Evidence that the number of hematopoietic stem cells per animal is conserved in mammals

Hematopoietic stem cells (HSCs) are the functional units of blood cell production. Through self-renewal (replication) and differentiation, HSCs maintain hematopoiesis throughout an animal's lifetime. HSC number and kinetics have been studied extensively in mice and are genetically regulated. Less is known about HSC behavior in larger mammals or humans, in whom hematopoietic demand (ie, number of mature cells required) is significantly higher. To understand the adaptation to increased size and longevity, we have previously investigated HSC behavior in cats (which make the same number of red cells in 8 days as humans in 1 day or mice in a 2-year lifetime)1 and have demonstrated that the frequencies of HSCs differ by 100-fold in mouse and cat marrow.2-8 In this study, we determined the total number of HSCs. Surprisingly, it appears that the total number of HSCs in a cat is similar to, and within-estimation variability overlaps with, the total number of HSCs in a mouse.

To compute the total number of HSCs in cats, HSC frequency (6 HSCs/10⁷ nucleated marrow cells [NMCs] in adolescent cats, as determined by limiting-dilution competitive transplantation6) was multiplied by the total number of NMCs. Because the number of NMCs per cat was unknown, the value was derived by observing the distribution of iron Fe 59 (⁵⁹Fe)–transferrin to the erythroid marrow of 4- to 9-month-old cats, using the methods of previous murine7 and human8 studies. In this way, a direct comparison of the results between species was feasible.

In a preliminary study, we confirmed that ⁵⁹Fe had at½ in the circulation of 39 minutes.9We demonstrated that infused ⁵⁹Fe was undetectable in blood by 16 hours (which is 25 t½ s), and we assumed complete redistribution to marrow at this time. Heparinized plasma (1.5-2.5 mL) was incubated with 3 μCi (0.111 MBq)⁵⁹Fe as ferric chloride at 37°C for 30 minutes to allow transferrin saturation9 and then was infused into cats intravenously. Studies were performed in 8 adolescent cats (4- to 10-month-old cats were used in studies to determine the HSC frequency6). Marrow and blood were collected at baseline, 2 minutes after infusion, and at 16 to 23 hours. The number of NMCs and radioactivity (Auto-Gamma Counter; Packard Instrument, Meriden, CT) were calculated in 1-mL samples. The equation

 =$erythroblasts in 1 mL marrowtotal erythroblasts in the cat$

=$NMCs in 1 mL marrowtotal NMCs in the cat$

was used to compute the total number of NMCs. Total cpm expected in marrow at 16 to 23 hours was approximated as the total cpm delivered to the circulation at time 0. This was computed as [(cpm of 1 mL of blood at 2 min) – (cpm of 1 mL of blood at 0 min (background)] × the blood volume (or wgt [g] × 7%).

The number of NMCs per cat was determined by ⁵⁹Fe distribution, using methods similar to those in previous murine7 and human8 studies, and the results are in Table 1. The total number (mean ± SD) of NMCs was 1.9 ± 0.9 × 1010. To calculate the number of hematopoietic stem cells per cat, this was multiplied by HSC frequency as shown in Table2, yielding 11 400 ± 5400 HSCs. When the total number of HSCs in a mouse was calculated using a comparable methodologic approach (ie, ⁵⁹Fe distribution analysis to determine the total number of NMCs and limiting-dilution competitive transplantation assays to determine HSC frequency), the result was similar (11 000-22 400 HSCs; Table 2). Therefore, despite the increased hematopoietic demand in cats versus mice, it appears that the number of HSCs per animal, ie number of units for blood cell production, are equivalent.

When experimentally derived estimates are multiplied, measurement error increases; thus, potential sources of error should be assessed. Of note, ⁵⁹Fe studies used male and female domestic 4- to 9-month-old cats (mean weight, 2.5 kg), whereas the HSC frequency estimate was derived from studies of 4- to 10-month-old female Safari (domestic × Geoffroy F₁) cats (mean weight, 3.6 kg). However, if the latter animals had proportionately more NMCs because of their increased size, the estimates of total HSCs per cat would be higher—more completely overlapping murine estimates.

There are 2 direct implications of the finding that the total number of HSCs in cat and mouse is equivalent. First, the proliferation or differentiation capacity of individual feline HSCs must vastly exceed that of individual murine HSCs. It is thus likely that feline HSCs are regulated differently, and with more complexity, so that their use in maintaining hematopoiesis is more efficient. This latter adaptation would be conceptually similar to adjustments of transcriptional and translational control mechanisms through evolution. Although the number of protein-encoding genes in the human genome (30 000-40 000) is only twice that of Caenorhabditis elegans orDrosophila melanogaster, human proteins, because of alternative splicing and other regulatory mechanisms, vastly outnumber proteins in worms and flies.10 

A second implication of our findings relates to human hematopoiesis. If the total number of human HSCs were also 11 200 to 22 400, one could calculate the frequency of human HSCs. Skarberg,8 using⁵⁹Fe distribution analyses, estimates that there are 2.1 × 1010 human NMCs/kg. Assuming that the subjects of these studies weighed 70 kg, the total number of human NMCs would be 1.5 × 1012, and the frequency of human HSCs would be 0.7 to 1.5/10⁸ NMCs (Figure 1). This frequency is 20-fold less than that estimated by NOD/SCID repopulating cell assays,11 a surrogate assay of HSC in which the persistence of human marrow cells transplanted into immune incompetent mice is measured, and may explain why it is difficult to enrich and purify HSCs from human marrow.

Table 3 summarizes and extends the concept that insight into human hematopoiesis can be derived from comparative observations of mice and cats. It appears, for example, that the number of marrow cells per kilogram is conserved and is 1.4 × 1010, 0.6 × 1010, and 2.1 × 1010 in mouse, cat, and human, respectively. Thus, the frequency of HSCs inversely correlates with size and with the total number of marrow cells. Consistent with this concept, the frequency of hematopoietic stem cells among marrow cells in infants and young children would be higher than in adults, as is suggested by previous studies in mice12 and clinical observations in humans. One could argue that in large animals such as elephants, HSCs would be extremely infrequent and that perhaps other adaptive mechanisms may be necessary to ensure adequate hematopoiesis. Table 2 also demonstrates that the total number of HSC replications per animal lifetime is relatively conserved, an observation supportive of the Hayflick hypothesis that somatic cells undergo a finite and fixed number of cell divisions before senescence.13 If one estimates that human HSCs replicate 100 times in 80 years, HSCs would replicate, on average, only once per 42 weeks. Although extremely low, this rate is equivalent to that (once per 50 weeks) derived from the analysis of telomere shortening in human hematopoietic cells.14 

We thank Allan Dimaunahan for help in the preparation of the manuscript.