Bone Marrow Transplantation (2012) 47, 1489–1498; doi:10.1038/bmt.2011.245; published online 19 December 2011

Allogeneic cellular and autologous stem cell therapy for sickle cell disease: ‘whom, when and how’

J Freed1,5, J Talano2,5, T Small3, A Ricci3 and M S Cairo4

  1. 1Department of Pediatrics, Hackensack University Medical Center, Hackensack, NJ, USA
  2. 2Department of Pediatrics, Medical College of Wisconsin, Milwaukee, WI, USA
  3. 3Department of Pediatrics, Columbia University, New York, NY, USA
  4. 4Department of Pediatrics, Medicine, Pathology, Microbiology and Immunology, Cell Biology and Anatomy, New York Medical College, Valhalla, NY, USA
  5. 5These authors contributed equally to this work

Correspondence: Dr MS Cairo, Pediatrics, Medicine, Pathology, Microbiology & Immunology and Cell Biology & Anatomy, New York Medical College, Munger Pavilion Room 110A, Valhalla, NY 10595, USA. E-mail:

Received 10 October 2011; Accepted 10 October 2011
Advance online publication 19 December 2011

This manuscript has been supported in part by a grant from the Pediatric Cancer Research Foundation.



Sickle cell disease (SCD) is an autosomal recessive inherited hematological disorder characterized by chronic hemolysis and vaso-occlusion, resulting in multiorgan dysfunction and premature death. The only known curative therapy for patients with severe SCD is myeloablative conditioning and allo-SCT from HLA-matched sibling donors. In this state of the art review, we discuss current and future considerations including patient selection/eligibility, intensity of conditioning regimens, allogeneic graft sources, graft manipulation, mixed donor chimerism, organ function and stability and autologous gene correction stem cell strategies. Recent novel approaches to promote mixed donor chimerism have included the use of matched unrelated adult donors, umbilical cord blood donors, haploidentical familial donors and the utilization of nonmyeloablative, such as reduced intensity and reduced toxicity conditioning regimens. Future strategies will include gene therapy and autologous gene correction stem cell designs. Prospects are bright for novel stem and cellular approaches for patients with severe SCD, and we are currently at the end of the beginning for utilizing cellular therapeutics for the curative treatment of this chronic and debilitating condition.


allo-SCT; sickle cell disease; pediatrics


Genetics, epidemiology and pathophysiology of sickle cell disease (SCD)

SCD is a rare recessive inherited disorder, secondary to a point mutation that results in the replacement of valine for glutamic acid at the sixth position of the β chain of human Hb (Figure 1). Worldwide, there are an estimated 270000 patients affected with SCD.1, 2 The polymerization of the deoxygenated form of Hb S induces a major distortion of the shape of the human sickle cell RBC resulting in a decrease in sickle cell RBC deformability, a change in the rheology and consequential vaso-occlusion.3 The most challenging aspect of the disease is the episodic and unpredictable nature of the vaso-occlusive events.4

Figure 1.
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The Hb switch. The fetal (γ) and adult (β) globin chains are expressed from genes on chromosome 11. SCD is caused by mutation of the β chain to the sickle (βS) chain. Genome-wide association studies have identified loci on chromosome 2 (BCL11A) and chromosome 6 (HBS1LMyb) that modify HbF expression. These modifiers affect the expression switch from γ to either β or βS globin. They may affect HbF levels either directly or indirectly. Targeted therapy could reverse the fetal-to-adult switch, and hence reduce disease severity. Orkin SH et al.14

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There are a significant number of clinical complications that can occur in patients with SCD. Sickle cell-induced vaso-occlusive events can occur anywhere in the circulation but most often occur in bones within the chest, back, abdomen or extremities. These episodes can last for days to weeks. Acute chest syndrome affects about 40% of patients with SCD. Acute chest syndrome is more common in children, and when it recurs it can cause chronic respiratory disease.4 Stroke can occur in 10% of SCD patients during childhood with silent central nervous system damage occurring in 5–9 times as many patients, and both can lead to cognitive impairment. Priapism can occur in 10–40% of men with SCD, which can result in permanent erectile dysfunction. Patients with SCD are predisposed to severe infections.5 This can be attributed to a variety of immunological causes.6, 7, 8, 9, 10, 11, 12 The dominant defect is attributed to poor splenic function. Other defects include depletion of IgM memory B cells, reduced levels of T cell subsets CD4+ and CD8+, defects in the C′, impaired opsonization and phagocytosis, deficiency of factor B and skewed TH2 phenotype, resulting in decreased TH1 function.6, 7, 8, 9, 10, 11, 12 This leads to the patient's vulnerability to encapsulated organisms, which can include pneumococcus sepsis, salmonella osteomyelitis and E. coli urosepsis. Despite this year being the 100th anniversary of the original clinical description of SCD and that over the last 30–40 years there has been extensive genetic characterization and elucidation of the biochemical properties of sickle Hb, long-term cure for most patients with SCD still remains elusive.13, 14


