EXPERIMENTAL HEMATOLOGY: SOURCES AND LESSONS
From the first experimental proof of the existence of hematopoietic stem cells provided by the classical experiment of Till and Mc Culloch[1] and from its consequences[2] (1961), a new discipline - experimental hematology - has developed. The first approach of experimental hematology is to characterize the functional heterogeneity of stem and progenitors cells by in vivo and in vitro functional assays; the second approach consists of searching for an immuno-phenotype characterizing each of the different sub-populations of stem and progenitors cells[3-6]. Although important advances have been made in terms of enrichment of stem cells by means of immuno-phenotypical properties, the initial functional characterization is still the only way to prove the existence of the stem cell entities[7]. This functional definition could not be avoided; this can be illustrated by two major breakthroughs in stem cell biology: (1) induction of pluripotent stem cells from somatic cells[8-10]; and (2) initiating hematopoiesis from human embryonic stem cells[11].
Taken together, almost four decades of research on stem cells that exhibit a hematopoietic differentiation potential allowed an understanding of the functional heterogeneity of stem and progenitor cells, proposed a long time ago as the “generation-age hypothesis”[12]. This heterogeneity is the main factor leading to a very complex situation that does not allow simplification without losing some essential notions.
The first reports revealing this heterogeneity dealt with the phenomenon of hematopoietic reconstitution after engraftment. Two phenomena; i.e. the kinetics of red blood cell repopulation (erythrocyte repopulating ability-ERA) and the kinetics of granulocyte repopulation (granulocyte repopulating ability-GRA) were reported[13-15]. It was evident that these phenomena resulted from the activity of two distinct cell populations that are more immature than morphologically recognizable precursors of these two lineages, but less immature than the multi-lineage progenitors called “colony forming unit-spleen (CFU-S)” detected by the assay of Till and McCulloch[16-21]. The development of in vitro assays for clonogenic progenitors showed that these two repopulating activities result from two distinct populations of committed progenitors: those of granulocyte monocyte lineage (CFU-GM) and those of erythroid lineage (CFU-E, BFU-E)[21]. But these “repopulating activities;” i.e. “committed progenitors,” are different from CFU-S[22], whose population is capable, if transplanted after lethal irradiation, to protect animals from acute radiation-induced lethality (“radio-protective ability”)[23]. The CFU-S population has also been shown to be heterogenous; relatively less primitive CFU-S produced colonies 8 to 9 d after injection of hematopoietic cells and the other relatively more primitive CFU-S produced colonies 12 to 14 d after the injection. In fact these sub-populations of CFU-S are overlapping[24]. Furthermore, the “late” colonies growing 12 to 14 d from more primitive multipotential progenitors contain more primitive cells, which are responsible for short-term engrafted clone maintenance, known under the generic terms “pre-CFU-S” or “marrow repopulating ability-MRA”[25-27]. Actually, this is the first population that could be considered as a real stem cell population according to current standards. Even more primitive stem cells have subsequently been found, allowing long-term maintenance of hematopoiesis after engraftment[28].
The previous paragraphs summarize 25 years of work, which enabled realization that hematopoietic stem cells and progenitors are organized as a continuum of descendant cell populations having a decreasing proliferative capacity and decreasing self renewal ability, starting from the most primitive stem cells to the last progenitors preceding precursors. In animal experimental models, the reconstitution of hematopoiesis after engraftment and consequent repopulation of peripheral blood results from successive waves of repopulation. This phenomenon stems from the heterogeneity of stem and progenitor cells since less primitive cells take less time to develop morphologically recognizable hematopoietic cells and vice versa for more primitive progenitors and stem cells. Some results suggest, however, that long term reconstitution could stem from short term reconstituting stem cells that are activated and exhausted in a successive manner[29-30]. This question does not interfere with the phenomenon of initial reconstitution after transplantation, for which the mechanism is well established and accepted. In summary, the works of experimental hematology imply that for a rapid and long term hematopoietic repopulation, a sufficient number of both stem cells and committed progenitors (of all categories) should be injected. With the development of in vitro cultures for the detection of human committed progenitors, as well as in vivo xenogenic transplantation models for the detection of human stem cells, the main points initially established in animal models have been confirmed for hematopoietic stem and progenitors cells issued from three main “human” sources: bone marrow, peripheral blood after mobilization and placental (cord) blood[5,31].
