¹Department of Clinical Biochemistry, Istituto Superiore di Sanità, Rome, Italy

²Department of Cell Biology, Istituto Superiore di Sanità, Rome, Italy

³Department of Biomorphology, University G D'Annunzio, Chieti, Italy

⁴Department of Obstetrics and Gynecology, The Mount Sinai Medical Center, New York, NY, USA

Correspondence to: Dr A R Migliaccio, Department of Clinical Biochemistry, Istituto Superiore di Sanità, 00161 Rome, Italy

Our aim was to evaluate the number of progenitor cells circulating in an -thalassemic fetus during its infusion in utero with paternal CD34⁺ and adult red cells and to compare those values with those circulating in normal and non-thalassemic anemic fetuses of matched gestational age. The treatment of the -thalassemic fetus has been described elsewhere. Fetal blood was obtained from normal and anemic fetuses by fetal blood sampling for diagnostic or therapeutic purposes according to a protocol approved by the human subject committee. The number of progenitor cells in fetal blood was estimated on the basis of the number of colonies they gave rise to in semisolid cultures. The -thalassemic fetus, as did the other fetuses analyzed, contained high numbers (10⁶-10⁷ depending on the age) of progenitor cells, values which were higher than the number (10⁴-10⁵) of paternal progenitor cells being transplanted. Progenitor cells with adult characteristics (adult kinetics of differentiation) were detected rapidly (10 min) after the CD34⁺ cell infusion, but were not detectable 2-3 weeks after the transplant. These results indicate that adult progenitor cells do not have a numerical advantage when transplanted into -thalassemic fetuses.

Bone Marrow Transplantation (2002) 30, 75-80. doi:10.1038/sj.bmt.1703599

-thalassemia; progenitor cells; in utero transfusion; in uterotransplantation; fetal therapy

Hemoglobin (Hb) is a tetrameric protein composed of two - and two -chains joined by a heme prostatic group. The pattern of globin chain expression changes during fetal development. In humans, the first erythroid cells synthesize embryonic Hbs (Gower I, ₂₂, Gower II, ₂₂ and Portland, ₂₂).¹ In contrast, definitive erythroid cells, which circulate from 10 to 30 weeks gestation, synthesize mainly fetal Hb (Hb F, ₂₂), together with a small component (10-15%) of adult Hb (Hb A, ₂₂). At birth, approximately equal amounts of Hb A and Hb F are synthesized and the final adult erythroid pattern (adult Hb with <1% fetal Hb) is reached a few months after birth (reviewed in Ref. ²).

-Thalassemia (-thal) is a genetic disorder caused by mutations in one or more of the four alleles that encode the chain of hemoglobin Hb (two alleles on each of the chromosome 16) (reviewed in Ref. ³). The most severe of these disorders (-thal-1) is caused by deletion of all the four globin genes. As a consequence, thal-1 fetuses do not synthesize any chains and their red cells contain, from approximately 10 weeks gestation, Hb Barts, a tetramer of -globin chains, which does not transport O₂ efficiently. Therefore, unlike -thalassemia in which clinical manifestations occur after birth, -thal-1 leads to hydrops fetalis often resulting in spontaneous abortion or pre-term birth between 16 and 36 weeks of gestation. Liveborn infants often have significant neurological deficits due to chronic in utero hypoxia.

The recent improvements in ultrasound-guided chorionic villous biopsy and molecular PCR genotyping have made it possible to diagnose genetic defects in the first trimester of pregnancy. In cases of fetuses affected with -thal-1 when the parents opt to continue the pregnancy, some have advocated intrauterine fetal red cell transfusions in an attempt to prevent the neurodevelopmental problems mentioned above.^4,5,6,7 The success of this treatment depends on how early the transfusions are begun and how frequently they are repeated. Since in the case of -thal-1, fetal anemia (and, thus hypoxia) can occur as early as the end of the first trimester, any attempt to prevent the hypoxia-induced neurodevelopmental problems by in utero red cell transfusion should begin at the end of the first or beginning of the second trimester and the frequency of transfusions should be sufficient to prevent significant fetal anemia.