Standard of care (non-transplant) therapy for patients with SCD

In industrialized countries, patients with SCD now survive into their fifth and sixth decades (Figure 2) in large part due to many advances in supportive care in the management of SCD, such as penicillin prophylaxis, chronic transfusion regimens and screening examinations, as detailed in Table 1.15

Figure 2.
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Probability of OS stratified by sex in patients with SCD and normal African Americans. Platt OS et al.5

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Hydroxyurea has emerged as an important therapeutic option for children and adolescents with SCD. The percent of Hb F is known to be protective against clinical severity in patients with SCD.15, 16 In comparison, increased leukocyte counts have been associated with a poor clinical outcome because of the increased viscosity caused by hyperleukocytosis.17, 18 Hydroxyurea not only increases the percent of Hb F, presumably by suppressing the marrow and allowing for new marrow recovery and new RBC production with early Hb F-containing RBC precursors, but also decreases the leukocyte count, and thus has many characteristics that make it an ideal drug for patients with SCD. Initial studies in adults have suggested that hydroxyurea significantly reduces the number of veno-occlusive events.19, 20 Subsequent studies demonstrated similar results in both infants and children with SCD.21, 22 In the recently completed multicenter randomized Baby Hug study by Wang et al.,23 hydroxyurea was shown to be safe and effective in infants aged 9–18 months of age with SCD. Hydroxyurea as an alternative therapy for secondary prevention of stroke coupled with phlebotomy for treatment of iron overload has been proposed and recently tested in a multicenter randomized clinical trial (SWITCH), stroke with transfusions changing to hydroxyurea, but was recently terminated for safety and futility reasons (National Heart, Lung, and Blood Institute (NHLBI) 3 June, 2010 press release), as the patients switched from chronic transfusion regimens to hydroxyurea had a significantly higher rate of second strokes. Although case reports of cancer occurring in SCD patients on hydroxyurea exist,24, 25, 26 these are isolated cases, and in order to truly determine whether the risk is increased in SCD patients on hydroxyurea a registry of SCD patients on hydroxyurea would be helpful.


Myeloablative conditioning (MAC) and allo-HSCT from HLA-matched sibling donors in patients with SCD

Allo-HSCT has been shown to be effective in other hemoglobinopathies such as thalessemia as the only curative option for the specific underlying disease. Johnson et al.27 first demonstrated the cure of SCD by a myeloablative and HLA-matched sibling allo-HSCT in a patient with SCD who was receiving an allo-HSCT for acute leukemia. Walters et al.28 subsequently reported on the successful use of HLA-matched sibling MAC allo-HSCT in a larger series of patients with SCD. A total of 22 patients with severe symptoms of SCD received allo-HSCT from a fully HLA-matched sibling donor, after receiving a MAC regimen consisting of BU, CY and anti-thymocyte globulin (ATG) (Table 2).28 The EFS and the disease-free survival (DFS) rates at 4 years were 91% and 73%, respectively.28 Panepinto et al.29 subsequently reported on 67 patients in the Center for International Blood and Marrow Transplantation registry, and demonstrated that the 5-year overall survival (OS) and DFS rates were 97% and 85%, respectively (Table 2). Bernaudin et al.30 reporting results from France demonstrated similar results in 87 allo-HSCT SCD recipients who received allo-HSCTs from HLA-matched sibling donors, after MAC with BU and CY. The 6-year OS was 93.1% and the EFS was 86.1%, respectively (Table 2). These studies demonstrate that HLA-matched sibling allo-HSCTs after MAC offer very high survival rates with few-transplant-related complications (Table 2).

