In vitro biology of human hematopoietic stem and progenitor cells

Abstract

Hematopoietic stem and progenitor cells from human umbilical cord blood have been the focus of both basic and clinical research during the last 20 years. It has been clearly demonstrated that such sells possess higher proliferation and expansion potentials, as compared to their adult counterparts, and their capacity to reconstitute the hematopoietic system of mammals has also been shown. Different in vitro systems have been used to characterize the biology of these hematopoietic cells and some culture methods are being currently used to expand the numbers of such cells for clinical purposes.

Keywords:

• Cord blood
• In vitro
• Progenitor cells
• Stem cells

Hematopoietic Stem and Progenitor Cells in Cord Blood

Hematopoietic stem (HSC) and progenitor (HPC) cells reside in the bone marrow, where they proliferate and differentiate, giving rise to mature blood cells. However, by mechanisms that are still not fully understood, such primitive cells can be mobilized into peripheral blood, thus, it is possible to find them in circulation. Such a mobilization occurs not only in adult subjects, but throughout the entire life of the individual and, in fact, since the third month of prenatal life. Accordingly, HSC and HPC can be observed in umbilical cord blood (UCB). This was first reported by Knudtzon in 1974, who described the presence of relatively mature myeloid progenitors in UCB. About 10 years later, Ogawa and colleagues documented the presence of more primitive hematopoietic cells, and in the late 1980s, Broxmeyer et al.1 showed that UCB contains vast amounts of both primitive and mature hematopoietic cells. To date, UCB is recognized as a major source of HSC and HPC both for research and clinical application.

One milliliter UCB contains between 1000 and 10 000 multipotent progenitors (CFU-MIX), about 8000 primitive erythroid progenitors (BFU-E) and between 5000 and 14 000 myeloid progenitors (CFU-G/M); thus, the total number of HPC is around 23 000 per ml. When comparing the relative levels of stem and progenitor cells in UCB and bone marrow, it has been found that no significant differences exist in the values of total progenitos; however, important differences in the frequency of particular HPC subpopulations have been observed. That is to say, whereas the levels of relatively mature progenitors are similar in both sources, the frequency of primitive progenitors, including multipotent, erythroid and bipotent granulo-monocytic, is significantly higher in UCB than in marrow.

The presence of more primitive hematopoietic cells, including long-term culture-initiating cells (LTC-IC) and SCID-repopulating cells (SRC), has also been observed in UCB. Pettengel and colleagues reported that LTC-IC can be found at frequencies similar to those in bone marrow, i.e. 1/15 000 mononuclear cells; whereas Wang et al.2 have estimated that the frequency of SRC in UCB (1 per 9×10⁵ mononuclear cells) is three-fold higher than in bone marrow (1 per 3×10⁶ mononuclear cells).2 It is noteworthy that the presence of these latter cells has not been documented in adult peripheral blood under unperturbed conditions. Although it seems reasonable to suggest that they are, indeed, present in adult circulation, their frequency might be too low to allow their detection by standard methods.

Immunophenotype

HSC express a particular pattern of cell surface antigens that includes CD34, CD49f, CD90, CD117, and CD133. They do not express CD38, CD45RA, CD71 or any lineage-specific antigen (they are lineage-negative). Subpopulations of HPC from UCB have been clearly identified based on the expression of certain antigens. Studies by Mayani and Lansdorp3 indicate that UCB CD34⁺ cells can be separated into functionally distinct populations based on the expression of CD45RA and CD71. CD34⁺ cells expressing low/undetectable levels of both antigens are enriched for multipotent (CFU-MIX) progenitor cells, which correspond to almost 50% of the CFC in this particular subpopulation. CD34⁺ CD45RA⁺ CD71⁻ cells are enriched for myeloid progenitors, which correspond to 90% of the CFC, whereas CD34⁺ CD45RA⁻ CD71⁺ cells are enriched for erythroid progenitors (>70% of the total CFC). Among UCB CD34⁺ CD45RA⁻ CD71⁻ cells, 20–30% also express CD90 (Thy-1) and this subpopulation is highly enriched for high proliferative potential CFC (HPP-CFC) and LTC-IC.3 Interestingly, all of the CD34⁺ CD90⁺ cells also express CD117, although at low levels.

In vitro Proliferation, Expansion and Differentiation

The proliferation potential of a hematopoietic cell has been defined as its ability to divide and generate new daughter cells (regardless of the cell lineage and maturation stage of the cells produced); the expansion potential, on the other hand, has been defined as the capacity of a cell to divide and generate new cells with biological features similar to those of the original cell. These two biological features depend upon intrinsic factors (transcription factors, cell cycle regulators, telomerase, signal transduction molecules, cytokine receptors, etc.). However, the ability of a cell to exhibit such potentials depends on extrinsic factors that include all the cell types and their products that form part of the microenvironment in which the cell develops. In vitro, proliferation and expansion of hematopoietic cells also depend on variables such as type of culture medium, medium change schedule, temperature, cell density, presence or absence of serum, presence or absence of stromal cells, etc.