The successful treatment of SCID by in utero transplantation,^8,9 had suggested that this therapy could be more effective than conventional post-natal bone marrow transplantation for all those genetic diseases, including -thal-1, that can be cured with such treatment.¹⁰ In fact, not only could it effect a cure before the phenotypic manifestations of the disease have occurred, but might also be safer than 'conventional' bone marrow transplantation because the fetus is immunologically immature and, therefore, less likely to reject the transplant. In addition, it will obviate the need for the myelosuppressive regimen necessary for post-natal engraftment, which is responsible for most of the morbidity of the therapy.¹¹

Based on these assumptions, in 1995 a fetus with -thal-1 was treated by intrauterine red cell and paternal CD34⁺ cells transfusion⁷ in Denver and in New York, under a protocol approved by the institutional Human Subjects Committee, the National Heart Lung and Blood Institute, and Food and Drug Administration of the United States of America. The red cell transfusions did indeed prevent abnormal neurological development. Microchimerism, however, was detected in the fetus soon after the paternal CD34⁺ cell transfusion, but full engraftment never occurred. More recently, it has became evident that even in the case of the SCID fetuses transplanted in utero,^8,9 the donor stem cells did not engraft and only the mature T cells were of donor origin. It is debated whether the lack of engraftment in utero transplantation is due to lack of space in the developing hemopoietic organs or to rejection of the donor cells by the recipient's immune system (ie NK cells).

Here we compare the number of paternal progenitor cells (CFC) transplanted with the number of fetal CFC in the circulation at the time of treatment or, when sampling of the fetal blood had not been possible, with the CFC numbers observed in non-thalassemic fetuses (anemic and non-anemic) of comparable gestational age. The results indicate that the paternal CD34⁺ cells transplanted in utero did not have a numerical advantage over the number of cells already circulating in the fetus.

Materials and methods

Human subjects

The treatment of the homozygous -thal-1 male fetus is described in detail in Ref. 7, and is briefly summarized in Table 1. Age-matched controls were represented by patients referred to the Prenatal Diagnosis Unit at the New York Hospital-Cornell Medical center for fetal blood sampling (a total of 31 specimens from 17-39-week-old fetuses) for either diagnostic (two suspected fetal infection, one HLA typing, seven at risk of genetic disorders such as trisomy 8, 13 and 21 or Fanconi anemia) or therapeutic (four alloimmune thrombocytopenia and 17 red blood cell alloimmunization) purposes. In these cases, fetal blood sampling was performed from the umbilical vein at the insertion of the umbilical cord into the placenta under continuous ultrasound guidance with a 20- or a 22-gauge needle as described.¹² This technique is reasonably safe when performed after 17 weeks of gestation.¹³ The blood from four younger fetuses (12-13 weeks old) was obtained from the heart just before first trimester multifetal pregnancy reduction. The blood was confirmed to be fetal in origin by comparison of the fetal erythrocyte mean cell volume with that of the mother. Further controls were represented by blood from adults (the mother and the father of the -thal-1 patient), from a 2-month-old normal infant and from 20 full-term delivery cord bloods. Permission to obtain fetal blood was granted through informed consent and approved by the Institutional Review Board.