In addition to symptom alleviation, HSCT has also been shown to stabilize or reverse the organ damage due to SCD. In a long-term follow-up study of patients who received HLA-matched related allo-HSCT, pulmonary function tests were stable in 22 of 26 patients, worse in 2 and not studied in 2.31, 32 Linear growth measured by median height s.d. score improved from −0.7 before HSCT to −0.2 after HSCT.31, 32 Radiological improvement of a patient with avascular necrosis of the humeral head has been reported,33 as well as correction of splenic reticuloendothelial dysfunction.29, 34

The effect of HSCT on reversal of cerebral vasculopathy has been variable. Many studies have found that patients who successfully engraft do not experience any sickle-related central nervous system complications, and have evidence of stabilization of central nervous system disease on magnetic resonance imaging.31, 35, 36 In addition to stabilization, a few studies have found improvement in areas of previous abnormality; however, this was in an extremely small subset of patients.31, 32, 37 One study compared the vessel diameter on magnetic resonance imaging of patients with SCD treated with HSCT vs those treated with either chronic transfusion or hydroxyurea.38 They found a 12% increase in the lumen of 22 vessels in patients who underwent HSCT vs an 8% increase in the lumen of 42 vessels in the transfusion/hydroxyurea patients. However, worsening of cerebral large vessel disease and stroke has also been reported after HSCT.39


Alternative allogeneic donor sources for allo-HSCT in patients with SCD

We and others have demonstrated that unrelated umbilical cord blood (UCB) is an excellent alternative allogeneic donor source for some childhood malignant and nonmalignant conditions.28, 40, 41, 42, 43 However, the preliminary results of unrelated UCB transplantation (UCBT) as an alternative allogeneic source for children and adolescents with SCD, although limited in scope, have been disappointing.44, 45, 46 Furthermore, the NHLBI BMT Clinical Trials Network Trial 0601 of reduced intensity conditioning (RIC) before UCBT in symptomatic patients with SCD recently closed to accrual in the arm, utilizing unrelated cord blood grafts, secondary to increased graft rejection (Memorandum BMT Clinical Trials Network, 6/21/2010).

Ruggeri et al.47 examined the efficacy of unrelated UCBT in children with SCD (n=16). OS and DFS were 94% and 50%, respectively. The 2-year probability of DFS was 45% in patients who received grafts with nucleated cell dose >5 × 107/kg and 13% with lower cell doses. Primary graft failure was the predominant cause of treatment failure occurring in seven patients with SCD. These results suggest that only UCB units containing an expected infused nucleated cell dose >5 × 107/kg should be considered for transplantation for hemoglobinopathies, which further limits the available UCB units for this population of patients. Additional studies are required to enhance improvement in engraftment of UCB in patients with SCD, including double UCBT, ex-vivo UCB expansion, and the combination of UCB and other stem cell sources.


Lessons learned in patients with thalassemia

Allo-HSCT from multiple allogeneic donor sources has been proven to be successful. Thalassemia is a hemoglobinopathy that like SCD can lead to elimination of the chronic signs and symptoms of that disease. Bernardo et al.48 transplanted 17 thalassemia patients with a conditioning regimen of treosulfan/thiotepa/fludarabine/ATG followed by unrelated adult donor BMT. A 2-year probability of survival and thalassemia-free survival was 95% (95% confidence interval, 85–100%) and 85% (95% confidence interval, 66–100%), respectively.

Familial haploidentical (FHI) T-cell depletion allo-HSCT in patients with thalassemia is another approach of overcoming the paucity of well-matched unrelated donors. Lucarelli et al.49 and colleagues originally designed a conditioning regimen consisting of hydroxyurea (30mg/kg per day) and azathioprine (3mg/kg per day) between days −45 to −11, before fludarabine 20mg/m2 per day × 6 days, BU 14mg/kg per total dose and CY 60mg/kg per total dose in 33 poor-risk class 3 thalassemia patients prior to allo-HSCT. Graft rejection was reduced to only 8% compared with the previous 30% reported from the same group without hydroxyurea, azathioprine and fludarabine.50 Further improvements were achieved in 22 poor-risk thalassemia patients by modifying the conditioning regimen to add thiotepa 10mg/kg per day, rabbit ATG 12.5mg/kg per total dose, expanding the use of hydroxyurea and azathioprine to day −59 and increasing CY to 200mg/kg per total dose. Grafts primarily from a maternal donor (N=20) were depleted of T cells using the CliniMACS system (Miltenyi Biotec, Auburn, CA, USA) to achieve a median of 14.2 × 106 CD34+cells/kg, with a controlled add-back of 2x105 CD3+/kg and CY added as acute GVHD prophylaxis. Engraftment was achieved in 16/22 patients without acute GVHD and with OS of 90%. These results suggest that a similar approach could be investigated in high-risk patients with SCD.


