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. Author manuscript; available in PMC: 2022 May 1.
Published in final edited form as: Curr Opin Hematol. 2021 May 1;28(3):164–170. doi: 10.1097/MOH.0000000000000640

Heterogeneity of Fetal Hemoglobin Production in Adult Red Blood Cells

Eugene Khandros 1, Gerd A Blobel 1
PMCID: PMC8211413  NIHMSID: NIHMS1713287  PMID: 33631783

Abstract

Purpose of review

Small amounts of fetal hemoglobin can be expressed in a subset of adult red blood cells called F-cells. This review examines potential mechanisms and clinical implications of the heterogeneity of fetal hemoglobin expression.

Recent findings

Although the heterocellular nature of fetal hemoglobin expression in adult red blood cells has been noted for over 70 years, the molecular basis of this phenomenon has been unclear. Recent discoveries of novel regulators of fetal hemoglobin as well as technological advances have shed new light on these cells.

Summary

Fetal hemoglobin reactivation in adult RBCs through genetic or pharmacological approaches can involve both increasing the number of F-cells and cellular fetal hemoglobin content. New technologies enable the study and eventually the improvement of these parameters in patients with sickle cell disease and β-thalassemia.

Keywords: sickle cell disease, β-thalassemia, fetal hemoglobin, F-cell

Introduction:

The human β-globin locus consists of embryonic (ε, HBE), fetal (Gγ and Aγ, HBG2 and HBG1), and adult (β, δ, HBB and HBD) globin genes all of which are under the control of a powerful distal enhancer, called the locus control region (LCR). Among the two developmental switches, embryonic to fetal and fetal to adult, the latter has garnered the most attention because of its clinical importance. Thus, after birth, the γ-genes are silenced concomitantly with the activation of the adult β- and δ-globin genes to produce adult hemoglobin HbA1 (α2β2) and HbA2 (α2δ2). This developmental switch is governed by transcription regulatory complexes at the β-globin locus (Figure 1). However, silencing of the fetal globin genes in adult RBCs is incomplete such that small amounts of HbF (α2γ2) are found in healthy adults, and even higher levels may be found in patients with sickle cell disease (SCD) and several types of β-thalassemia.[1,2] Increasing the expression of HbF in adults is of major clinical interest in β-thalassemia, where it may compensate for lack of HbA production, and in SCD, where HbF provides a potent anti-sickling effect by blocking deoxygenation-induced HbS polymerization.[3,4] Higher HbF levels in patients with SCD reduce morbidity and improve survival (for excellent recent reviews of SCD and the advances in therapeutic HbF induction, please see [5–7]). Impressive examples for the impact of elevated HbF levels on outcomes in SCD patients are given by those who co-inherit genetic mutations leading to hereditary persistence of fetal hemoglobin (HPFH). In cases where adult HbF levels represent 30% or more of total hemoglobin sickling and associated manifestations are abated.[8,9] Yet it is important to realize that the benefits of HbF expression follow a continuum and levels lower than 30% can be beneficial.

Figure 1: Structure of the human β-globin locus on chromosome 11 and developmental globin switching.

Figure 1:

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The locus consists of the LCR transcriptional enhancer and the β-like globin genes arranged in order of developmental expression. The postnatal switch from fetal (HbF) to adult (predominantly HbA) globins occurs as a result of changes in LCR-promoter interactions.

The field of hemoglobin switching experienced a much-needed boost when, through genome wide association studies, BCL11A was identified as a key γ-globin gene repressor.[10,11] A connection between BCL11A and fetal hemoglobin levels was entirely unexpected not in the least because of its wide expression pattern. With the subsequent recognition of ZBTB7A (LRF) as γ-globin repressor, the two major direct transcriptional silencing factors were discovered,[12–19], and their direct involvement buttressed by naturally occurring HPFH mutations affecting their binding to the γ-globin genes.[13,20] Biochemical studies and CRISPR-based genetic screens discovered a host of coregulatory complexes and “upstream” regulators that indirectly converge on BCL11A and/or LRF, or function by unknown mechanisms.[21–30] Inhibition or genetic targeting of some of these regulators, or genetic editing of their essential DNA binding sites, are currently being investigated as novel therapeutic approaches for hemoglobinopathies (reviewed in [5**,7**,31*]).