Progenitor cell assay

A standardized progenitor cell assay (variance between blind replicate determination of the same sample <10%) was developed as part of the Placental/Cord Blood for Transplantation Program of the New York Blood Center¹⁴ and was used here for the evaluations of progenitor cells in fetal blood. Aliquots (5 and 10 l) of fetal, perinatal and post-natal blood were directly mixed with standardized ready-to use semisolid methylcellulose (0.8% wt/v, Eastman-Kodak, Rochester, NY, USA) media (2.4 ml per tube) dissolved in Iscove's modified Dulbecco's medium (Gibco, Grand Island, NY, USA) containing fetal bovine serum (30% v/v, Gemini BioProd, Calabasas, CA, USA) and deionized albumin (1% v/v, Sigma, St Louis, MO, USA), 7.5 ´ 10^-5 M -mercaptoethanol (Sigma) and 1% (vol/vol) antibiotic-antimycotic solution (penicillin, streptomycin, fungizone, Gibco). The cultures were stimulated with a mixture of pure recombinant human growth factors including stem cell factor (SCF, 10 ng/ml), interleukin 3 (IL-3, 2 ´ 10^-10 mol/l), granulocyte-macrophage colony-stimulating factor (GM-CSF, 4.5 ´ 10^-10 mol/l), granulocyte colony-stimulating factor (G-CSF, 2 ´ 10^-10 mol/l) and erythropoietin (EPO, 1.5 U/ml). The growth factors were a gift from Amgen (Thousand Oaks, CA, USA) and Genetics Institute (Cambridge, MA, USA). Each culture was analyzed in duplicate (1 ml/35-mm dish) and incubated at 37ºC in a 5% CO₂/O₂ atmosphere until scoring. The number of colonies containing >500 cells was enumerated by eye with the help of an inverted microscope according to standard morphological criteria.¹⁵ Erythroid, granulo-monocytic and mixed (granulocytic and erythroid) colonies were scored individually, but their number was pooled in this analysis because it had been previously shown that, in the case of cord blood transplantation, the total colony number correlates with the speed of engraftment.¹⁴

Globin chain content determination

The globin chain content of fetal and adult blood was analyzed by triton urea electrophoresis as previously described by Alter et al.¹⁶

Statistical analysis

Analysis of variance (Anova test) and regression analysis was performed with Origin 3.5 for Windows (Microcal Software Inc, Northampton, MA, USA).

Results

Circulating progenitor cells in normal and anemic non-thalassemic fetuses

The number of progenitor cells circulating in human fetuses of increasing gestational age is presented in Figure 1. The blood of normal fetuses, and of fetuses sampled for hemopoietic disorders whose Hct values were normal, contained a relatively constant number of progenitor cells (60 ± 13 CFC/l of blood) although a tendency, which did not reach statistical significance, toward a decrease in CFC content with gestational age was observed (Figure 1). The blood of five of the eight anemic non-thalassemic fetuses analyzed, contained a number of CFC per l higher than the blood of normal fetuses of comparable gestational age. On average, the difference in the number of circulating progenitor cells in normal and anemic non-thalassemic fetuses (140 ± 20 CFC/l of blood) was statistically significant (P < 0.01).

Since the increase in blood volume during human gestation is known,¹⁷ it is possible to calculate from the frequency of progenitor cells per l of blood presented in Figure 1 and the total blood volume presented in Ref. 17, the total number of progenitor cells circulating in the blood at any given gestational age. The total number of progenitor cells circulating in the blood increases exponentially with age from as low as 2 ´ 10⁵ CFC at 12 weeks, to 5 ´ 10⁸ at 34 weeks of age.

Globin chain content of RBC and numbers of circulating progenitor cells in the -thalassemia patient

The -thal-1 fetus was transfused on several occasions with packed normal red cells and transplanted at 14, 19 and 23 weeks of age with paternal CD34⁺ cells (see Table 1). The Hb Barts content of its blood during these transfusions was previously described.⁷ For practical (i.p. and not i.v. site of injection) and ethical (sampling of blood from a small fetus for reasons not strictly linked to its well-being) reasons, blood was not available for this study before 23 weeks of gestation. The globin chain content of the fetal blood at 23, 27, 30 and 34 weeks was further analyzed in this study by triton urea electrophoresis (not shown) and the globin chain ratios obtained are summarized in Table 1. The injection of adult red cells maintained the /non- ratio between 37 and 59%, against ratios observed in the blood of normal fetuses of 75-95%. Another indicator that adult red cells were circulating in the fetus is represented by the /+ ratio which remained between 35 and 60%, values significantly higher than those expressed by red cells from fetuses of comparable age (from 5 to 35%, depending on the age).

