Past, present, and future efforts to enhance the efficacy of cord blood hematopoietic cell transplantation

Background: the beginning of cord blood transplantation

Cord blood (CB) is a clinical source of transplantable hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs) for hematopoietic cell transplantation (HCT)^1–3. Until the late 1980’s, the cellular source to treat patients with malignant and non-malignant hematopoietic and other disorders by HCT was mainly bone marrow (BM). Mobilized peripheral blood (mPB), where clinicians utilize known agents (e.g. granulocyte colony-stimulating factor^4,5 and/or HSC/HPC retention signal antagonists such as AMD3100^6–8) to release high numbers of HSCs and HPCs into the bloodstream for easy collection, was only in its early stages. In the 1980’s, umbilical CB, usually a discarded material except for routine newborn blood tests, was studied for HSC and HPC biology in a national collaboration⁹. This scientific work was performed at the Indiana University School of Medicine (IUSM). The authors⁹ demonstrated that there were likely enough HSCs and HPCs for CB clinical transplantation and tha collected cells could be stored for days at room temperature, shipped by overnight express mail to a distant site, and cryopreserved for future CB HCT. The first proof-of-principle CB storage bank for HLA-matched siblings, set up in the Broxmeyer laboratory^9,10, led to the first CB HCT at the Hôpital St. Louis, Paris, in the transplant center directed by Eliane Gluckman as part of an international study¹¹. CB cells were collected and sent from a distant obstetric unit to the Broxmeyer laboratory, where they were tested, cryopreserved, and tested again after thawing of a small separate part of the frozen unit before being hand-delivered to Paris for the clinical HCT. On 6 October 1988, the CB unit was infused into a 6-year-old boy with Fanconi anemia who had been first conditioned by a modified regimen specifically for patients with Fanconi anemia that had been previously developed by Dr. Gluckman, utilizing HLA-matched sibling CB from his sister. This first CB HCT¹¹ was curative for the hematological manifestations of Fanconi anemia; the recipient is alive and well over 31 years later. A total of six more CB HCTs were done in Paris, Baltimore, and Cincinnati using HLA sibling CB cells cryopreserved in the Broxmeyer laboratory to treat Fanconi anemia^10,12,13 and juvenile chronic myelogenous leukemia (first CB transplant to treat a leukemia)¹⁴. Additional information on CB HCT has been reported^1–3,15,16 and on the functionality of CB HSCs and HPCs^17–30. Information presented in the original scientific paper was produced years in advance of the clinical transplant, but the scientific⁹ and clinical¹¹ papers were both published in 1989, as we waited until we knew the first clinical CB HCT¹¹ was successful before submitting the scientific paper⁹. Cryopreserved CB can be stored for at least 23 and a half years^18,31,32 with little or no loss of HPCs (comparing thawed cells to pre-freeze numbers). CB HCT was extended to partially HLA-disparate and unrelated donors^1–3. Over 35,000 clinical CB HCT procedures have been performed to date to treat both children and adults with the same malignancies and non-malignancies treated by BM HCT. Advantages of CB HCT are ease of collection and storage of CB without significant risks for the delivering mothers, the ready and quick availability of HLA-typed frozen CB units in public and private CB banks (should such units be rapidly needed for transplantation), and elicitation of relatively low acute and chronic graft versus host disease (GVHD) in recipients after CB HCT, even with unrelated partially HLA-disparate donor cells, compared to that elicited by BM or mPB. Problems include fewer HSCs and HPCs in CB collections than BM or mPB, in part resulting in delayed engraftment of neutrophils, platelets, and immune cells compared to BM and especially with mPB. While not life-threatening, this delay in engraftment with CB prolongs hospital stays, incurring additional health costs^3,33.

Efforts to address slower time to donor blood cell recovery have focused on ex vivo (in cell culture) expansion of HSCs (with the idea and possibility that increased numbers of HSCs infused will ameliorate slower time to recovery) or enhancing the homing capacity of HSCs to optimize engraftment. Few such efforts have been tested in the clinical setting^34–40 and only in a few selected transplant centers. This review focuses on enhancing the efficacy of “limited” numbers of HSCs and HPCs in CB collections for CB HCT. A clear distinction must be made between phenotypically recognizable and functional HSCs and HPCs. There are rigorous criteria to phenotypically identify human and mouse HSCs and subsets of HPCs by their cell surface proteins, entailing specific antibodies and flow cytometry. However, phenotype does not necessarily recapitulate functional status. For functional analysis, one must perform specific engraftment studies in vivo in mice for mouse and human HSCs and colony forming assays in vitro for HPCs^41,42. Recent information on collection, ex vivo expansion, and homing of CB HSCs/HPCs for the potential enhancement of CB HCT follows.