The majority of clinical studies on the expression levels of hemoglobin forms have relied on bulk approaches such as high-performance liquid chromatography (HPLC) in hemolysates. There has long been a realization, however, that HbF in adult cells is not evenly distributed but rather enriched in a subset of cells called “F-cells”. In SCD, a clinically important variable related to HbF anti-sickling properties is thus not only the total amount of HbF in a population but also its distribution.[32] This brief review focuses on the heterocellular distribution of HbF and its implications for the treatment of hemoglobinopathies.

Identification and initial characterization of F-cells

The presence of a distinct population of erythrocytes in patients with SCD was first noted 70 years ago. Transfusion studies of SCD donor erythrocytes into healthy recipients showed the majority of transfused cells displayed shortened survival but also, surprisingly, revealed a sub-population with near normal life span that varied from donor to donor.[33] Singer and colleagues used the resistance of fetal hemoglobin to alkaline denaturation to show that it is present in SCD erythrocytes.[34] In subsequent transfusion experiments with SCD erythrocytes, they showed that while the fraction of donor RBCs declined quickly, the percentage of fetal hemoglobin declined much slower, reflecting the presence of a healthier subset of RBCs expressing HbF in the blood of SCD patients.[35] They also demonstrated enrichment of fetal hemoglobin in cells that are more resistant to mechanical injury, likely reflecting decreased sickling as well as the later-characterized greater stability of HbF under mechanical stress.[36]

In parallel to biochemical approaches, microscopy based observations of F-cells began with the acid elution technique of Kleihauer and Betke for detection of fetal cells in maternal blood smears.[37] A semi-quantitative version of this technique allowed the observation of heterocellular distribution of HbF in RBCs from individuals with SCD, β-thalassemia and aplastic anemia, compared to a pancellular distribution in examined individuals with HPFH.[38] This approach also showed that F-cells in adults contained a combination of HbA and HbF, rather than only HbF. Both biochemical and microscopic methods were used to further characterize SCD RBCs to show that F-cells were less likely to be found in the population of irreversibly sickled cells – permanently deformed cells that drive SCD pathophysiology,[39,40] and that the total hemoglobin content of F- and non-F cells was the same while HbF and HbS varied reciprocally.[41] These techniques also led to the discovery of a case of HPFH in which HbF was distributed in heterocellular fashion even though erythroid cells had the same genetic makeup.[42]

The development of antibodies specific for γ-globin allowed more quantitative examination of F-cells. Boyer and colleagues used quantitative immunodiffusion approaches to examine HbF expression in F-cells from healthy individuals.[43] They estimated that HbF in healthy adult F-cells represents about 14–28% of total cellular hemoglobin (4.6–9 pg/cell). Variations of these techniques were soon developed that allowed better quantification of HbF production throughout erythroid development and comparisons between different disorders.[44–46]

The advent of flow cytometry approaches for more accurate quantification of F-cells and their HbF content yielded additional insights.[47,48] In a study of HbF expression in a large cohort of children with various SCD genotypes (including SS, Sβ0, Sβ+, and SC) as well as patients with HbS/HPFH, Marcus and colleagues examined a broader range of HbF expression by HPLC and quantitative flow cytometry.[49] Importantly, they noted that the % HbF by HPLC increases logarithmically with a linear increase in % F-cells for all SCD genotypes, with the implication that the amount of HbF per F-cell is not fixed as a linear relationship would indicate, and can vary among individual patients – this suggests that both the F-cell number and HbF content can be amenable to manipulation. Recently the cellular variation in HbF expression in SCD patients has also been studied by flow cytometry to determine thresholds to prevent sickling at the single cell level.[50]