RIC and allo-HSCT in pediatric recipients

A major limitation of MAC and allo-HSCT is the risk of TRM or treatment-related toxicities associated with MAC regimens. Organ toxicities are more likely to occur and be more severe in symptomatic patients with SCD who have impaired organ function or have been exposed to multiple RBC transfusions before MAC and allo-HSCT.51, 52, 53 MAC facilitates durable engraftment of donor cells, but is limited by toxicities of the conditioning as well as allogeneic transplant-related complications.54, 55 RIC regimens have been examined in patients with malignant diseases who could not tolerate MAC regimens. Pulsipher et al.56 examined the use of a RIC regimen consisting of BU, fludarabine and ATG in 47 pediatric patients with hematological malignancies. With a variety of graft sources including matched sibling donor, matched unrelated adult donor (MUD) and UCB sustained engraftment occurred in 79–98% and full donor chimerism occurred in 76–88% of patients. TRM was only 11%, although relapse was 43%. Roman et al.23 and Del Toro et al.57 reported some success with RIC regimen in children with hematological malignancies. RIC regimens have also been examined in patients with nonmalignant disorders.58 The use of a RIC regimen has also been examined prior to UCBT. Bradley et al.59 investigated the use of a RIC regimen before UCBT in 21 patients; 7 with nonmalignant conditions. The 5-year OS for all patients was 60%. TRM was only 14%, and the incidence of acute GVHD and chronic GVHD was 28% and 17%, respectively.

The ongoing NHLBI BMT Clinical Trials Network 0601 trial utilizes an RIC regimen, which includes alemtuzumab, fludarabine and melphalan as a conditioning regimen. GVHD prophylaxis consists of CYA/FK506, MTX and steroids. However, the major difficulty with this approach is identifying an 8/8 HLA MUD, as there are lower percentages of African-American and Hispanic-American donors in the international BM registries. Approximately, only 20–25% of patients identified as a potential BMT candidate have an 8/8 HLA MUD available. Clearly, other strategies of increasing the donor pool or other alternatives are desperately needed for identifying allogeneic donors for SCD.


Donor chimerism after allo-HSCT in patients with SCD

Walters et al.60 demonstrated that stable mixed chimerism after MAC and allo-HSCT in patients with SCD may be sufficient to cure the disease and prevent further SCD-related symptoms or complications. In all, 50 patients survived free of SCD after receiving MAC and HLA-identical sibling marrow transplants, and of these 50, 13 developed stable mixed donor–host chimerism. Eight patients had a chimerism between 90–99%, but five patients had a lower proportion of donor chimerism, ranging from 11–74%. All five of these patients had Hb levels >11. In the three patients whose donors had a normal Hb phenotype, the Hb S percentage ranged from 0–7%, and in the two patients whose donors had sickle cell trait, the Hb S percentage was 36 and 37%. None of the patients experienced painful events or other clinical complications related to SCD. These results strongly support that a persistent mixed donor–host chimerism is sufficient to alleviate clinical and laboratory manifestations of SCD. Andreani et al.61 investigated the donor origin of mature erythrocytes in four patients with persistent mixed chimerism after transplantation for hemoglobinopathies. The percentage of donor-derived nucleated cells ranged from 15–71%; however, the percentage of donor-derived erythrocytes ranged from 73–100%. These results suggest that the majority of the erythrocytes were donor-derived even in the patients with minimal total donor-derived nucleated cells. This suggests that perhaps only a small proportion of donor-engrafted cells may be needed to prevent further symptoms or complications of SCD.

As stable mixed chimerism appears sufficient to eliminate all of the symptoms of SCD,60, 61 it is possible that RIC might be an effective alternate method of conditioning, even if it only results in mixed donor chimerism. Krishnamurti et al.62 reported on stable donor engraftment after RIC with BU, fludarabine, equine ATG, and TLI. Six of seven patients with SCD demonstrated long-term engraftment.