Figure 2 shows the number of total nucleated cells per ml of blood and the frequency of progenitor cells per l and per 10⁴ mononuclear cells in the blood of the -thal-1 fetus.

At 23 weeks, the blood of the -thal-1 fetus contained 20 times fewer mononuclear cells than normal fetuses (<10⁵ per ml vs the 2 ´ 10⁶ found in the blood of normal fetuses). The number of total nucleated cells significantly increased, (>5 ´ 10⁵ cells per ml) at 30 and 34 weeks, just before birth. Soon after birth, following a double exchange transfusion (Table 1), the mononuclear blood cell counts became very low to return to normal values 2 weeks after birth (Figure 2).

The frequency of progenitor cells per l of blood remained consistently two-fold lower than those observed in normal fetuses (Figure 2, middle panel). This frequency dramatically increased around birth (~100 CFC/l of blood) (Figure 2), although the values remained within the range observed in normal neonates. In contrast, due to the low number of nucleated cells present in its blood, the frequency of progenitor cells per 10⁴ mononuclear blood cells of the -thal-1 fetus was higher than the frequency observed in normal fetal blood. Values ranged from as many as 800 progenitor cells per 10⁴ mononuclear cells at 23 weeks (almost one CFC for every 12 mononuclear cells) to 80-200 CFC per 10⁴ mononuclear cells at 25, 30 and 34 wk (one CFC per 50-200 cells). It would be important to analyze the CFC content in the blood of more -thal-1 fetuses to see if this is a general feature of these subjects.

Fetal and adult progenitor cells mature in vitro with different kinetics, as determined on the basis of the day of culture in which maximal number of colonies are scored and optimal levels of globin chains are synthesized by the cells within the colonies: maximal number of fetal colonies and of globin chain incorporation are detected as early as 8-10 days after the initiation of the culture.^15,18,19 In contrast, adult colonies, whose presence is below detectable levels on day 10, undergo optimal growth as late as at day 14-16 of culture.²⁰ Although it is theoretically possible to discriminate between recipient's and donor's CFC circulating in the fetal blood by single colony PCR genotyping (either for the HLA⁷ or for the -globin²¹ locus), these experiments are not trivial and involve numerous PCR reactions. Therefore, fetal and adult progenitor cells were discriminated in this study on the basis of their different kinetics of maturation. At 23 weeks, the blood of the -thal-1 fetus, that had been transfused 4 weeks earlier with paternal CD34⁺ cells, was sampled either before or 15 min after the injection of additional paternal CD34⁺ cells (Table 1). No difference was observed in the number of CFC which grew at 10 or at 15 days in cultures of blood harvested before the transfusion (~550 colonies/10⁴ nucleated cells in both cases). In contrast, two-fold more colonies were scored at day 15 than at day 10 in cultures of blood sampled after the transfusion (~250 vs 600 colonies/10⁴ nucleated cells, respectively). Furthermore, while the erythroid colonies from the -thal-1 fetus are usually small and pale, many of the erythroid colonies observed at day 15 in cultures of blood harvested after the transfusion were large and bright red. These results suggest that at least 50% of the progenitor cells circulating 15 min after the transfusion were of adult origin.

Discussion

In utero stem cell transplantation therapy is potentially more effective than conventional post-natal bone marrow transplantation for those genetic diseases that can be cured with such treatment. In fact, it could effect a cure before the phenotypic manifestations of the disease have occurred and might be safer than 'conventional' bone marrow transplantation because it does not require the myelosuppressive regimen necessary for post-natal engraftment, which is responsible for most of the morbidity of this therapy.^10,11 In addition, the immunologically immature fetuses may be less likely to reject the transplant, thus improving chances for engraftment.

