I. Embryonal vasculature formation recapitulated in transgenic mammary tumor spheroids implanted pseudo-orthotopicly into mouse dorsal skin fold: the organoblasts concept

Some of the unsolved problems in vascular biology

Tumor vascular biology research has been beleaguered with multiple controversial experimental data and unanswered questions despite the technological progress and new concepts in network biology. The importance of understanding interactions among individual elements of a system has only recently gained popularity. Much of the information on mapping of mammalian interactome networks waits to be experimentally validated, although advancements have been made in small model organisms (yeast, Saccharomyces cerevisiae; fruit fly, Drosphila melanogaster; and worm, Caenorhabditis elegans)¹. Richness and redundancy enabling the natural selection of evolving biological systems make their analysis difficult and call for new methods and ways of thinking^2,3. Unsolved problems include lineage relationship between endothelial stem cells (SCs) and hematopoietic stem cells (HSCs)^4,5; the role of platelets in embryonal development^6,7; lineages of immune cells⁸, tolerance of tumors by the host immune system⁹ and reprogramming of energy metabolism by tumors^5,9. The question of biological significance of the cellular heterogeneity within tumors also remains unanswered.

Vasculogenesis versus angiogenesis

Traditionally vasculogenesis and hematopoiesis were subjects of independent studies and some fundamental issues have remained unclear in each case. The origin of the precursors of endothelial cells (ECs) has created controversy in understanding tumor growth related vasculogenesis, whereas the illusive origin of HSCs had been a daunting problem in understanding embryonal development. As opposed to de novo vasculature formation during embryogenesis (vasculogenesis), tumor vasculature has been thought to emerge by expansion of the host vasculature through postnatal sprouting of endothelial cells (angiogenesis). Therefore, it was proposed that inhibiting angiogenesis would stop tumor growth¹⁰. When that conceptually simple objective turned out to be impossible to reach, a notion of neo-vasculogenesis was born, suggesting that tumor vessels form from circulating bone marrow-derived endothelial precursors rather than from mature ECs¹¹. However, the views on tumor neo-angiogenesis remained polarized^3,12.

Erythropoiesis

Descriptions of the two main types of erythropoiesis, i.e. primitive and definitive, has kept changing over the years. Extremely large nucleated cells and smaller enucleated ones, distinguished first, were later referred to as nucleated and anuclear, respectively^13,14. Each wave of erythropoiesis was reported to generate more potent progenitors (progenitors of more numerous cell types)¹⁵. Lately, five distinct classes of hematopoietic cells in murine conceptus emerged from studies on their activity in transplantation or clonogenic assays in vitro: primitive; pro-definitive (myeloid progenitors); meso-definitive (lymphoid-myeloid progenitors); meta-definitive (neonatal repopulating HSCs); and adult-definitive (adult repopulating HSCs). Maturation of primitive, nucleated precursors from yolk sac in circulation has also been reported¹⁶. These classes of cells appeared to be generated independently of each other in distinct hematopoietic sites¹⁷. In other words, the origins of HSCs seemed to be multiple. Conclusions on the embryonic location of HSCs capable of long-term repopulation depended on the assays used to test them. Those from yolk sac repopulated neonate recipients better than adults, whereas those from aorta-gonad-mesonephros (AGM) repopulated adult recipients better than neonates¹⁸. Evidently, the successful repopulation requires a certain level of developmental compatibility. After birth, bone marrow (medulla ossea) was proposed to be the proper niche where the formation of blood elements would continue for the lifetime as needed. Therefore, extramedullar hematopoiesis observed in liver^19,20, adipose tissue²¹, cerebellar hemagioblastoma²², meningioma²³, or subdural hematoma²⁴ was perceived as unusual. However, in our study, it appeared essential for tumor growth and for regeneration of normal tissues.

Erythrocytic enucleation

The debate over the mechanism leading to the elimination of the nucleus from maturing erythroblast during erythropoiesis, expulsion of nucleus or karyolysis, dated back to the late nineteenth century²⁵. The current consensus on achieving enucleation by nuclear extrusion came from the ultrastructural studies published in 1967 that addressed the controversy by looking at either peripheral blood from dogs intentionally made anemic²⁶ or at erythroid clones grown in the spleen of irradiated mice transplanted with syngeneic marrow cells²⁵. In normal peripheral blood or non-manipulated spleen, the enucleation event was too rare to study by transmission electron microscopy (TEM). Ultrastructural analysis of a developing embryo are not available in the literature, even for an organism as well studied as the mouse. The Edinburgh Mouse Atlas Project offers no TEM images (http://www.emouseatlas.org/emap/home.html). In our model, the enucleation event was frequent enough to reveal its mechanism and we found no evidence for nuclear extrusion. Instead, we observed several variants of generating erythrocytes by nucleo-cytoplasmic conversion. Thus, depending on the model, the same method could generate conflicting results. Interestingly, the analysis of chromosomal topology showed that at certain stages of erythroblast differentiation, two chromosomes were unidentifiable²⁷. Below, we use the term "erythrosome" as a synonym for "erythrocyte" because the latter is not a cell.