The concept of ex vivo expansion is derived directly from this knowledge. It is based on a very attractive idea to increase the number of cells and progenitors (aimed to accelerate hematopoietic reconstitution) in order to insure a secure and favorable long-term outcome of transplantation. As a matter of fact, for clinicians, the first objective of an ex vivo expansion is shortening the period of post transplantation agranulocytosis. The duration of this period varies between 1 and 4 wk depending on the source of transplanting cells [peripheral blood after mobilization, bone marrow, and placental (cord) blood].
On the basis of experimental hematology data from animal models[13-22], duration of this period depends mostly on the number of relatively mature progenitors present in populations of transplanted cells. On the other hand, experimental data demonstrate that, for long-term reconstitution, the presence of more primitive stem cells is required[25-28]. Accordingly, the ideal ex vivo expansion should allow amplification of both committed progenitors and stem cells.
IMPLEMENTATION IN CLINICAL HEMATOLOGY
From this experimental work, clinical hematology adopted the principle of hematopoietic stem cell transplantation. This practice started with bone marrow cells but other sources were preferred subsequently: hematopoietic progenitors and stem cells mobilized to peripheral blood as well as those from placental (cord) blood. The first bone marrow transplantations were allogenic, aimed to reconstitute the hematopoietic system of humans irradiated in a nuclear accident[32]. Since then, hematopoietic cell transplantation as a clinical discipline yielded a tremendous amount of knowledge, not only related to stem cell biology, but also for immunology (e.g. discovery of the HLA system). In spite of this fact, the development of clinical transplantation sometimes neglects some fundamental points of experimental hematology. We discuss one of these points in this review.
Total CD34+ cell dose issue
Since the beginning of clinical transplantation practice, the total number of viable cells has been considered as a main parameter in transplantation. Though polymorphonuclear cells, monocytes, and lymphocytes do not provide hematopoietic reconstitution after transplantation, the total cell number is still considered as a first rate qualifying and prognostic factor in transplantation, especially for placental (cord) blood cells. Indeed, in most papers describing transplantation of cells issued from hematopoietic sources in the steady state, there is a correlation between engraftment, kinetics and the total cell number in the graft[33]. This correlation results from the fact that the concentration of stem and progenitor cells in hematopoietic tissues in the steady state, or after some standard therapeutic protocols, is more or less constant. Thus, the increase in total cell numbers also means an increase in stem and progenitor cell numbers. In addition, this parameter is easily and rapidly determined. Taken together, its usefulness has been confirmed. Of course, it would be wrong to consider that, due to this correlation between total cell number and transplantation outcome, the engraftment is achieved by total cells instead of stem cells.