However, because of the risk of graft rejection, particularly in the heavily RBC-transfused patients with SCD, a high level of immunoablation is important for successful donor engraftment. The regimen of fludarabine and melphalan in combination with alemtuzumab would provide a RIC regimen while potentially preserving immunoablation. This conditioning regimen was used in 44 patients with malignant disorders.63 Only two patients experienced graft failure, and the incidence of acute GVHD and chronic GVHD were 6.5% and 0%, respectively. In this study, the alemtuzumab was given 4–8 days before SCT, which may account for the low risk of acute GVHD and chronic GVHD. However, in an immunocompetent host, such as a patient with SCD, this could increase the risk of graft rejection. Owing to this theoretical possibility, Shenoy et al.64 modified the regimen to dose alemtuzumab on days −21 to −19, prior to allo-HSCT in patients with nonmalignant disease. A total of 16 patients, 7 of whom had SCD, between the ages of 2 and 20 were treated with this regimen, all receiving either unrelated donors matched at 8–10/10 loci, HLA-identical sibling donors or UCB matched at 4–6/6loci. Graft failure occurred in 5% of patients, although two of these patients had received a lower dose of melphalan in an attempt to further reduce the intensity of the conditioning. The OS and DFS are 100% and 71%, respectively. Bhatia et al.65 also demonstrated the successful use of an RIC in patients with SCD. A total of 18 patients received BU, fludarabine and alemtuzumab prior to receiving either sibling or unrelated allo-HSCT. The OS and EFS were 83% and 78%, respectively. The median one and two year donor chimerism for whole blood and CD71 were 93/90% and 94/95%, respectively.

Hsieh et al.66 performed RIC allo-HSCT in ten adult patients (ages 16–45 years) with high risk SCD. The conditioning regimen consisted of alemtuzumab with low dose TBI (3 Gy) followed by sirolimus for GVHD prevention. Nine patients showed stable lymphohematopoietic engraftment at levels that sufficed to reverse the SCD phenotype. The mean donor–recipient chimerism for T cells (CD3+) and myeloid cells (CD14+15+) was 53.3% and 83.3%, respectively. J Bolaños-Meade et al.67 reported on ten adult patients with SCD who underwent a RIC haploidentical BMT with post-transplant high-dose cytoxan. The ages ranged from 16–33 years. The conditioning regimen included ATG, fludarabine, cytoxan (14.5mg/kg on days −6 and −5) and 2 Gy TBI. GVHD prophylaxis consisted of post-transplant CY (50mg/kg on days +3 and +4), tacrolimus and mycophenolate mofetil. Four patients rejected their graft (one primary and three secondary). At a follow-up of 406 days, all patients were alive and six patients were off immunosuppresion. This confirms that RIC regimens can allow for sufficient donor whole blood and RBC engraftment to ameliorate SCD. However, rejection remains a major hurdle to overcome. These studies support the use of RIC regimens in selected patient populations with symptomatic SCD (Table 3).


FHI T-cell depleted (TCD) family donor allo-HSCT in patients with malignant disease

FHI TCD allo-HSCT has been an excellent allogeneic stem cell source for children and adults with malignant disease.68, 69 Aversa et al.70 reported 100% engraftment and no acute GVHD/chronic GVHD in 43 patients with hematologic malignancies, and utilizing CD34 selection of FHI PBSCs. Aversa et al.70 also has reproduced these results in a recent phase II trial and demonstrated high rates of engraftment and low rates of acute and chronic GHVD.71 Also, Evans et al.72 recently demonstrated long-term fetal microchimerism in PBMCs in healthy women, suggesting a potential selective advantage to utilizing maternal FHI donors to promote tolerance and/or decrease severe acute GVHD.73


