesis and the presence of the permissive environment that attracts them to the BM tissue. HSC and other nonhematopoietic stem cells are actively chemoattracted by factors secreted by BM stromal cells and osteoblasts (e.g. SDF-1), hepatocyte growth factor (HGF)) and colonize marrow by the

It is assumed that these non-hematopoietic pluripotent stem cells are deposited in the BM during early embryogenesis and subsequently may be mobilized in stressed situations and circulate in the peripheral blood. Similarly due to the stress of delivery these cells may also

Interestingly various terminologies like MAPCs, MIAMI, RS cells etc. (Table 1) disappeared from the literature after initial publications and excitement except VSELs. Ratajczak and group have made tremendous contribution to the field of VSELs biology. At present various laboratories across the world are providing evidence to support the pluripotent property and potential of VSELs isolated from cord blood and bone marrow [38]. Possible reason being the method to isolate VSELs by flow cytometry described by Ratajczak and group

VSELs were identified by Ratajczak and group in 2006 by multi parameter sorting in adult murine BM. They express several morphological (e.g., relatively large nuclei containing euchromatin) and molecular (e.g. expression of SSEA-1, Oct4, Nanog, Rex1) markers

end of the second and the beginning of the third trimester of gestation [24].

could easily be replicated in various labs across the world.

**4. Very small embryonic like stem cells (VSELs)** 

to home into BM where they finally establish adult haematopoiesis.

regeneration during postnatal life.

be present in cord blood [18].

**Figure 2.** Developmental hierarchy of stem cells in bone marrow

The existing controversial literature that HSCs and MSCs can trans-differentiate into various lineages can be alternatively explained by the presence of these pluripotent stem cells. These PSCs interact closely with the MSCs by a process defined as emperipolesis [34]. The MSCs secrete SDF-1(Stromal Derived Factor-1) and other chemo-attractants thereby creating a homing environment for these pluripotent stem cells (express CXCR4). Thus isolated BM stem cells have always been contaminated with these PSCs which may have resulted in trans-differentiation and that HSCs/MSCs (being lineage restricted themselves) possibly do not account for the observed plasticity.