It has been clearly demonstrated that the developmental stage of a hematopoietic cell dictates their biological behavior in vitro. Primitive subpopulations of CD34⁺ cells, e.g. CD34⁺ Rh^low; CD34⁺ CD38⁻; CD34⁺ CD45RA⁻ CD71⁻ cells and CD34⁺ CD45RA⁻ CD71⁻ CD90⁺ cells, possess greater expansion potentials than their more mature counterparts. This has been shown both in bulk cultures of purified cells and in cultures containing one cell per well. Indeed, at the single cell level, it has been shown that one HSC from UCB can give rise to 37×10⁶ nucleated cells and over 60 000 CD34⁺ cells, whereas committed erythroid and myeloid progenitors produce lower numbers of both nucleated cells (4×10⁶ and 0·5×10⁶ cells, respectively) and CD34⁺ cells (1500 and 4000 cells, respectively).

It has been postulated that the vast majority of progenitor cells from UCB are in the G₀ phase of the cell cycle. Some investigators, however, argue that rather than being deeply quiescent (G₀), a significant amount of the circulating progenitors is in G₁ and might readily enter S-phase, particularly if exposed to certain cytokines. These observations are of relevance since the proliferation and expansion potentials of primitive cells seem to depend, at least in part, on the cell cycle phase in which they are at a particular moment. G₀ CD34⁺ cells from cord blood possess a 1000-fold higher capacity to generate progenitors (expansion) in vitro, than their G₁ counterparts.

As mentioned before, the microenvironment elements surrounding HSC and HPC influence the proliferation and expansion potentials of hematopoietic cells. Among the different cytokines that participate in hematopoiesis, those acting at the early stages of the hematopoietic hierarchy (i.e. SCF, FL, TPO, IL-6) have been found to be essential in favoring the expansion of HSC and HPC in vitro. Piacibello and colleagues have reported that culture of CD34⁺ cells in the presence of FL and TPO results in a 2×10⁶-fold expansion in the number of CFC. Addition of late-acting factors, such as erythropoietin, usually contribute to the production of large numbers of mature cells, however, they do not seem to affect HPC expansion. The participation of stromal cells in the in vitro proliferation and expansion of primitive hematopoietic cells has also been documented. They seem to be important in such processes,4 however, their actual roles have not been fully elucidated; some argue that a direct contact between hematopoietic and stromal cells is necessary for optimal growth of the former; in contrast, others sustain that their major function is to provide soluble cytokines and their presence may not be critical as long as the cultures are supplemented with cytokines.

It has been clearly shown that UCB-derived HPC possess higher proliferation and expansion potentials than their adult marrow counterparts. Similarly, several investigators have demonstrated that significant functional differences exist between hematopoietic cells from UCB and adult mobilized peripheral blood (MPB).5 Gilmore and colleagues reported that UCB-derived CD34⁺ cells were able to proliferate and expand in cultures supplemented with FL and TPO for up to 16 weeks, generating significant numbers of HSC/HPC; in contrast, under the same culture conditions, CD34⁺ cells from adult MPB were unable to expand. In keeping with these observations, Tanavde et al. also showed that in cultures supplemented with FL, SCF, and TPO, the expansion capacity of cord blood-derived CD34⁺ cells was far superior to that of their MPB counterparts. Furthermore, such expanded UCB cells showed in vivo repopulating potential in immunodeficient mice; in contrast a significantly decreased engrafting activity was seen in MPB cells. According to different studies, such functional differences seem to be due, at least in part, to the fact that cord blood cells (i) exit more rapidly from G₀/G₁ phase of the cell cycle, and (ii) possess longer telomeres than MPB cells.5

Differentiation of CD34⁺ cells along different hematopoietic lineages also depends on the action of specific cytokines. It is still unclear whether such molecules play a deterministic or a permissive role in the differentiation process of hematopoietic cells; however, it is clear that the presence of particular cytokines is fundamental for the generation of mature cells from primitive, multipotent progenitors. In this regard, important differences have been observed in terms of megakaryocytic differentiation between UCB and MPB cells. UCB- and MPB-derived progenitors seem to follow different megakaryocytic maturation pathways, since their immunophenotypes show distinct CD34 and CD41 expression patterns. Furthermore, it has been reported that, in contrast to their PB counterparts, cord blood megakaryocytes do not complete their maturation process in vitro.

Ex vivo Expansion for Clinical Application

Ex vivo expansion of circulating HSC and HPC can be achieved by culturing UCB cells in liquid cultures supplemented with recombinant cytokines. Although significant expansion levels have been documented using different experimental systems, two major points remain controversial and represent important goals to be achieved in the near future: the generation of hematopoietic cells that retain multipotentiality and self-renewal capacity, and the production of such cells in sufficient numbers for their clinical application.