Increasing hematopoietic stem cell numbers in single cord blood collections

Hypoxia is associated with HSC/HPC functions in these cells’ in vivo microenvironment⁴³. A means to enhance the efficacy of HCT is through hypoxic collection and processing of HSCs such that the collected cells are never exposed to ambient air oxygen (~21% oxygen) levels^44,45. The BM environment, in which HSCs/HPCs reside, has oxygen levels ranging from 1–5%, with some areas possibly being slightly higher or lower depending on proximity to the vasculature^46–49. Isolating HSCs/HPCs under ambient air (~21% oxygen) exposes these cells to hyperoxic conditions, which within minutes decrease HSC numbers through the differentiation of HSCs to HPCs and not because of HSC cell death^44,45. Studies dating from the 1970’s compared culturing of HSCs and HPCs in low (~5% oxygen), in vivo physiological oxygen versus high (~21% oxygen) ambient air oxygen. Culturing human and mouse BM, human CB, and mouse fetal liver at low oxygen in vitro increased numbers of detectable functional HSCs/HPCs^50–56. When cultured in low oxygen (48 mmHg, 6.8% oxygen), clonal growth of granulocyte macrophage progenitors (CFU-GM) from mouse BM was enhanced with increased colony numbers and size compared to a more conventional oxygen environment (135 mmHg, 19% oxygen)⁵⁰. Culturing erythroid progenitors (BFU-E) and more mature erythropoietic precursors (CFU-E) from mouse BM or fetal liver at 5% oxygen increased erythropoietin sensitivity of cells and CFU-E colony numbers⁵⁵. Human low-density CB cells cultured at 5% oxygen had increased CFU-GM, BFU-E, and multipotential progenitors (CFU-GEMM) and were readily expanded ex vivo⁵⁶. Human BM cultured at 5% oxygen had increased CFU-GM numbers⁵¹. Human BM Lin^– CD34⁺ CD38^– cells (enriched for HSCs) cultured at 1.5% oxygen for 4 days had more functional SCID repopulating cells (an assay for functional human HSCs) than comparable human BM cells cultured at 20% oxygen for 4 days (~5.8-fold increase) or freshly isolated Lin^– CD34⁺ CD38^– cells (~4.2-fold increase)⁵³, events associated with stabilization of hypoxia-inducible factor (HIF)-1α, increased surface angiogenic receptors, and VEGF secretion within cultures.

However, in all of these reports, hematopoietic cells were first collected under ambient (~21%) oxygen levels before being placed in culture under lower oxygen and thus the collected cells were already exposed to extra physiological oxygen stress/shock (EPHOSS) or hyperoxia, which induces the production of mitochondrial reactive oxygen species (ROS), increased HSC differentiation, increased functional HPC numbers and cell cycling, and increased mitochondrial mass/activity. EPHOSS effects are mediated by a p53-cyclophilin D-mitochondria permeability transition pore axis and involve HIF-1α and the hypoxamir miR-210⁴⁵. Collecting and processing of mouse BM and human CB under low oxygen (3% oxygen, where cells are never exposed to ambient air) resulted in ~two- to five-fold increases in functional HSC numbers (assessed by engraftment in NSG immune-deficient mice) compared to cells collected or processed under ambient air^44,45. Methods to mimic the effects of low oxygen are being examined. Cyclophilin D inhibitor, cyclosporin A (CSA, used to alleviate GVHD in human HCT), resulted in increased mouse BM and human CB HSC numbers and engraftment capability⁴⁵. However, CSA is difficult to work with. It is hard to get into solution and manifests batch-to-batch variations so that each batch needs to be titrated. Combinations of antioxidants and epigenetic enzyme inhibitors within the flush/collection fluids increased numbers of mouse BM HSCs with increased engrafting capacity in a competitive in vivo assay⁵⁷, but effects of antioxidants and epigenetic enzyme inhibitors have not yet been verified with human CB cells.