Parallels between tumor and embryo

Similarities and differences in the antigenic composition of tumors and embryonal tissues, as well as in patterns of isozymes distribution, have been studied for years^28,29. Reappearance in tumors of embryonic and fetal antigens, as well as isozymes, has been shown to be a common phenomenon, yet similar alterations have sometimes been observed in non-neoplastic tissues under dietary and hormonal influences^29,30. In cases of carcinoembryonic antigen (CEA), the concept ultimately did not apply, contradicting the antigen’s name, as CEA-cross-reactive antigens were located in normal human tissues including blood³¹. Also, "patterned vascular channels without EC", i.e. vascular mimicry, were observed in embryos as well as in growing tumors^32,33. The analogy between the two types of growing tissues was further enforced by showing that tumor cells could be epigenetically reprogrammed into normal cell types^34,35. Here, we present an unprecedented collection of original images documenting the full range of growing dynamic complexity of the tumor-induced vasculature formation, reminiscent of the embryonal one.

Definitions of stem cells

The hallmark of the classic definition of adult TSCs is the potential of the cells to self-renew and differentiate³⁶. The 2002 amendment to that definition³⁷ emphasized additional aspects of the concept, namely: a shift from the cellular view to a system view including self-organizing processes, stemness as a capability rather than as a cellular property, dependence on the growth environment, within-tissue plasticity, and the functionality of the TSCs and the tissue. The stemness as an attribute of a system, rather than of a particular cell lineage, questioned the usefulness of presumed protein markers for identifying stem cells (SCs)^37–39. With regard to both embryonal and cancer SCs (ESCs and CSCs, respectively) the views remain opposing. On the one hand, neither ESCs nor CSCs fulfill all those criteria⁴⁰; on the other hand, both do but with certain qualifications. In the case of the ESCs, self-renewal and differentiation is likely to occur sequentially for two reasons. (1) The redundancy during development could serve as an evolutionary safety measure; in the context of the early embryo, that could mean generating a certain number of identical cells before their differentiation begins⁴¹. (2) Heterogeneity with respect to cell cycle activity was detected even in such an iconic cell lineage as the HSCs of adults⁴². In case of the CSCs, their role in tumorigenesis appeared to depend on microenvironment as discussed below and in one of our accompanying articles⁴³.

The goal and significance of this study

The goal of our study was to visualize the cellular mechanisms of vasculature formation associated with the growth of malignant tissue. We carried out the ultrastructural in situ analysis of initially avascular breast tumor spheroids co-implanted with homologous tissue graft, into the mouse dorsal skin folds^44,45. A complementary study on such spheroids implanted without the graft is the subject of a separate report⁴³. In contrast to the reductionism that dominated the ultrastructural studies of the second half of the last century, we analyzed tissue sections instead of isolated tissue components. The high resolution was necessary to investigate distinct physiology and functions of individual cells whereas the tissue context unveiled interactions among the cells. The results revealed interdependence between erythrogenesis and vasculogenesis localized at the site of tissue growth or repair and other new qualities of the emerging system. The data illustrated a great variability and increasing complexity of the vasculature morphogenesis, i.e., formation of blood and vessels, as both were evolving from primitive forms while enabling the tissue building process. Like in the embryo¹⁷, the new tumor vasculature was developing independently of the existing one. New erythrosomes and other blood elements formed extramedullary, and new vessels were assembling around them before initiation of the blood flow occurred. Vessel "sprouts" progressed toward other vessels not away from them. By suggesting local tissue cells as progenitors of the new vasculature, those observations related to the concept of tissue stem cells (TSCs).