Several models of marrow engraftment without myelosuppression have been recently developed in mice.²³ Engraftment under such conditions is achieved when the transplanted cells have either (1) a proliferative;²⁴ (2) differentiative;²⁵ or (3) numerical²⁶ advantage. Fetal and adult stem cells have different biological properties (reviewed in Ref. 2). In particular, fetal progenitor cells display a unique phenotypic and functional profile including higher cell cycling rates (<30% vs 10% of the adults), lower doubling time (20 h vs 32 h for adult cells), faster kinetics of in vitro differentiation (10 days vs 14-16 days), longer average telomeric length (12.8 ± 0.35 kb vs 8.4 ± 0.3 kb),²⁷ and the need for fewer growth factors to achieve optimal differentiation in vitro (a single vs at least three to four growth factors). Each one of those differences could provide either an advantage or disadvantage to the adult cells with respect to the fetal ones and adult cells will ultimately compete with the fetal ones depending on the balance established among all of those differences in vivo, a balance hardly predictable on a theoretical basis. On the other hand, the small size of the recipient supports the notion that adult cells might have a numerical advantage when transplanted in utero. Because of the limited number of stem cell niches that may became available in a fetus, the ratio between the number of adult cells injected and the number of the corresponding fetal cells already present in the circulation may represent one of the most important factors for the efficient engraftment of adult cells in a fetus.

Several investigators have shown that the human fetal blood contains high numbers of progenitor cells, either phenotypically defined as CD34⁺ cells or functionally defined as CFC.^12,28,29 The number of progenitor cells calculated according to both definitions correlates with the number of repopulating cells in humans: the CD34⁺ content predicts the engraftment of adult grafts,³⁰ while the CFC content predicts the outcome of transplants with umbilical/cord blood grafts.¹⁴ However, the total number of hemopoietic progenitor cells, either CD34⁺ or CFC, present in fetal blood during ontogenesis has never been calculated. Since in the mouse, expression of CD34 is acquired by the marrow repopulating cells only after birth,³¹ and in humans the CFC frequency in newborn blood correlates with the frequency of a fraction of all the circulating CD34⁺ cells, CD34^dim (JAM Visser and P Rubinstein, personal communication). The number of progenitor cells present in the fetal blood was determined in this study as CFC content. The data presented indicate that the blood of the -thal-1 fetus, unlike the blood of fetuses with anemia due to other causes, contains a number of progenitor cells comparable to the number observed in normal blood. They also indicate that the paternal cells injected into the fetus were detectable soon (15 min) after the transplant and were viable after the injection. However, in every single transfusion performed the theoretical number of adult CFC injected was lower than the corresponding theoretical number of fetal cells present in the circulation (Table 2). Since in the mouse, engraftment in the absence of myelosuppression is achieved by injecting a number of stem cells at least 10²-10³ higher than those present in the marrow of the host,²⁶ it is concluded that the paternal CD34⁺ cells did not have a numerical advantage when injected into the -thal-1 fetus. It is possible that, as suggested by Golfier et al,³² the stem cells present in fetal bone marrow may represent, due to their fetal-like biological properties, a better source of stem cells than adult CD34⁺ cells for in utero transplantation.

Acknowledgements

The authors are grateful to Nancy Hamel for technical assistance. This study was supported by grant Telethon No. E. 1172, Fondi Ricerca Corrente, Ministry of Health, Italy and institutional funds from Istituto Superiore di Sanità, Rome, Italy.

1 Huehns ER, Dance N, Beaven GH et al. Human embryonic haemoglobins. Nature 1964; 201: 1095-1097.

2 Migliaccio AR, Papayannopoulou Th. Erythropoiesis. In: Steinberg MH, Forget BG, Higgs D, Nagel RL (eds) Disorders of Hemoglobin: Genetics, Pathophysiology, Clinical Management Cambridge University Press: Cambridge, 2001, 52-71.