Ex vivo expansion of functional hematopoietic stem cells

Small molecules, including, but not limited to, diethylaminobenzaldehyde (DEAB), LG1506, StemRegenin 1 (SR1), UM171, BIO (GSK3β inhibitor), NR-101, trichostatin A (TSA), garcinol (GAR), valproic acid (VPA), copper chelator, tetraethylenepentamine, and nicotinamide, are reported agonists for experimental ex vivo expansion of human HSCs and HPCs^58–65. Clinical studies with a few of these small molecules have been reported^35–40. Verification of these clinical studies will take time. SR1 and UM171 are efficient HSC expansion compounds^58,61. SR1, a purine derivative, was identified in a chemical compound screen for candidates promoting ex vivo expansion of human HSCs/HPCs⁵⁸. SR1 binds aryl hydrocarbon receptor and antagonizes AhR signaling in CB HSCs/HPCs, but the exact molecular mechanisms remain unclear. SR1 has been tested in a phase I/II clinical trial⁴⁰. However, the investigators transplanted both SR1-expanded and -unexpanded CB into patients, so it is too early to determine if SR1-expanded cells contain long-term repopulating HSCs. UM171 promotes ex vivo expansion of long-term repopulating HSCs in experimental models⁶¹, but the clinical trial using UM171 has not yet been published.

Mechanisms behind mouse and human HSC expansion may be different. Neither SR1 nor UM171 stimulates mouse HSC ex vivo expansion^58,61. Thus, mouse studies to evaluate these molecules are not possible. In contrast, overexpression of HOXB4 or co-culturing of recombinant HOXB4 significantly promoted the expansion of both human CD34⁺ and mouse HSCs^66,67. Activation of OCT4 was found to enhance ex vivo expansion of CB HSCs/HPCs by regulating HOXB4 expression⁶⁸. Angiopoietin-like proteins support mouse and human HSC expansion in culture⁶⁹. Overexpression of MSI2, an oncogene, antagonizes aryl hydrocarbon receptor signaling and expands human HSCs to levels similar to those seen with SR1⁷⁰, even though SR1 does not promote mouse HSC expansion⁷¹.

Readout of HSC expansion is related to the culture systems used, which is one reason why the reproducibility of published research may not be easily confirmed. In our experience, serum-free medium such as SFEM (StemSpan^™ Serum-Free Expansion Medium, Catalog #09650, Stemcell Technologies) or Stemline (Stemline II HSC expansion medium, Catalog #S0192, Sigma-Aldrich) with 100 ng/mL SCF, TPO, and Flt3L can efficiently maintain CD34⁺ HSC and HPC numbers for 7–10 days. Antagonizing retinoid acid receptor (RAR) or PPAR-gamma (PPAR-G) signaling maintains CD34⁺ CD38^– stem and progenitor cell populations when CD34⁺ starting cells are cultured in serum and cytokine-containing RPMI-1640 medium, thus facilitating the expansion or maintenance of HSCs in culture^72,73. As SFEM medium is effective in maintaining CD34⁺ CD38^– cell populations, RAR or PPAR-G antagonists do not further enhance the expansion of human HSC production when CD34⁺ cells are cultured in SFEM medium. PPAR-G expression was repressed when CD34⁺ cells were cultured in SFEM medium^72,73. Most recently, a simple HSC ex vivo expansion method was reported by replacing recombinant human serum albumin (HSA) with polyvinyl alcohol (PVA)⁷⁴. It was suggested that potential contaminants in recombinant proteins might induce inflammatory responses, thus dampening HSC stemness maintenance. By this minor manipulation, the authors reported a massive 900-fold enhancement in functional HSC numbers after 28-day ex vivo culture⁷⁴. This incredibly efficient expansion system needs to be confirmed by other labs. However, such massive increases in functional HSC numbers may not be needed for enhancement of CB HCT. A few fold increase in these cells may suffice to enhance time to engraftment, although the excess expanded cells can be frozen and stored for additional CB transplants.

N⁶-methyladenosine (m⁶A) modulates the expression of a group of messenger (m) RNAs critical for stem cell fate decisions by modulating their stability⁷⁵. Suppressing m⁶A reader, Ythdf2, which promotes targeted decay of mRNA, promotes ex vivo expansion of mouse BM and human CB HSCs⁷⁶. Conditional knock-out of mouse Ythdf2 increases functional HSC numbers without changing lineage differentiation and without apparent manifestation of hematological malignancies. Knockdown of human YTHDF2 resulted in a 10-fold increase in cytokine-mediated ex vivo expansion of human CB HSCs, a 5-fold increase in HPCs, and a greater than 8-fold increase in serial transplantation⁷⁶. This was associated with enrichment in mRNAs encoding transcription factors in HSCs previously shown to be critical for stem cell renewal. This procedure thus targets multiple effectors rather than one^76,77.