The results of the morphological analysis of the vasculature formation in our model were consistent with the amended functional definition of the TSCs concept. They are not in harmony with the common understanding of the cell lineages as hierarchical and irreversible progress of differentiation. Moreover, the new findings suggest a hypothesis that the adult vasculature formation represents a partial recapitulation of the embryonal organogenesis involving coordinated activity of some cells capable to form all tissue types of a particular organ de novo. Such a perspective is not entirely new. The term "organoblast" was used in 1997 in reference to pediatric salivary gland tumors that had morphological characteristics of the organ with maturation arrested at a primitive state of development⁴⁶. Later, mouse mammary SCs in the form of in vitro grown mammospheres ("organoids") were shown to regenerate morphologically normal mammary epithelial ducts after implantation into mammary fat pads surgically cleared of the endogenous epithelium⁴⁷.

Our results also demonstrate the potential of immunocytochemistry to reveal further molecular-level details of the cellular interactions as they relate to regional tissue growth. This approach is suitable to validate concepts derived from proteogenomics as well as effects of pharmacological treatments. It is far more relevant to medical problems than in vitro studies and cheaper than conducting clinical trials.

Acronyms

AGM: aorta-gonad-mesonephros

ATP: adenosine tri-phosphate

BMP4: bone morphogenetic protein 4

CEA: carcinoembryonic antigen

CIB1: calcium & integrin binding protein 1

CSCs: cancer stem cells

ECM: extracellular matrix

ECs: endothelial cells

ER: endoplasmic reticulum

ESCs: embryonal stem cells

GFP: green fluorescent protein

H2B: histone 2B

HSCs: hematopoietic stem cells

OLAW: Office of Laboratory Animal Welfare

RT: room temperature

SCs: stem cells

TEM: transmission electron microscopy

TIMPs: with tissue inhibitors of metalloproteinases

TSCs: adult tissue stem cells

VVOs: vesiculo-vacuolar organelles

Animals

Host and graft donor, female, athymic nude mice, 8–9 weeks old, were purchased from Harlan. The donor mouse with GFP-labeled ubiquitin was from The Jackson Laboratory (Stock Number: 004353; Strain Name: C₅₇BL/6-Tg(UBC-GFP)₃₀Scha/J). The mice were housed in the SKCC animal care facility The mice were housed in the SKCC animal care facility with controlled 12/12 hr light/dark cycle and temperature maintained at + 22°C. The mice were on Tekand global 14% protein rodent diet (Harlan) with access to water ad libitum. For surgery, they were anesthetized (7.3 mg ketamine hydrochloride and 2.3 mg xylazine/100 g body weight, inoculated i.p.) and placed on a heating pad. Immediately before tissue harvesting the tumor hosting mice as well as the graft donors were euthanized with pentobarbital overdose (100 mg/kg i.p.).

Cell lines

The parental murine breast cancer cell line, N202.1A⁴⁸ was stably transfected to express GFP under histone H2B promoter⁴⁹. The two cell lines, N202.1A parental and N202.1A+H2B-GFP (obtained from Drs. J. Lustgarten and P. Borgstrom) were used to form tumor spheroids by culturing 2×10⁵ cells per well for 2–3 days prior to implantation.

Chambers

A week after establishment of mouse dorsal skin chambers, the tumor spheroids were implanted on a pad of homologous tissue, namely, minced breast fat pad from a lactating mouse (pseudo-orthotopically) as described earlier⁴⁵. The tumors were incubated in the chambers for one or three weeks (Table 1). Their final size was about 1–3 mm in diameter. The blood circulation in tumor vessels was monitored in vivo by light microscopy before disassembling the chambers⁵⁰.

Antibodies

The GFP-specific rabbit polyclonal IgG (ab290) was from Abcam; CIB1-specific rabbit polyclonal IgG (11823-1-AP) was from ProteinTech Group, Inc.; CD34-specific rat monoclonal IgG_2a (sc-18917) and non-reactive goat polyclonal IgG (sc-34284) were from Santa Cruz.

Tissue processing

The tumors with some surrounding tissues were dissected out and cut in halves perpendicularly to the host skin surface while immersed in cold fixative (4% paraformaldehyde in 0.1 M Na cacodylate pH 7.4). The skin region served later as a reference to distinguish between edges of the tumor facing the skin and those facing the glass window of the chamber. The halves were then separated and processed independently for TEM and immunocytochemistry.