3 Chui DHK, Wayne JS. Hydropsis fetalis caused by -thalassemia: an emerging health care problem. Blood 1998; 91: 2213-2222. MEDLINE

4 Carr S, Rubin L, Dixon D et al. Intrauterine therapy for homozygous -thalassemia. Obstet Gynecol 1995; 85: 876-879. MEDLINE

5 Abuelo DN, Forman EN, Rubin LP. Limb defects and congenital anomalies of the genitalia in an infant with homozygous alpha-thalassemia. Am J Med Genet 1997; 68: 158-161. Article MEDLINE

6 Naqvi A, Waye JS, Morrow R et al. Normal development of an infant with -thalassemia. Blood 1997; 90: 132a, (Abstr).

7 Hayward A, Ambruso D, Battaglia F et al. Microchimerism and tolerance following intrauterine transplantation and transfusion for -Thalassemia-1. Fetal Diagn Ther 1998; 13: 8-14. MEDLINE

8 Flake AW, Roncarolo MG, Puck J et al. Treatment of X-linked severe combined immunodeficiency by in utero transplantation of paternal bone marrow. New Engl J Med 1996; 335: 1806-1810. MEDLINE

9 Wengler G, Lanfranchi A, Frusca T et al. In utero transplantation of parental CD34 haematopoietic progenitor cells in a patient with X-linked severe combined immunodeficiency (SCIDX1). Lancet 1996; 348: 1484-1487. MEDLINE

10 Flake AW, Zanjani ED. In utero transplantation for thalassemia. Ann NY Acad Sc 1998; 850: 300-311.

11 Flake AW, Zanjani ED. In utero hematopoietic stem cell transplantation: ontogenic opportunities and biological barriers. Blood 1999; 94: 2179-2191. MEDLINE

12 Eddleman KA, Chervenak FA, George-Siegel P et al. Circulating hematopoietic stem cell populations in human fetuses: implications for fetal gene therapy and alterations with in utero red cell transfusion. Fetal Diagn Ther 1996; 11: 231-240. MEDLINE

13 Hohlfeld P, Forestier F, Kaplan C et al. Fetal thrombocytopenia: a retrospective survey of 5194 fetal blood samplings. Blood 1994; 84: 1851-1856. MEDLINE

14 Migliaccio AR, Adamson AW, Stevens CE et al. Cell dose and speed of engraftment in placental/umbilical cord blood transplantation: graft progenitor cell content is a better predictor than nucleated cell quantity. Blood 2000; 96: 2717-2722. MEDLINE

15 Peschle C, Migliaccio AR, Migliaccio G et al. Identification and characterization of three classes of erythroid progenitors in human fetal liver. Blood 1981; 58: 565-572. MEDLINE

16 Alter BP, Goff SC, Efremov GD et al. Globin chain electrophoresis: a new approach to the determination of the G/A ratio in fetal haemoglobin and to studies of globin synthesis. Br J Haematol 1980; 44: 527-534. MEDLINE

17 Zhang J, Bowes WA Jr. Birth-weight-for-gestational-age patterns by race, sex, and parity in the United States population. Obstet Gynecol 1995; 86: 200-208. MEDLINE

18 Gianni AM, Comi P, Giglioni B et al. Biosynthesis of Hb in individual fetal liver bursts: gamma-chain production peaks earlier than beta-chain in the erythropoietic pathway. Exp Cell Res 1980; 130: 345-352. MEDLINE

19 Comi P, Giglioni B, Pozzoli ML et al. Biosynthesis of globin chains in fetal liver and adult marrow cultures. Comparative analysis of individual colonies derived from early, intermediate or late erythroid progenitors. Exp Cell Res 1981; 133: 347-356. MEDLINE

20 Peschle C, Migliaccio G, Covelli A et al. Hemoglobin synthesis in individual bursts from normal and adult blood: all bursts and subcolonies synthesize G gamma and A gamma globin chains. Blood 1980; 56: 218-226. MEDLINE