The possibility of detecting CD34+ cells enabled researchers and technologists to approach a non-differentiated cell population containing most hematopoietic progenitors and stem cells[34], but this was not specific. Vascular endothelial cells, perivascular dendritic cells, hair follicle “stem” cells, spindle shaped cells of eccrine glands cells, for example, also express CD34[35]. This molecule, (also known as, e.g. “podocalyxin-like protein”, “thrombo mucin”, “gp135”, etc.) belongs to a family of proteins (“CD34-family”) that have overlapping expression patterns[36]. CD34-family proteins (CD34, podocalyxin, and endoglycan) have a serine-, threonine-, and proline rich extracellular domain that is extensively O-glycosylated and sialylated (90-170 kDa). The function of CD34 family members has not yet been definitively elucidated. However, several roles have been ascribed to these proteins; for example, the proliferation-promoting effect, differentiation-blocking effect on progenitor cells, enhancement of trafficking and migration of hematopoietic cells, and a role in cell morphogenesis[36]. Despite this expression pattern, nonspecific to hematopoietic tissue, and an elusive physiological role, the CD34 protein has become, in the minds of many in the biomedical community, the main marker endowing hematopoietic stem and progenitor cells. Furthermore, most clinicians and biologists who are not directly involved in stem cell research have a tendency to add the term “stem cells” each time they say or write “CD34+”. This tendency has been a permanent source of misunderstanding and confusion and it heavily affects experimental and clinical hematology. It should therefore be repeatedly stressed that the fact that the majority of hematopoietic stem and progenitors cells express CD34+ does not mean that all CD34+ cells are stem cells or progenitors. The CD34+ cell population is very heterogeneous[34]. For example, in the CD34+ population of placental (cord) blood, 30% to 50% are progenitors (CFU-GM, BFU-E, CFU mix, and CFU-Mk) and only a small percentage are primitive stem cells. Approximately one half of the CD34+ cell population does not exhibit either progenitor or stem cell functional properties. Some stem cells do not express CD34+ in a steady state[37] and expression of this molecule could be reversible and not related to functional capacities of stem cells[38]. Here again, the CD34+ cell count in different cell populations derived from hematopoietic tissues in a steady state or mobilized in peripheral blood has been confirmed as a useful parameter of the graft concerning the kinetics of engraftment[39-44], although only a small fraction of these cells have stem cell characteristics. The dose of CD34+ cells correlates well with the dynamics of hematopoietic reconstitution compared to total cell number. This results from the fact that the proportion of progenitors in stem cells inducing “transitory” engraftment in the CD34+ population is higher than in other sub-populations. It is also relatively stable for the tissue in question. In addition, it is easy to get the count of CD34+ cells by immuno-staining and flow-cytometry. Thus, the number of CD34+ cells became a main parameter of graft quality control. Since rapidity of hematopoietic reconstitution correlates with the number of CD34+ cells per kilo of patient weight, this approximation induced a “mental shortcut” in clinical hematology; the term “CD34+ cells” is frequently equated with the term “stem cells”. On the contrary, experimental hematology considers the term “stem cell” as a functional entity (or state)[7]. Even a very complex and sophisticated procedure aimed to isolate “stem cells”, based on several immuno-phenotypic markers and combined with metabolic properties, only enabled a high degree of enrichment and not a completely pure stem cell population[45]. For example, Lin- CD34+ CD38- fraction from placental (cord) blood only contains 1%-2% of stem cells detectable by a functional ex vivo assay[46]. In addition, if steady state is disrupted, as it is in ex vivo expansion cultures, for instance, the relationship phenotype/function is less evident or even non-existent[47-51].
Clinical vs experimental
In general, it is more difficult to follow the specific effect of one variable in clinical rather than experimental situations. The individual variations of cellular parameters in humans are larger than in rodents. The treatment of humans should be effected within the requirements of clinical trials. In addition, the preparation of the graft is restrained to only accepted and validated procedures. After all, the interference of different human pathologies, as well as previous treatments and therapeutic approaches, could have a big impact on the effects of transplantation. These are only some of the reasons why it is sometimes difficult to reproduce the same effect on humans that was demonstrated in animal experimentation. The apparent absence of correlations in some clinical trials, however, between two variables that correlated in animal trials, does not mean that the principle is automatically erroneous.
This should be considered in the issue of hematopoietic reconstitution after transplantation. Many papers have been published demonstrating a positive correlation between the total number of cells and the number of CD34+ cells and hematopoietic reconstitution. Determination of hematopoietic progenitors on the basis of their colony-forming capacity in culture is less practical and more time consuming than determining CD34 expression; therefore, the number of hematopoietic progenitors has not been systematically taken into consideration in analysis of hematopoietic reconstitution[33,39-44]. However, in some reports, these parameters were properly analyzed. These analyses almost always showed that the best correlation is between committed progenitors and rapidity of hematopoietic reconstitution[52-54] in comparison with total cells and CD34+ cells. Other studies have shown the absence of correlation between the total cell number, CD34+ number and clinical and hematologic outcomes[55]. This confirms a relative progenitor and stem cell source-dependent value of these parameters [unfortunately, the progenitor (CFC) number analysis was not shown]. Furthermore, short term repopulating cells, previously demonstrated in animal models, also exists in human grafts. In bone marrow grafts, for example, these short term repopulating cells have clearly demonstrated a hematopoietic reconstitution inferior to 100 d[56]. These stem cells, found in sub-populations CD34+ and CD34- and CD34+ HLA-DR-, are not correlated with a long term hematopoietic reconstitution (between 100 d and a year post transplantation). This late reconstitution, however, is correlated with CD34+ cell number, due to the presence of very primitive stem cells inside this heterogeneous population, as mentioned above[56].
