Developmental Hematology

Stem Cell Biology

Pluripotential stem cells sustain marrow function throughout a person's lifetime. A unique characteristic of these cells is that their direct offspring include at least one identical daughter cell, thus perpetuating the population. In contrast, progenitor cells are more differentiated and give rise only to cells more differentiated than themselves. The fate of any particular developing cell is determined in large part by its microenvironment.

The developmental changes in the number, function, and location of HSCs are of interest to transplantation biologists and gene therapists. The proliferative capacity of HSCs differs with the anatomic source of the cells and with the age at which the cells are harvested. The sensitivity of these cells to recombinant cytokines also changes with age. Improving the understanding of the ontogeny of these cells may be helpful in optimizing their clinical use.

Embryonic and fetal HSCs are capable of repopulating adult organisms. In contrast, transplanted adult stem cells have a lower capacity for self-renewal, sometimes resulting in late graft failure. This might be because stem cells harvested from adults continue to express the adult differentiation program, even if transplanted into a neonatal environment, indicating an irreversible change in gene expression. Other explanations for the decrease in proliferative potential may have to do with DNA damage over time and loss of telomere repeats with each stem cell division, limiting the replicative potential.

Ongoing research focuses on optimizing stem cell harvesting techniques. Cell-surface markers, which are dependent on cell maturity and gestational age, are often used to identify and separate HSCs with the use of monoclonal antibodies and fluorescence-activated cell sorting analysis. For example, CD34, a cell-surface sialomucin, is an antigen commonly used to select HSCs and early erythropoietic progenitor cells. Combining CD34 positivity with the absence of lineage-specific markers allows the selection of a population highly enriched for cells desired for transplant. Research is also focused on optimizing stem cell harvest sites. Both bone marrow and umbilical cord blood (UCB) are rich in stem cells and have long been used as sources of progenitor cells. The collection of stem cells from peripheral blood by stimulated apheresis, with ex vivo expansion of select populations, is now also an option.

UCB can be harvested as a source of HSCs and used for transplant in patients with marrow failure, malignancy, or immunodeficiency. Hematopoietic progenitor cells in UCB tend to have a high proliferation index compared with cells harvested from adult marrow. Because of the immaturity of the UCB cells, HLA matching is less stringent for UCB, allowing more efficient donor unit identification compared with a bone marrow registry. This feature also allows the improved matching ability for patients in minority ethnic populations since bone marrow registry matches are frequently in short supply. Promising strategies to increase cells available for transplant include combining multiple units of UCB and ex vivo expansion of UCB units.

The first successful use of UCB for HSC transplant purposes was in 1989, between HLA-identical siblings, for severe aplastic anemia from Fanconi anemia. Since then, thousands of UCB transplants have been performed to treat malignancies such as acute lymphoblastic leukemia, acute myeloid leukemia, and chronic myeloid leukemia; bone marrow disorders such as Fanconi anemia; immunodeficiencies such as severe combined immune deficiency; metabolic disorders; and hemoglobinopathies.

UCB can be stored in public or private commercial banks. Donation of harvested cells to public UCB banks allows storage in a central repository available to all individuals in need of a transplant. Private banking is expensive and less standardized. Banked cells are available for future use by individuals or a family, but there is a low likelihood of any one individual needing an autologous UCB transplant.

Delaying umbilical cord clamping by 60 seconds increases the blood volume of the neonate and increases the endowment of iron. In theory, this practice might result in insufficient UCB remaining for banking. However, the Canadian Task Force on Preventive Health Care suggests that delayed cord clamping has significant benefits for the neonate and that sufficient blood typically remains in the umbilical cord and placenta after delayed clamping for UCB banking. The task force suggests that both techniques can generally be accomplished.

Primitive and Definitive Erythropoiesis

During development, two types of RBCs are formed: primitive and definitive erythrocytes. The liver is the primary organ of hematopoiesis during fetal life, but primitive RBCs are first formed in the yolk sac. Large primitive RBCs are produced in blood islands of the yolk sac days after implantation of the embryo. These cells enter the newly formed vasculature of the embryo, where they continue to divide and differentiate for several days. This process is only minimally responsive to Epo. Primitive erythroblasts are large (>20 μm), nucleated, CD34 negative, and contain predominantly embryonic hemoglobin. Hemoglobin synthesis continues until cell replication ceases. In mice, a transition to definitive erythropoiesis occurs at embryonic day 13.5 (full gestation is 21 days). Definitive erythropoiesis is characterized by smaller (<20 μm) CD34-positive erythroblasts, which produce fetal and adult hemoglobins, extruding their nuclei when mature. Unlike primitive erythropoiesis, this process is dependent on Janus kinase signal transduction and Epo stimulation. Primitive erythroblasts normally undergo apoptosis, becoming extinct during fetal life, whereas definitive erythroblasts are able to self-renew.