Recent reports have shown that when primitive hematopoietic cell subsets are cultured in liquid media supplemented with recombinant cytokines, there is an increase in the number of CD34⁺ cells (including those that do not express CD38), with a concomitant increase in the levels of CFC and LTC-IC. However, no increase was observed in the numbers of SRC, suggesting that the relationship between stem cell surface phenotype and function may not be reliable for cultured cells. In keeping with these results, McNiece and colleagues reported that ex vivo expansion of UCB CD34⁺ cells (cultured for 14 days in liquid media supplemented with SCF, TPO and G-CSF) resulted in the generation of increased numbers of myeloid progenitors and mature cells that were able to engraft primary sheep but lacked secondary and tertiary engrafting potential. This study suggested that although ex vivo expanded cells may be able to provide rapid short-term engraftment, the long-term potential of expanded grafts may be compromised.

In contrast to the above study, a recent report by Verfaillie’s group indicates that human UCB cells capable of engrafting in primary, secondary and tertiary xenogeneic hosts (mice and sheep) can be preserved after ex vivo culture in a non-contact system, in the presence of the AFT024 stromal cell line and the recombinant cytokines FL, SCF, TPO and IL-7; thus, suggesting that true stem cell activity is maintained in culture. The discrepancies observed in the results obtained in this latter study and those mentioned above may be due to differences in culture conditions, namely, the presence or absence of stromal cells and the cytokine combination used.

Two common points of discussion between laboratories working on the ex vivo expansion of hematopoietic cells relate to whether stromal cells must be present, or not, in culture, and what should be the optimal cytokine combination that has to be used.6 Although some basic culture principles have been identified, no consensus exists so far, and every single laboratory keeps working out the particular culture conditions that best suite their specific needs and interests. However, if expanded hematopoietic cells are going to be taken to the clinic, standard, reliable, and reproducible culture systems need to be established.

Interestingly, and based on the existing evidence, McNiece has suggested that clinical protocols may require transplantation of two cell fractions: an expanded fraction of UCB cells to provide rapid short-term engraftment and an unmanipulated fraction of cells to provide stem cells for long-term engraftment. An important point that has relevance in the application of ex vivo expansion of hematopoietic cells into the clinic is the fact that the cell fraction to be expanded does not have to be a pure population (>95%) of CD34⁺ cells. Indeed, enriched fractions consisting of 40–70% CD34⁺ cells can be significantly expanded and provide sufficient numbers of both progenitor and mature cells for transplantation.

Two separate studies have recently been reported in which ex vivo expanded UCB cells were administered into patients. In both studies, the main goal was to assess the feasibility of using UCB expanded cells in patients with hematological diseases, breast cancer, and some other metabolic disorders. The culture period consisted of 10–12 days and the cells were cultured in the presence of recombinant cytokines (SCF, TPO and G-CSF in one study, and PIXY321, FL and EPO in the other). The expansion observed in terms of CFC and CD34⁺ cells was quite variable, and although infusion of expanded cells into patients did not significantly alter myeloid, erythroid or platelet engraftment, both studies concluded that this procedure is feasible and safe. Randomized phase two clinical trials will determine whether this approach is beneficial to patients.7,8

More recently, Delaney and colleagues described the use of the Notch-ligand Delta-1 as a means to induce the expansion of HSC and HPC.9,10 They enrolled 10 subjects with acute leukemia in a phase one clinical trial consisting of infusing two UCB units in each patient, one unmanipulated and one ex vivo-expanded. Sixteen days before the transplant, the UCB unit chosen for ex vivo expansion was thawed, CD34⁺ cells were enriched and Delta1-cultures were established. On the day of transplant, cultures were collected and cells were infused into patients 4 hours after infusion of the unmanipulated unit. At collection time, there was a 164±48-fold expansion in CD34⁺ cells, and an average fold increase of total cell numbers of 562±134. The infused CD34⁺ cell dose derived from expanded cord blood grafts averaged 6×10⁶ CD34⁺ cells per kg of body weight of the patient; this compared very favorably to the number observed in unmanipulated cord blood grafts (0·24×10⁶ CD34⁺ cells per kg). Two major findings were observed. First, there was a significant reduction in the time to myeloid engraftment in those patients that received one unmanipulated and one expanded unit (16 days; range 7 to 34 days), as compared to a cohort of 20 patients undergoing double cord blood transplantation with two unmanipulated units (26 days; range 16–48 days). Second, engraftment of the expanded unit seemed to be only transient, since in the majority of the evaluable cases, the expanded unit was undetectable after 20–40 days post-transplant.

Thus, based on the results discussed herein, it seems clear that, although some issues remained to be clarified, ex vivo expansion and manipulation of UCB hematopoietic cells will play a role in UCBT and regenerative medicine.11