TEM

The tissues were transferred into a stronger fixative (4% paraformaldehyde/2.0% glutaraldehyde in 0.1 M Na cacodylate pH 7.4) to better preserve the ultrastructures before further cutting. They were cut into 1 mm thick slices in planes perpendicular to the plane of the first cut and to the skin surface, finally, into ~ 1 mm³ blocks, transferred into a fresh portion of the fixative in which they were cut and incubated for 2 hrs at 4°C. The fixed tissue blocks were washed with 0.1 M Na cacodylate – HCl buffer pH 7.4 (3 × 15 min.) and post fixed in 1% OsO₄ in 0.1 M Na cacodylate buffer, pH 7.0 for 60 min. on ice, washed with water and stained with 1% uranyl acetate at room temperature (RT) for one hour. The blocks were embedded in EMbed-12 (EM Sciences, Cat. No 14120). The resin embedded tissues were cut into 60 nm sections, using a Leica Ultracut UCT ultramicrotome, and stained with lead citrate⁵¹ or viewed without further contrasting.

Immunocytochemistry

During cutting into ~1 mm³ blocks as described above, the tissues were kept in the mild fixative to protect antigenic epitops (4% paraformaldehyde in 0.1 M Na cacodylate pH 7.4). The tissue blocks were vitrified by infiltrating the pieces with 50% PVP (polyvinylpyrrolidone) containing 2.3 M sucrose in 0.1 M Na-cacodylate buffer, pH 7.4, for 2 hrs or over night, mounted on metal pins and frozen in liquid nitrogen, as described by Tokuyasu⁵². Frozen tissues were cut into 70 nm sections, using a Leica Ultracut UCT ultramicrotome with the cryo-attachment. The sections were picked from the knife with 2.3 M sucrose and floated on 1% ovalbumin (Sigma, Cat No.A5378) in 0.1 M Na-cacodylate buffer for at least one hour before incubation with specific or non-reactive antibody (50 µg/ml) at RT for one hour. Sections were then rinsed eight times with 0.1% ovalbumin in the same buffer and incubated for one hour with 10 nm Au coupled to protein A (from Dr G. Posthuma; Cell Microscopy Center, university Medical Center Utrecht, The Netherlands). The eight rinsing steps were repeated before fixation of the immune complexes with 1% glutaraldehyde. After rinsing three times with water, the immunostained cryosections were contrasted with a mixture of uranyl acetate and methyl cellulose (25 centipoises, Sigma M-6385) in water, at a final concentration of 1.3% each, for 10 min. Excess liquid was removed and the sections were dried at RT.

Viewing

All sections were viewed and the images captured at 100 kV using a Morgagni 268 electron microscope equipped with MegaView III digital camera. Analyzed tissue sections were first examined at low magnification and coordinates for each hexagonal sector of a grid covered with tissue were recorded automatically. Subsequently all sectors were explored at least once at variable high magnifications. Interesting images were captured at the magnification best suited to document a particular phenomenon or structure, including colloidal Au grains. The images were transmitted from the microscope camera to an iTEM imaging platform from Olympus Soft Imaging Solutions and archived in a designated database. In some cases, the final images were assembled by multiple image alignment (MIA) to increase the surface area without losing the resolution. We used the graphics editing program, Adobe PhotoShop, to add cell type-specific color-coding shown in the twin set of images included in the Supplement.

Cancer stem cells

Judging by the activities of cells evolving in our new experimentally created environment, we retrospectively assumed the presence of their precursors, in agreement with the functional TSCs definition. The question was which cells participated in the formation of the tumor vasculature and how did the vasculature form? At the outset, the components of the tumor niche (tumor spheroid, graft and local host tissues) did not have an ongoing vasculature formation. In both variants of the model, with and without homologous tissue graft, the new qualities emerged after the implantation (this report and the accompanying article⁴³, respectively). New tissues formed locally and integrated into the host animal. Some of the graft cells formed the new vasculature for the pseudo-orthotopically implanted tumor, whereas in the absence of the graft, some of the tumor cells eventually evolved to do the same. In both types of the tumor niche (pseudo-orthotopic and ectopic) some local cells differentiated to form vasculature at the site of the tissue growth. Regardless of the origin (lineage) of those cells, one could think of them as SCs - based on the definition of such cells³⁷.