Increased HbF and F-cell production in other hematopoietic disorders

Although the primary interest in HbF production was rooted in the treatment of hemoglobinopathies, it was quickly recognized that most other conditions with elevated HbF also exhibit heterocellular expression in F-cells. Increased F-cell numbers were found in diverse conditions of erythropoietic stress, such as recovery from iron deficiency anemia, hemolytic crises, transient erythroblastopenia of childhood, chemotherapy, stem cell transplantation, and phlebotomy [51–54]. One potential explanation is that the increase in F-cells is driven by a change in the cytokine milieu under conditions of hypoxia or erythroid stress. Erythropoietin, one of the primary drivers of RBC production in response to anemia, has been shown to increase the number of F-cells in primate models and in vitro human erythroid cultures,[55–57] although combination therapy in SCD patients has shown more mixed results. [58–60] Additionally, stem cell factor and TGF-β have been implicated in the regulation of F-cell production.[61–64] Another potential explanation is the expansion of stress erythroid progenitors that are more likely to produce fetal hemoglobin.[65] Further studies are needed to determine whether different sources of erythropoietic drive or cytokine levels converge on a common mechanism of F-cell production.

Cellular origins of F-cells

Since F-cells were first identified, there has been significant interest in their origin. Two main hypotheses for their origins posited that F-cells may (1) arise from a residual distinct population of fetal or fetal-like hematopoietic stem or progenitor cells or that (2) F-cells arise stochastically from adult erythroid progenitors. Several in vivo and in vitro lines of evidence have argued against a separate fetal stem or progenitor cell. Disorders of clonal hematopoiesis such as polycythemia vera (PV) and paroxysmal nocturnal hemoglobinuria (PNH) provided natural experiments to test this hypothesis: in all patients studied, the stem cells of the PNH or PV clone gave rise to both F-cells and non-HbF containing cells, demonstrating that the F-cells do not come from a separate stem cell pool.[66–68] Additional in vitro experiments by multiple groups examined F-cell production in CFU-E and BFU-E colonies arising from single cells.[57,69–71] These studies demonstrated that single erythroid progenitors could give rise to colonies with a mix of F- and non-F cells, and that F-cells were not restricted to a subset of CFU-E or BFU-E progenitors. Interestingly, single clonal burst-forming cells could give rise to a mixture of all HbF+ or HbF− subclones, suggesting that at some point after the BFU-E stage the erythroid progenitors commit to expressing HbF or fail to do so. Quantitative analysis of these colonies was consistent with F-cell formation as a stochastic process, with the probability influenced by the differentiation stage of the progenitors (with BFU-E more likely to give rise to F-cells).[53,57,69] Based on these findings, Stamatoyannopoulos and Papayannopoulou proposed that stimuli such as erythropoietin under conditions of erythropoietic stress can increase F-cell formation through alterations in cell cycle and differentiation time. [50,66]