FHI TCD allo-HSCT in patients with SCD and indications for allo-HSCT

We have created a multicenter, multidisciplinary consortium of pediatric SCT centers, each having a substantial SCD patient population with the intent of investigating FHI TCD allo-HSCT in high-risk patients with SCD. We have adopted the conditioning regimen that Lucarelli et al.49 piloted in the FHI TCD allo-HSCT study in a high-risk thalassemia population (Figure 3). We have included the addition of TLI in order to potentially reduce the rejection rate in this SCD population. Selected high-risk patients with SCD defined in the eligibility criteria will be enrolled on this study. The eligibility criteria includes: (1) clinically significant neurologic event (stroke) or any neurological deficit lasting >24h that is accompanied by an infarct on cerebral magnetic resonance imaging; (2) minimum of two episodes of acute chest syndrome (defined as new pulmonary alveolar consolidation involving at least one complete lung segment associated with acute symptoms including fever >38.5, chest pain, tachypnea, intercostal retractions, nasal flaring, use of accessory muscles of respiration, wheezing, rales, or cough not attributable to asthma or bronchiolitis) in the preceding 2 year period prior to enrollment that have failed, been noncompliant or declined hydroxyurea treatment; (3) recurrent painful events (at least 3 in the 2 years prior to enrollment). Pain occurred in typical sites associated with vaso-occlusive painful events, and cannot be explained by causes other than SCD and (4) two consecutive abnormal transcranial Doppler studies (2 months apart) with mean velocities in the middle carotid artery, internal carotid artery or anterior carotid artery of >200cm/s requiring chronic transfusion therapy. Patients will receive a myeloimmunosuppressive conditioning regimen (Figure 4) and will receive FHI TCD (CD34 selected) PBSC transplantation using the CliniMACS device (IND no. 14359).

Figure 3.
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Childhood and adolescent and young adult FHI allo-SCT SCD consortium.

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Figure 4.
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Myeloimmunosuppressive conditioning regimen (hydroxyurea and azathioprine day −59 to day −11, fludarabine day −17 through day −13, BU day −12 through day −9, thiotepa day −8, CY day −7 through day −4, ATG day −5 through day −2, TLI day −2) followed by FHI T cell depleted (CD34 selected) PBSCT in patients with SCD.

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Autologous gene replacement/correction- a stem cell approach for patients with severe SCD

Despite some success in allo-HSCT for patients with poor-risk SCD, as mentioned above, there continues to be limitations with this approach. These complications have stimulated interest in the use of gene replacement/correction stem cell therapy. There have been many attempts to correct the sickling globin gene via the transfer of a regulated globin gene in autologous hematopoietic stem cells using viral vectors. To date, remarkable success has occurred in treating patients with X-linked SCIDS and adenosine deaminase deficiency.74 Over 30 patients with SCIDS have been treated with gene therapy, although there remains a problem with insertional mutagenesis and oncogenesis. In 2000, a lentivirus was used as the vector to cure a mouse model of thalassemia intermedia.34 This breakthrough vector allowed high levels of gene transfer without rearrangement of gene sequences35 and increased β gene expression.75 Since this discovery, human trials are underway. The major hurdles in using gene addition therapy in treating SCD remain in obtaining therapeutic levels of β globin gene expression and preventing insertional mutagenesis.76


Gene replacement therapy/homologous recombination (HR)

A different approach to preventing insertional mutagenesis and oncogenesis is that of gene replacement therapy of the sickle gene with normal Hb A via HR. This method allows one to create a break of the ds DNA upstream and downstream to the genetically mutated site dissociating it from the DNA, and then replacing this previously mutated site with the genetically corrected DNA. Townes et al.77 reported that by replacing one allele of sickled β embryonic stem cells, then transplanting these cells into blastocysts, hematopoietic cells are able to produce corrected RBCs. However, the low yield of HR in mammals (1 per 106) makes this technique very difficult. By using customized zinc-finger nucleases, hypothetically, there is a possibility of increasing the likelihood of HR at the mutation site by 10000-fold.


Induced pluripotent stem cells (iPS) cells

The main limitation after correcting patient-derived somatic hematopoietic cells is the difficulty in expanding these cells while maintaining multipotency. This decrease in self-renewal ability affects long-term engraftment.

In recent years, there has been accelerated interest in the study of pluripotent stem cells because of their limitless proliferation potential and possible contribution in the study of genetic diseases and gene manipulation.78 However, obtaining embryonic stem cells presents with an ethical conundrum. In 2006, Takahashi/Yamanaka et al.79 evinced that somatic cells could be reprogrammed into pluripotent stem cells called iPS cells, which had similar characteristics to embryonic stem cells. These iPS cells had the capability of proliferating in vitro, differentiating into all three germ layers and forming teratomas and embryoid bodies. They expressed similar cell surface Ag-ic markers, and had a similar morphology and chromatin methylation patterns as embryonic stem cells. iPS cell production was accomplished by the transduction of only four transcription factors Oct4, Sox2, Klf4 and c-Myc via a retroviral vector.79 As then, multiple different vectors have been used, including adenoviral and lentiviral vectors, plasmids and piggyback transposon system with and without a non-viral vector system.76 Challenges in deriving iPS cells using viral vectors include the potential for insertional mutagenesis or oncogene reaction from viral integration.80 Transcription factors cMyc and KLF4 are also well-known oncogenes. Alternative methods currently investigated encompass reprogramming with less factors, protein reprogramming, mRNA reprogramming,81 small molecule reprogramming and choosing cell types with high plasticity to generate iPS cells. The inception of iPS cells paves the way for science to find new ways of replacing damaged tissues, studying genetic diseases and correcting their genetic mutation, autologous transplantation and tissue engineering.78