The amorphous continuously growing tumor mass was out of touch with tissue morphogenesis-controlling mechanisms but depended on the ability of the tumor cells to induce formation of vasculature either by CSCs or by local TSCs. Thus, tumors were subjected to selective pressure because those incapable of either inducing local nonmalignant SCs to form tumor-supporting vasculature or of generating their own vasculature⁴³ could not grow. The ability of the tumor to generate its own vasculature in the ectopic environment implied a conditional existence of CSCs, i.e. evident in one in vivo environment but not in the other (and not in vitro). Any single cell of the spheroid population could potentially establish new tumors in the orthotopic environment. However, large numbers of those cells would be necessary in a variety of ectopic environments, whether encountered by metastatic tumors or created experimentally⁴³. One should not be surprised at finding HSCs markers among CSCs and ESCs because some of the features perceived in the literature as defining the stemness might be attributed to tissue growth, shared by malignant and non-malignant tissues. Our results showed that growing tissue sectors were capable of generating their own vasculature (blood and vessels) independently of the existing circulatory system and therefore recapitulated the embryonal vasculogenesis. However, the newly created blood-containing vascular segments did eventually fuse with the host vasculature whereas the embryonal vasculature does not fuse with the maternal one.

The vascular "sprouts" grew towards existing vessels, not away from them (Figure 11 & Figure S11 [A, C] and Figure 13 & Figure S13). Yet the new tumor-supporting vasculature observed here originated from the graft, not the tumor. To understand the paradox, one needs to keep in mind that the implanted components, tumor spheroid and graft, were related; both originated from the breast. When implanted separately, each behaved differently. The graft (injured lactating breast tissue) first produced new normal mammary epithelial ducts (results not shown) whereas the tumor initially used some of its own cells to feed the others, a fraction of which evolved into megakaryocytes and ECs⁴³. However, when implanted together, breast tumor and breast tissue reconstituted the primary tumor in its native environment, as intended. Self-organizing potentials of the two influenced each other and a new dynamic balance emerged because of such interaction. At the same time, the host was responding to the injury inflicted by the surgical procedure associated with the implantation. That enabled the comparison of the tumor and the scar tissue vasculature formation side-by-side. The involvement of non-malignant TSCs in the formation of the tumor vasculature suggested that normal and malignant tissues share the cellular mechanism of vasculature formation. The developing nerves accompanying the forming vessels also indicated that those vessels were normal, yet they were as close to a muscle as to the tumor (Figure 11 & Figure S11). The growing undersupplied region likely initiated the process because without new substrates, the growth would be energetically prohibitive, regardless of the genetic makeup. The oxygen diffusion range and the maturity level of cells in the microenvironment shaped the initial vascular pattern. The presence of calmyrites in non-malignant cells suggested that anaerobic metabolism was not unique for tumors either but could function in normal cells as an alternative, less-efficient pathway functional during mitosis and therefore more frequently used by tumors⁴³. Morphologically, the tumor vascular pattern appeared abnormal because it never matured due to perpetual tumor growth⁶⁴. Vascular tortuosity had been noticed to be a feature of young tissues^65,66, presumably in anticipation of growth. The blood flow would help to establish the hierarchical pattern for optimal functionality.

HSC lineages

Traditionally, the ontogenetic tree symbolizing cellular differentiation pathways, for instance hematopoiesis, consisted of arrows arranged in certain sequence and often branching but always pointing in the same direction, therefore signifying progress and irreversibility. Such a concept of phenotypic changes does not reflect great flexibility in terms of the functions of differentiated cells for which compelling evidence has already been accumulated, including in vitro-induced pluripotent SCs^67,68, and is fast growing as the stem cell research prospers. The reliance of erythroblast and EC differentiation on natural selection, i.e. differentiation of HSCs by cytoevolution illustrated here for the first time, suggested "broken lines" for lineages (the dead ends and some stage skipping). The process was characterized by great variability of forms and perishing of nonfunctional ones that nevertheless must have contributed to the progress in differentiation of others by changing the microenvironment. The dead ends were demonstrated also in vitro⁶⁹. Another interesting point related to cell lineages that emerged here as examples of the cell types thought of as mature that could trans-differentiate into other phenotypes. Megakaryocytes could become pericytes or ECs, depending on circumstances, and collagen-producers could become ECs. To trans-differentiate might be more practical than to migrate long distances and deal with the forms that are no longer useful. The requirement for continuous regulation to maintain a differentiated phenotype was postulated previously⁷⁰. A network of arrows pointing in different directions, including reverse, would represent the actual cellular differentiation pathways better than the traditional tree. "A good analogy likens cell types to metabolites in metabolic networks"⁴⁰.