Epigenetic reprogramming using VPA has been utilized to experimentally expand human CB HSCs ex vivo⁵⁹. This required the coordination of cellular reprogramming with remodeling of mitochondria and activation of p53 that apparently limits ROS levels⁷⁸, which induce HSC differentiation^44,45. DEK, a nuclear heterochromatin remodeling agent, which can be secreted from the cell and act as a cytokine that manifests its effects through the chemokine receptor CXCR2 (the only known non-chemokine to bind CXCR2), enhances cytokine-mediated ex vivo expansion of human CB and mouse BM HSCs and HPCs⁷⁹.

One challenge for ex vivo expansion is the lack of markers labeling functional HSCs during and after ex vivo culture. Some signaling pathways might stimulate the expansion of CD34⁺ cells, most of which are progenitors. Markers including CD90 or CD49f have often been used to isolate HSCs from fresh human CB and BM samples. However, CD34⁺ CD90⁺ CD49f⁺ phenotypic HSCs do not necessarily reflect functional HSCs, especially under stress conditions such as ex vivo expansion⁴¹. The only currently available way to confirm the expansion of human HSC numbers is through transplantation using sublethally irradiated immune-deficient mice. A logistical problem is that ex vivo clinical studies will likely have to be performed in very select centers with expertise for these procedures. Also, the economics associated with ex vivo expansion must be taken into account, as it will likely add significant additional costs to the clinical HCT procedure. While it does not appear that ex vivo expansion procedures have damaged HSCs or caused pre-leukemia/leukemia, anytime HSCs are manipulated ex vivo, there is potential for long-term detrimental effects, which may involve gene expression pattern changes and epigenetic modifications that might result in long-term counter-productive outcomes.

During ex vivo expansion, we must also keep in mind the different physical characteristics of the in vivo HSC BM niche that help maintain HSC homeostasis (e.g. interactions that HSCs have with the other cells within their BM niche and lower oxygen concentrations within the BM). Taking these factors into account, CB HSCs/HPCs have been expanded in the presence of mesenchymal stem/stromal cells and have been proven to be safe in a clinical study³⁶. In addition, hypoxia culturing (5% oxygen) after cells were collected in ambient air potentiated ex vivo expansion of CB HSCs/HPCs⁸⁰.

Although ex vivo expansion is a promising means to overcome limited numbers of CB HSCs collected for transplantation, there is still much work to be done in this area. More mechanistic insight is required regarding the regulation of HSC stemness.

Improving hematopoietic stem cell homing to enhance cord blood hematopoietic cell transplantation

After infusion into peripheral blood, HSCs home to the BM microenvironment by sensing chemical gradients of chemoattractants⁸¹. The BM microenvironment provides a unique matrix bedding and conducive signaling environment supporting long-term engraftment and balances HSC proliferation and differentiation^82,83. HSC homing is crucial for successful clinic outcomes⁸⁴.

Directing HSC migration and homing from the peripheral circulation to the BM involves interactions between chemokine ligand CXCL12/stromal cell-derived factor (SDF)-1 and its receptor CXCR4^85,86. CXCL12 is highly expressed by BM stromal cells padding the stem cell niche. Gradients of CXCL12 provide directional cues and orchestrate HSC migration towards the BM. CXCR4 is a seven-transmembrane G-protein-coupled chemokine receptor expressed on the surface of HSCs. Knockouts of CXCL12 or CXCR4 result in severe hematopoietic defects^87–89. Sphingosine-1-phosphate (S1P) and ceramide-1-phosphate (C1P) also provide homing gradients guiding HSCs to BM niches^90–92. Strategies to enhance HSC homing are classified into three categories: regulation from the cell membrane, regulation in the cytoplasm, and regulation in the nucleus.

Concluding remarks

Enhancing CB HCT efficacy will not only reduce the time of donor cell recovery but also make it possible to use more banked CB units that contain fewer HSCs/HPCs. There are a number of new ways to potentially enhance the efficacy of CB HCT¹³⁴. However, most are laboratory efforts. How to get these new methods into clinical trials is a problem that needs to be solved. We believe that simpler is always better. The simpler the procedure, the more likely that it will be clinically translated. There are just not enough clinical CB HCTs available to set up phase I/II clinical trials to test these new procedures. Most investigators doing such trials are “wed” to their personal favorite procedure. If, in the future, we can deal with this problem and find means for additional clinical efforts, it is possible that several new procedures can be used together¹³⁴. This, however, adds additional logistical problems versus the use of one procedure. Clinical trials are costly, and it is not clear where the money to pursue such trials will come from, or even if they can be supported at all, since current trials are funded by companies to test their own products. A gathering of interested scientists and clinical investigators who can think-tank this problem is desperately needed and strongly encouraged.