By taking advantage of the regenerative features of pluripotent stem cells, investigators are now able to derive an unlimited amount of iPS cells obtained from sickled somatic cells, then attempt to correct the mutated gene by HR and then eventuate these genetically corrected iPS cells into hematopoietic stem cells. This allows for an unlimited supply of gene-corrected hematopoietic stem cells, which can be used for autologous transplantation.82, 83 In 2007, by using a humanized sickle cell knock-in mouse, Hanna et al.84 derived hematopoietic progenitors from mouse fibroblast iPS cells, which were able to reconstitute the hematopoietic system of sickle cell mice and correct their disease phenotype (Figure 5). These mice demonstrated stable engraftment up to 12 weeks after transplant. Compared with untreated mice, the transplanted mice showed decreased markers of hemolysis. This technique produced cells that are fully immune compatible to the host, thus avoiding immunosuppressive therapy. Currently there are many studies deriving gene-corrected hematopoietic stem cells using human cell lines obtained from patients with SCD to generate iPS cells, with the ultimate goal of autologous transplantation (Figure 6).

Figure 5.
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Derivation of autologous iPS cells from HbS/HbS mice and correction of the sickle allele by gene targeting.84 (a) Scheme for in vitro reprogramming of skin fibroblasts with defined transcription factors combined with gene and cell therapy to correct sickle cell anemia in mice. (b) Representative images of various steps of deriving hbs/hbs iPS line no. 3. (c) Southern blot for c-Myc viral integrations in (i) ES cells, (ii) hbs/hbs iPS line no. 3 and (iii) its derived subclone hbs/hbs iPS no. 3.3 obtained after infection with adeno-Cre virus and deletion of the viral c-Myc copies. *indicates endogenous c-Myc band. Arrows point to transgenic copies of c-Myc. (d) hbs/hbs iPS no. 3.3 displayed normal karyotype 40xy (upper left), was able to generate viable chimeras (upper right), and formed teratomas (bottom). (e) Replacement of the hbs gene with a hbA globin gene in sickle iPS cell line no. 3.3. Homologous recombinants were identified by PCR to identify correct 5′ and 3′ end replacement. PCR with primers 3 and 4 followed by Bsu36I digestion was used to distinguish hbs and hbA alleles. The correctly targeted clone no. 11 displayed a pattern identical to that previously obtained for the correctly targeted ES cell clone.

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Figure 6.
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Studies deriving gene-corrected hematopoietic stem cells using human cell lines obtained from patients with SCD to generate iPS cells, with the ultimate goal of autologous transplantation.

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Regenerative medicine is an emerging field with the increased potential to cure genetic diseases in the near future. However, there remain many complications, most notable being the increased risk of oncogenesis. As this field continues to grow, we are imminently approaching the possibility of auto-SCT to cure patients with SCD with complications.



MAC and HLA-matched sibling donor allo-HSCT is still the gold standard and only known curative therapy in patients with SCD. More novel approaches are being investigated to promote permanent mixed donor chimerism in these patients, including RIC and the use of alternative allogeneic donors (MUDs, UCBT and haploidentical) and alternatively autologous gene correction/replacement stem cell therapies. Over the next 5 years, pilot studies utilizing RTC and alternate allogeneic grafts including MUDs, UCBT and haploidentical should be completed. Randomized phase III trials should be in development within 5 years to compare some of these approaches with standard supportive care, with end points of quality of life, organ function/stability and neurocognitive stability or improvement. The future is bright for the use of HPCT allogeneic and autologous stem cell therapy for patients with severe manifestations of SCD. We are at the end of the beginning of a new era for cellular curative therapies for this chronic and debilitating genetic condition.


Conflict of interest

The authors declare no conflict of interest.



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We thank Erin Morris, RN, for her outstanding contribution to the preparation of this manuscript, and to all the brave children and adults with SCD who suffer from this chronic condition.

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