Megakaryocytes are known to develop from bipotent erythroid/megakaryocytic progenitor^7,53,71,72. However, their morphogenetic role in establishing vascular pattern in the absence of ECs is new (Figure 1, Figure 2, Figure 11 & Figure S1, Figure S2, Figure S11). Addressing one of the unanswered questions in vascular biology^6,7 it demystifies the presence of platelets in the early embryo and demonstrates that megakaryocytes and erythroblasts develop before ECs. Together they are capable of forming primitive vascular patterns without ECs. Given the chemical nature of CEA, (200 kDa glycoprotein containing about 50% carbohydrate) and its presence in tumors and in pregnancy, one could suspect that the presumed proteoglycans of ECM (Figure 1 & Figure S1) indeed contain CEA³¹. The second dichotomy in the pathway corroborated the controversial derivation of ECs from hemangioblasts⁷³. At a certain phase in vasculature morphogenesis, the three cell types co-localized as triplets and acted in concert. However, as the assembly of vessels progressed, megakaryocytes started positioning themselves abluminally suggesting their ability to trans-differentiate into pericytes (Figure 9 & Figure S9).

The presence of eosinophils (Figure 15 & Figure S15), neutrophils (Figure 1 & Figure S1), dendritic and plasma cells (not shown) in that environment, although relatively rare, suggests that they too differentiate locally and assist tumor growth. That possibility deserves further studies because, if confirmed, it would explain why the host immune system could tolerate the tumors. If primary tumors are similar enough to the tissue of origin to prompt local TSCs for vasculature formation, one could hardly rationalize the need for its rejection by the host. If, however, the tumor loses the tissue-specific characteristics, it would become alienated and dormant until an angiogenic switch occurred^43,74, which would not necessarily be immunogenic (e.g. medical implants are non-immunogenic). In further support of this theory, the lymphopoietic potential of mouse embryonic HSCs cells was indeed observed in vitro and in vivo^17,75.

Figure 15. Rare findings.

[A & B]: Erythrosome labeled with ubiquitin-detecting antibody, internalized by non-labeled macrophage (Mouse 5). [C]: Eosinophil (Mouse 3). [D]: Myelinated nerve fiber engulfed by Schwann cell (Mouse 2). [E]: Forming vessel located between muscle and nerves (Mouse 2). [F]: Empty vein or lymphatic vessel with an internal valve (Mouse 2).

Extramedullar erythrogenesis and tissue morphogenesis

The central role of the extramedullar erythrogenesis at every level of the dynamic complexity of vasculature formation was understandable because the ongoing tissue growth requires energy and nutrients. The complex issue of the origin and maturation of HSCs in developing embryos and their potential migration among AGM, yolk sac, umbilical artery, vitellin artery, placenta and fetal liver⁷⁶ was echoed in the model used here. Without the guidance from the mature bone marrow niche, the erythrogenesis was recapitulating the embryonal stages. Calmyrites contributed a new criterion for grading the maturity level of erythropoiesis.

Our observations offered new understanding of vasculature morphogenesis in relation to tissue morphogenesis. For example, plasticity of cells became well recognized but often perceived as "puzzling, surprising, intriguing, provoking", etc. as single TSC types or even clones gave rise to multiple phenotypes unexpectedly. Epithelium evolved from bone marrow progenitors⁷⁷, hematopoietic cells from endothelial or neuronal cells^75,78,79, blood from brain⁸⁰ or muscle^81,82, muscle from HSCs^82,83, and HSCs from stromal-vascular fraction of adipose tissue^21,84, causing the confusion. The common presence of the hematopoietic component in those heterogeneous differentiated populations derived from TSCs was striking. It suggests that progenitor cells committed to a specific tissue type also have hematoblastic potential. One can anticipate more reports on finding erythropoietic cells during procedures intended to isolate tissue-specific cells. That reasoning does not contradict trans-differentiation of cell types other than those involved in erythropoiesis. For example, neuronal cells were shown to change their phenotype to endothelial cells when co-cultured with (ECs)⁸⁵. Historically, the HSC lineage has shaped the traditional belief in terminal differentiation but, as discussed below, that lineage turned out to be a special case rather than the archetype from which to extrapolate to other tissues. Yet supposedly all connective tissues (and possibly others) derive from HSCs and therefore, transplantation of HSCs rather than mesenchymal SCs would be the choice therapy for genetic deficiencies of connective tissues, making HSCs a panacea for multiple related illnesses⁸⁶. However, our results suggest the opposite. All tissues, at all stages of life, might have hemato-lympho-vaso-neuro-adipo-poietic potentials as long as they have the potential to grow or to repair themselves. However, execution of that potential would depend on morphogenesis-controlling factors and the availability of proper substrates. Various types of hematopoiesis (primitive, definitive and subclasses of those) reflect the differentiation levels of the tissues harboring the process. In an emergency, even endothelium could become hemogenic⁸⁷.