Switch of the Primary Site of Erythropoiesis

Humans have four main sites of embryonic and fetal erythropoiesis—yolk sac, aorta (ventral aspect), liver, and marrow. In rodents, the spleen is also an important site of hematopoiesis, but there is no evidence for this in healthy humans. Studies using an in vitro embryonic stem cell differentiation system showed that endothelial cells, primitive hematopoietic cells, and definitive blood-cell colonies arise from a common fetal, liver, kinase-1 (Flk-1)-expressing progenitor. Between embryonic day 8 and embryonic day 11.5, runt-related transcription factor 1 is required for the formation of HSCs and their progenitors. Primitive progenitor cells first develop in the yolk sac, followed by the rise of definitive progenitors, also in the yolk sac. Another source of the early definitive progenitors is the ventral aspect of the embryonic aorta. Once circulation is established, progenitors from all lines are detected in the blood, then in the liver, and finally in the marrow.

Yolk Sac

The yolk sac is an extraembryonic structure that can be subdivided into the primary and secondary yolk sac. The primary yolk sac is transient and has no known hematopoietic function. In humans, it forms by proliferation and differentiation of primitive endodermal cells 7 to 8 days after conception. These endodermal cells give rise to mesodermal precursors (intermediate cells). The primary yolk sac then collapses into small vesicles, and the secondary yolk sac is formed from its remnants 12 to 15 days after conception. By 16 to 19 days, primitive erythropoiesis is found in the human yolk sac. The secondary yolk sac is an active site of protein synthesis, nutrient transport, and hematopoiesis. Primitive hematopoietic cells, adherent to surrounding endothelial cells, are first observed on day 16 in the mesodermal layer. These hematopoietic–endothelial cell masses have been described as blood islands . As maturation proceeds, these blood islands migrate toward each other, merging to form a network of capillaries. Small clusters of undifferentiated cells, the hemangioblasts, and clusters of primitive erythroblasts are observed in the small vessels present at this developmental stage. As differentiation proceeds, endothelial and hematopoietic cell lineages emerge. These cell types share common molecular markers and responsiveness to a cohort of growth factors and, depending on the microenvironment, can be derived from a common stem cell in culture.

After the sixth week after conception, definitive erythroblasts are found in the yolk sac. A decline in yolk sac hematopoiesis is observed after the eighth week. Yolk sac–derived hematopoietic cells have more restricted potential in vivo, as only RBCs and macrophages are present in the yolk sac, while progenitor cells in the liver develop into the full spectrum of hematopoietic cells. However, when yolk sac–derived stem cells are cultured in vitro or are transplanted, they are multipotent, illustrating the importance of the microenvironment in the development of committed cell lineages.

Aortogonadomesonephron

Another site of early erythropoietic activity in the developing human embryo is the ventral aspect of the aorta in the periumbilical region. At around the 23rd postconceptional day in humans, the multipotent hematopoietic progenitor cells in this region are more numerous than in the yolk sac or the liver. By day 40 of gestation, hematopoiesis in this site is concluded.

Liver

A short time after the onset of blood circulation (week 4 to week 5 of gestation), erythropoiesis begins in the liver. As in the yolk sac, primitive erythroblasts initially predominate. However, over the next 4 weeks, definitive erythrocytes become the predominant RBC form. During this time, the liver mass increases 40-fold, with hematopoietic cells constituting 60% of the liver from week 11 to week 12. ^. Meanwhile, other hematopoietic cell types are also produced in the liver. Early in this process (5 weeks), macrophages predominate, with approximately one granulocyte to every nine macrophages. In contrast to the yolk sac, during the period of peak hepatic hematopoiesis (week 6 to week 18), production of all hematopoietic cell lines (erythrocytes, macrophages, megakaryocytes, granulocytes, and lymphocytes) occurs. Between 18 and 21 weeks of gestation, hematopoiesis in the liver diminishes, but the liver continues as an erythropoietic organ until term.