Another important question is whether F-cells arise from adult erythroid cells by reversion to a fetal-like state. Fetal erythroblasts differ significantly from adult ones beyond just hemoglobin expression. At the transcriptome level, erythroblasts derived from fetal or adult sources exhibit approximately 600–3000 differentially expressed mRNAs and miRNAs,[72–74] as well as differences in DNA methylation,[75] epigenetic marks,[74] and chromosomal configuration at the β-globin locus.[73] The earliest evidence that F-cells do not represent reversion to a fetal phenotype came from observations that F-cells lose the fetal RBC i antigen and instead express the adult I antigen.[42,76] Likewise, F-cells express carbonic anhydrase, which is found in adult but not fetal RBCs.[77,78] Another potential explanation for F-cell formation is variation in the expression level of known HbF repressors such as BCL11A or LRF in erythroid progenitors. Grieco and colleagues used a system for culture of single-cell derived clonal human basophilic erythroblasts to study variation in γ-globin transcription and HbF regulators.[79] They found significant variability in γ-globin produced by clonal cultures from the same donor, with or without hydroxyurea. Clones with higher HbF expression showed lower transcript levels of BCL11A and KLF1, which could potentially account for higher HbF expression. More recently, we developed an approach for the purification of stage-matched HbF positive and negative erythroblasts from human erythroid cultures and demonstrated that there are unexpectedly few transcriptional differences in adult F-cells beyond the globins, and little overlap with the fetal erythroblast transcriptome.[80] Surprisingly, direct comparison of transcript levels of these regulators in sorted F-cells and non F-cells did not, however, detect any differences in the levels of BCL11A, KLF1, ZBTB7A, or any other known molecules involved in hemoglobin switching.[80] This lack of differences in HbF regulators between HbF positive and negative cells remained also when cells were exposed to experimental HbF inducers. A critical consideration here is that comparisons were made between HbF+ and HbF− cells grown under identical conditions, which contrasts with studies in which cells were either exposed to a drug or not. Hence, the former approach filters out drug induced changes irrelevant to HbF regulation. The reason for the differences in HbF levels remains to be established but seems to be related to changes, perhaps stochastic, at the β-globin locus and not to broad alterations in the nuclear environment. [80] However, it is also possible that variation in HbF regulators occurs earlier in erythroid differentiation, prior to HbF production, which would have been missed in this study since it relied on HbF levels as discriminating criterion. Alternative approaches such as single-cell analyses are needed to address this question.

Globin chain production in F-cells

F-cells express a combination of HbF and HbA, with HbA representing the majority of cellular hemoglobin under nearly all conditions studied; in patients with HbS-HPFH due to large deletions encompassing the adult globin genes, pancellular distribution of HbF results in HbF levels of about 30% in adults.[8] Several studies have demonstrated using biochemical, cytometry, and imaging approaches that F-cells have a total hemoglobin content similar to non HbF-expressing cells, and that any production of HbF comes at the expense of HbA.[38,43,81] There is however some evidence that the γ-globin and β-globin chain production does not occur at the same rate throughout erythroid development and that earlier erythroblast stages produce relatively more γ-globin, at least in healthy individuals.[82] The mechanism for the partial reversion of the maturational hemoglobin “switch” is unclear.

Transcription of the globin genes at the β-hemoglobin locus is controlled by the locus control region (LCR), an erythroid-specific enhancer that forms developmental stage-specific long-range chromatin loops with the promoters of the embryonic, fetal, or adult globin genes at the locus.[83–86] We have previously studied transcription in single human adult erythroblasts using RNA-FISH and showed that β-globin and γ-globin can be transcribed in the same cell in “bursts” of similar magnitude but in a fashion that indicates rapid switching of the LCR between the fetal and adult gene promoters, consistent with a mechanism by which fetal and adult genes compete for LCR activity.[87] For future mechanistic studies, it will be important to measure regulatory parameters of transcription and chromatin architecture in F-cells in response to different genetic and pharmacologic perturbations. We have observed, for example, that hydroxyurea increases the number of F-cells but not the γ-globin transcripts per F-cells, while the experimental HbF inducer pomalidomide increases both the percentage of F-cells and their γ-globin content.[80] Understanding how these drugs can achieve different HbF effects will be essential for successful use of rational combinatorial therapy.