Understanding the mechanisms regulating tissue morphogenesis could have therapeutic implications. For example, the observation that collagen is essential for the process could be useful. Assuming that ECM of tumor tissue might be unprotected with tissue inhibitors of metalloproteinases (TIMPs), as was shown to be the case in embryo⁸⁸, one could envision pharmacological intervention with proteolytic enzymes.

The organoblasts concept

The classic functional definition of the adult TSCs was amended in 2002 to include its flexible and regulated tissue self-organization capability based on cell-cell and cell-environment interactions³⁷. The amended definition allowed for the assumption that TSCs have tissue specificity and the ability to form vasculature de novo when needed. Conceptually one could view TSCs as histo-hematoblasts, as we did initially. Just like TSCs and SCs in general, the histo-hematoblasts was a concept, not a clone³⁷. Which cells would produce a new piece of tissue or its vasculature did not have to be predetermined during embryogenesis but later, when and where the growth was to take place. The location of tissue growth must be governed by organogenesis-controlling factors like BMP4^89–91. An example demonstrating the spatial correlation between hematopoiesis and regions of tissue growth is available for the mouse embryo⁹². Simply put, when traced back along their ontogenetic lineage, cells engaged in building new vasculature for the growing part of a tissue would not all turn out to belong to a single HSC clone. Such multi-origin does not imply their universal applicability as transplants; as mentioned above, the developmental compatibility was shown to matter to some extent¹⁸. Another practical aspect that follows is that a strategy to stop the vasculature formation in tumors should not target specific precursor cells but rather the process itself, regardless of the lineage of cells executing the tumor supporting function. To think of SCs as "some cells" might be helpful. Due to the self-organizing potential of tissues in higher organisms, at any phase of growth and development or life of an individual, some cells in the growing region of an organ might be capable of addressing the basic metabolic needs locally, and of establishing communication with the rest of the organism. Which cells of a given organ they are, may be neither predetermined nor critical but dictated by morphogenesis-regulating factors (malfunctioning in tumors) and local energy metabolism requirements (applicable to tumors).

The concept of histo-hematoblasts was rooted in the fundamental role of neo-hematopoiesis in tissue morphogenesis, namely, fueling metabolism of any tissue mass exceeding diffusion range of oxygen (100 – 200 µm^93,94; 150 µm⁶⁴) and presumably of nutrients. Neo-hematopoiesis appeared to be the core of the process, both figuratively speaking and literally. Emerging erythrosomes, known for their ability to secrete ATP⁵⁸, formed the central point to which other cells were chemotacticly attracted and around which endothelium evolved eventually with ATP-ase located in caveolae^60–63. What triggered the local (extramedullar) vasculature formation was the localized incremental tissue growth. Any tissue growth, (developmental, repair-related or malignant) would require new vasculature; whereas, proliferation of cells replacing those that perish while fulfilling their physiological functions would not. Examples are intestinal and skin epithelia, hair follicles and secretory cells of some glands (as discussed in⁸⁷). One could think of TSCs as "kits" containing potentials for all necessary elements of the tissue to be built. Chromaffin cells participated in assembling the new artery (Figure 11, Figure S11 & Figure 12, Figure S12) and cells from the immune system were present in that environment too. Developing nerves paralleled the forming vessels of similar dimensions (Figures 15, Figure S15 [E] and Figure 4 in⁴³). GATA-2 transcription factor required for hematopoiesis was also reported to be involved in the maintenance of the pool of ventral neuronal progenitors⁹². Moreover, existence of common neural and vascular patterning tools (signaling molecules like ephrins, netrins, slits, semaphorins and neuropilins) was in fact pointed out earlier^95,96. The correlation of vasculature formation with the local development of nerves, mentioned above and elsewhere^97–101, called for modifying the concept of histo-hematoblasts to the organoblasts concept, to cover the observed phenomena more adequately.