These discoveries confirm that human stem cell biology is not an exception with respect to other vertebrates. This information is in favour of the “expansion concept,” which postulates that ex vivo amplification of committed progenitors should accelerate hematopoietic reconstitution after transplantation. We could not analyze here all clinical trials that were recently reviewed dealing with the transplantation of bone marrow, peripheral blood, and cord blood hematopoietic cells after ex vivo expansion[57]. The initial inefficiency of this approach, however, was due to inefficient ex vivo protocols and/or to the study design rather than an erroneous concept. Some, however, demonstrated a positive effect on hematopoietic reconstitution after transplantation, decreasing the incidence of neutropenic fever, reduction of red blood cell transfusions, and the diminution of the duration of hospitalization[58,59]. A trial carried out with a combination of cytokines, showing a high pro-differentiation power, enabled a relatively modest expansion of total cells and progenitors. Although this trial did not provide an acceleration of hematopoietic reconstitution, it is important because transplanted cells failed to maintain short and long term reconstitution after aplasia[60]. With current knowledge, it could be proposed that the stem cells with short term and long term repopulating capacities have been exhausted in expansion cultures due to the culture conditions, especially to IL-3 and IL-1 association and the exposure of the culture to ambient oxygenation. Thus, this trial underlines the importance of the presence of primitive stem cells in a graft. Furthermore, it firmly demonstrates that the number of CD34+ cells only is not a universally appropriate parameter of the graft quality, since the primitive stem cells could be absent. Also, if a graft, as in this case an expansion product, is composed exclusively of committed progenitors without stem cells, it could only ensure a transient engraftment.
The first really successful expansion protocol[61,62] confirmed that hematopoietic reconstitution depends on the functional sub-populations of progenitor and stem cells that should be present in a graft. In addition, it presents a very interesting example of the phenomenon called “dissociation phenotype-function”. During the pre-clinical development of this expansion procedure, as well as in expansion for clinical trials, we found that the expansion of progenitors with a mean value of 27 fold was accompanied with an expansion of CD34+ cells of only 3.5 fold[62,63]. In terms of absolute number, we get almost twice the number of committed progenitors than CD34 cells. This means that, in the course of ex vivo expansion, the culture generated the progenitors that do not express CD34 antigen (see the studies related to the transient expression of CD34)[38]. Thus, the predictable value of the CD34+ cell count could be questioned for expansion products. Indeed, the results derived from clinical trials point to the absence of correlation between the number of CD34+ cells in a graft and the duration of post-transplantation agranulocytosis[62]. On the contrary, the number of committed progenitors was well correlated with the acceleration of post transplantation hematopoietic reconstitution[62].
Concluding remarks
On the basis of experimental data, the capacity of a CD34+ cell population to reconstitute hematopoiesis quickly after engraftment, as well as in the short- and long-term perspective, depends on the presence (in sufficient number) and proportion of functionally very different CD34+ sub-populations. This proportion should vary with respect to the source (e.g. bone marrow peripheral blood[64], cord blood[5,31]), pathology, treatment, processing procedure, the graft ex vivo treatment[62] and so on.
For all these reasons, the same number of CD34+ cells could give completely different results related to the rapidity of hematopoietic reconstitution and the short and long term maintenance of hematopoiesis. Considering this, it would not be expected that the number of CD34+ cells would become the universal “first rate” parameter for clinical transplantation, and that a universal CD34+ cell dose could be defined.
Also, to avoid further confusion in research and clinical practice, the heterogenous population of cells endowed by CD34+ antigen expression should not be named as “stem cells” or “progenitor cells”. These denominations only concern functionally characterized cell entities.