21 Chang JG, Lee LS, Lin CP et al. Rapid diagnosis of -thalassemia-1 of Southeast Asia type and hydrops fetalis by polymerase chain reaction. Blood 1991; 78: 853-854. MEDLINE

22 Migliaccio G, Migliaccio AR, Druzin MLI et al. Long-term generation of colony-forming cells in liquid culture of CD34⁺ cord blood cells in the presence of recombinant human stem cell factor. Blood 1992; 79: 2620-2627. MEDLINE

23 Harrison DE. Competitive repopulation in unirradiated normal recipients. Blood 1993; 81: 2473-2474. MEDLINE

24 Boggs DR, Boggs SS, Saxe DF et al. Hematopoietic stem cells with high proliferative potential. Assay of their concentration in marrow by the frequency and duration of cure of W/W^v mice. J Clin Invest 1982; 70: 242-253. MEDLINE

25 Ashany D, Elkon K, Migliaccio G et al. Functional characterization of early lymphoid precursor cells generated in serum-deprived culture stimulated with stem cell factor and interleukin 7 from normal and autoimmune mice. J Cell Physiol 1995; 164: 562-570. MEDLINE

26 Stewart FM, Crittenden RB, Lowry PA et al. Long-term engraftment of normal and post-5-fluorouracil murine marrow into normal nonmyeloablated mice. Blood 1993; 81: 2473-2474. MEDLINE

27 Vaziri H, Dragowska W, Allsopp RC et al. Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age. Proc Natl Acad Sci USA 1994; 91: 9857-9860. MEDLINE

28 Shields LE, Andrews RG. Gestational age changes in circulating CD34⁺ hematopoietic stem/progenitor cells in fetal cord blood. Am J Obstet Gynecol 1998; 178: 931-937. MEDLINE

29 Engel H, Kaya E, Bald R et al. Fetal cord blood as an alternative source of hematopoietic progenitor cells: immunophenotype, maternal cell contamination and ex vivo expansion. J Hematother 1999; 8: 141-155. MEDLINE

30 Krause DS, Fackler MJ, Civin CI et al. CD34: structure, biology and clinical utility. Blood 1996; 88: 2358-2361. MEDLINE

31 Sato T, Laver JH, Ogawa M. Reversible expression of CD34 by murine hemopoietic stem cells. Blood 1999; 94: 2548-2554. MEDLINE

32 Golfier F, Barcena A, Harrison MR et al. Fetal bone marrow as a source of stem cells for in utero or postnatal transplantation. Br J Haematol 2000; 109: 173-181. Article MEDLINE

33 Shpall EJ, Jones RB, Bearman SI et al. Transplantation of enriched CD34-positive autologous marrow into breast cancer patients following high-dose chemotherapy: influence of CD34-positive peripheral-blood progenitors and growthfactors on engraftment. J Clin Oncol 1994; 12: 28-36. MEDLINE

Figure 1 Number of progenitor cells per l of blood during the development of normal (open squares) and anemic non-thalassemic (closed squares) fetuses. The straight and dotted line indicate the best fitting second order polynomial curves for the two sets of data. The star indicates the mean (±s.d.) values observed in parallel cultures of full-term cord blood samples (20 specimens).

Figure 2 Number of total nucleated blood cell per l of blood (top panel), of progenitor cells per l of blood (middle panel) and of progenitor cells per 10⁴ mononuclear cells (MNC, bottom panel) during the development of the -thal-1 fetus (squares). The average values observed in normal fetuses are also indicated (circles) as a control. The black and white diamonds on the top indicate when the fetus was transfused with paternal CD34⁺ and normal red cells or with normal red cells alone, respectively. The numbers in parenthesis on the x-axis indicate the gestational age (in weeks), 0 indicates the time of birth and the numbers without parenthesis the age (in weeks) after birth.

Table 1 Schedule of in utero treatment of the fetus with homozygous -thalassemia

Table 2 Comparison of the theoretical number of fetal progenitor cells in the circulation and the theoretical number of adult progenitor cells injected in the -thalassemic fetus