The organoblasts (Organo-Bs) is designed to be a generic term for such organ-committed precursor cells. The plural of the term is deliberate here, like in the case of SCs, because those cells neither belong to a unique lineage nor are committed to a specific tissue type within an organ. In particular cases they would be for example: Nephro-Bs (kidneys), Hepato-Bs (liver), Osteo-Bs (bone), Dermo-Bs (skin), Pulmo-Bs (lungs), Entero-Bs (gut), Sarco-Bs (muscle), Spleno-Bs (spleen), Thymo-Bs (thymus), Ovario-Bs (ovary), Testo-Bs or Orchio-Bs (testes), Cerebro-Bs (brain) and Cardio-Bs (heart) and so on. Hemangio-Bs and Neuro-Bs would derive from the organoblasts of each organ independently to form vasculature, lymphatics and peripheral nerves. Accordingly, in the formation of any new tissue (or in the growing segment of an existing tissue), some of the growing cells would become hematoblasts or neuroblasts to connect them to the rest of the given organ in the system. Naturally, such a mechanism of vasculature morphogenesis would result in organ-specific variability in the endothelium. That would be consistent with published reports on the presence of tissue-specific markers on endothelium in particular organs, i.e. functional variability of endothelium in different organs^102–107 as well as ultrastructural differences. Brain vessels provided the most prominent example¹⁰⁸. The current definition of TSCs implies their potential to provide differentiated cells specific for a given tissue. The difference between TSCs and organoblasts (or organ SCs) is the ability of the latter to give rise to cells of specific tissue parenchyma as well as all the accessory cells necessary to maintain system homeostasis. All those cells would be locally differentiating from the organoblasts at the site of incremental tissue growth.

From the evolutionary perspective, flexibility is more valuable than rigid perfection of the complex systems. Phylogenetic analysis applied to mouse cell lineages have indicated that extensive cellular mixing homogenized the lineages in the embryo before gastrulation⁴¹. Therefore, up to that stage of development, the cells were functionally equivalent. Consequently, dissimilar lineages participated in morphogenesis of particular tissues during organogenesis. Such redundancy combined with cell plasticity and the natural selection at the cellular level might have been compensating for the high number of mutations happening at every mitosis. C. elegans can afford to lose an entire individual because one organism can produce ~1000 fertilized eggs, whereas mammals are too complex to take care of mutations that way⁴¹.

Tissue definition

The tissue definition based on the organoblasts concept would consist of two parts: (1) organ location (liver, heart, brain, stomach, pancreas, bone, striated muscle, ovary etc.), and (2) specific function, either unique for that organ, or system integrating (nerves, vasculature and lymphatics), or providing structural integrity. Tissues related to the latter two functions could be similar in different organs but not identical, as known for the endothelial surfaces. [Examples: brain grey matter, brain white matter, brain meninges, brain vessel; gut epithelium, gut smooth muscle, gut vessel; heart artery, heart vein, heart muscle; liver parenchyma, liver vessel; lung epithelium, lung endothelium, lung pulmonary pleura, lung costal pleura; lymph node capsule, lymph node nodule, lymph node hilum; peripheral nerve dendrites, peripheral nerve Schwan cells, peripheral nerve neurothelium; auricular cartilage, bronchial cartilage, articular cartilage, growth plate cartilage; bone marrow, bone cancellous, bone trabecular, bone periosteum, bone capillary; tumor parenchyma, tumor vessel, etc.]. There is more to organ development than differentiation of specific cell types. Dramatic qualitative changes of the tissue fabric in growing organisms occur by tissue remodeling; for example, bone morphogenesis involves formation of properly shaped cartilage first and its subsequent replacement with calcified tissue. In our model, free erythrosomes scattered in abundant collagen formed a scaffold that could support morphogenesis of the malignant tissue or scar.

The position of vasculature as a specific type of connective tissue equal in rank to other differentiated tissues, i.e. created during embryogenesis, deserves to be reassessed. Our results suggest extending the acceptance of the multiple origins of HSC precursors in the embryo to any tissue growth (malignant or reconstructive), also after birth. The vasculature formation (blood and vessels) is unique because it must be a part of any tissue growth, whether developmental, pathological or reconstructive and regardless of age, not just during embryogenesis. The novelty here was that not only vessels but also blood formed de novo at the location of growing tissue, not just in bone marrow and spleen. That could presumably occur in any organ. Under stress, a fraction of any tissue could feed the rest of the population if that meant survival of some cells. How the conversion of living cells into a meal could happen would depend on what kind of cells they were and on the microenvironment. Hematopoiesis appeared to have fundamental but accessory role in all tissues. That is why following HSC lineage was not a trivial issue. The implications go beyond cell lineages; they extend to organogenesis.