F-cells as a therapeutic marker in SCD

Although there was some initial debate about whether F-cells have improved sickling and survival,[88,89] this was directly tested by experiments tracking biotinylated F- and non-F SCD RBCs in vivo. F-cells indeed had significantly prolonged survival up to 6–8 weeks versus approximately 2 weeks for non-F cells.[90,91] Horiuchi et al also demonstrated that reticulocytes in SCD patients have lower HbF levels than mature RBCs, proposing that F-reticulocytes have a selective survival advantage.[92] With the increasing recognition that increased F-cell numbers are a desirable clinical goal, early studies of novel pharmacologic therapies for SCD, especially hydroxyurea, focused on F-cell production along with increases in HbF by HPLC. Veith and colleagues demonstrated that treatment with cytarabine or hydroxyurea led to a significant increase in HbF-expressing reticulocytes.[93] F-cells in SCD were subsequently evaluated in the major clinical studies of hydroxyurea therapy. A Multicenter Study of Hydroxyurea obtained data both on HbF percentage of total hemoglobin as well as % F-cells in SCD patients before and following hydroxyurea treatment.[94] They demonstrated a similar baseline logarithmic relationship between F-cells and HbF as Marcus et al [94], and showed that both parameters are increased by hydroxyurea treatment. In another study, SCD patients treated with HU had an increase in both the %F-cells (24.4 to 59.9%) and calculated HbF/F-cell (4.1 pg to 8.7 pg) leading to an increase in total HbF from 4 to 17.3%.[95] While hydroxyurea remains the only HbF inducer in clinical use, other high-potency HbF inducers such as UNC0638, pomalidomide, and decitabine have been developed and tested in human erythroid cultures.[96–98] Interestingly, even potent pharmacological HbF inducers alone or in combination still lead to heterocellular HbF expression despite presumed equal exposure of erythroid progenitors to the drug.[22*,80]

There has been an increasing realization that neither measurement of HbF in bulk using techniques such as HPLC or percentage of circulating F-cells provide a sufficiently accurate view of the in vivo anti-sickling effect.[32] Steinberg and colleagues proposed that the key measure is the number of F-cells reaching an anti-sickling threshold HbF content of 10 pg and using mathematical modeling estimated that a bulk HbF concentration of 30% is needed for 70% of RBCs to be protected from sickling.

Additional data to test these predictions will come from SCD patients receiving gene therapy for HbF induction. Frangoul et al recently reported the first two patients treated with CRISPR-Cas9 editing of the BCL11A erythroid enhancer, aimed at lowering BCL11A expression selectively in erythroid cells. [99**] Both patients demonstrated essentially pancellular HbF expression following treatment, with one patient expressing 93% HbF and the other 43% HbF by HPLC.[99] Esrick et al reported on 6 patients who were treated with a lentivirus expressing an shRNA targeting BCL11A in erythroid cells.[100**] Patients achieved F-cells ranging from 58.9–93.6%, HbF between 20.4 – 41.3%, and calculated HbF/F-cells of near or above 10 pg. As additional patients are treated in these studies, correlating clinical responses to F-cell parameters will allow a clearer determination of thresholds for anti-sickling effects.

Recent advances and future goals

Recent technological developments in imaging and flow cytometry have allowed more in-depth study of F-cells. Hebert and colleagues developed a quantitative HbF flow cytometry assay to determine the distribution of HbF per F-cell in erythrocytes of SCD patients with and without hydroxyurea treatment to begin to determine required thresholds of HbF per cell to reduce sickling both in vitro and in vivo.[50*] Imaging flow cytometry allows for simultaneous measurements of fluorescence parameters and cell features such as size and shape; Peslak et al successfully used this approach for quantification of SCD erythrocyte sickling in response to genetic and pharmacologic manipulation.[22*] Combination of this technique with HbF staining will allow more accurate determination of HbF thresholds needed to prevent sickling under different conditions. While F-cell counts of HbF/F-cell are not currently in use as clinical measures, as more data become available these parameters may be useful in understanding patient responses (or failure to respond) to HbF inducers.

Key Points:

  • HbF can be variably expressed in a subset of adult RBCs called F-cells, but not through retention of fetal cells or reversion to a fetal-like state

  • F-cell number and HbF content can be increased in multiple disease states, with erythropoietic stress, and following pharmacological treatment

  • F-cells in SCD can be resistant to sickling and hemolysis

  • Evaluation of novel SCD therapies should include characterization of F-cells and their globin content

Acknowledgements

Research funding was provided by the National Institutes of Health (HL119479, DK054937, DK058044, DK127405, DK106766, and HL007150) and the American Society of Hematology Research Training Award for Fellows.

Conflicts of interest

Work in the Blobel laboratory is supported by a sponsored research agreement from Pfizer.

References and Recommended Reading

* References of special interest

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