Adaptation to ex vivo culture reduces human hematopoietic stem cell activity independently of the cell cycle.

ABSTRACT: Loss of long-term hematopoietic stem cell (LT-HSC) function ex vivo hampers the success of clinical protocols that rely on culture. However, the kinetics and mechanisms through which this occurs remain incompletely characterized. In this study, through time-resolved single-cell RNA sequencing, matched in vivo functional analysis, and the use of a reversible in vitro system of early G1 arrest, we defined the sequence of transcriptional and functional events that occur during the first ex vivo division of human LT-HSCs. We demonstrated that the sharpest loss in LT-HSC repopulation capacity happens early on, between 6 and 24 hours of culture, before LT-HSCs commit to cell cycle progression. During this time window, LT-HSCs adapt to the culture environment, limit the global variability in gene expression, and transiently upregulate gene networks involved in signaling and stress responses. From 24 hours, LT-HSC progression past early G1 contributes to the establishment of differentiation programs in culture. However, contrary to the current assumptions, we demonstrated that the loss of HSC function ex vivo is independent of cell cycle progression. Finally, we showed that targeting LT-HSC adaptation to culture by inhibiting the early activation of JAK/STAT signaling improves HSC long-term repopulating function ex vivo. Collectively, our study demonstrated that controlling early LT-HSC adaptation to ex vivo culture, for example, via JAK inhibition, is critically important to improve HSC gene therapy and expansion protocols.

Characterization of a novel pesticide transporter and P-glycoprotein orthologues in Drosophila melanogaster.

Pesticides remain one of the most effective ways of controlling agricultural and public health insects, but much is still unknown regarding how these compounds reach their targets. Specifically, the role of ABC transporters in pesticide absorption and excretion is poorly understood, especially compared to the detailed knowledge about mammalian systems. Here, we present a comprehensive characterization of pesticide transporters in the model insect Drosophila melanogaster. An RNAi screen was performed, which knocked down individual ABCs in specific epithelial tissues and examined the subsequent changes in sensitivity to the pesticides spinosad and fipronil. This implicated a novel ABC drug transporter, CG4562, in spinosad transport, but also highlighted the P-glycoprotein orthologue Mdr65 as the most impactful ABC in terms of chemoprotection. Further characterization of the P-glycoprotein family was performed via transgenic overexpression and immunolocalization, finding that Mdr49 and Mdr50 play enigmatic roles in pesticide toxicology perhaps determined by their different subcellular localizations within the midgut. Lastly, transgenic Drosophila lines expressing P-glycoprotein from the major malaria vector Anopheles gambiae were used to establish a system for in vivo characterization of this transporter in non-model insects. This study provides the basis for establishing Drosophila as a model for toxicology research on drug transporters.

The Landscape of Early Clinical Gene Therapies outside of Oncology.

The field of cell and gene therapy (GT) is expanding rapidly and there is undoubtedly a wave of enthusiasm and anticipation for what these treatments could achieve next. Here we assessed the worldwide landscape of GT assets currently in early clinical development (clinical trial phase 1/2 or about to enter clinical trial). We included all gene therapies, i.e., strategies that modify an individual's protein make-up by introducing exogenous nucleic acid or nucleic acid modifiers, regardless of delivery. Unmodified cell therapies, oncology therapies (reviewed elsewhere), and vaccine programs (distinct therapeutic strategy) were not included. Using a December 31, 2018 cutoff date, we identified 336 gene therapies being developed for 138 different indications covering 165 genetic targets. In all, we found that the early clinical GT landscape comprises a very disparate group of drug candidates in terms of indications, organizations, and delivery methods. We also highlight interesting trends, revealing the evolution of the field toward in vivo therapies and adeno-associated virus vector-based delivery systems. It will be interesting to witness what proportion of this current list effectively translates into new medicines.

Inhibition of Mitochondrial Complex I Impairs Release of α-Galactosidase by Jurkat Cells.

Fabry disease (FD) is caused by mutations in the GLA gene that encodes lysosomal α-galactosidase-A (α-gal-A). A number of pathogenic mechanisms have been proposed and these include loss of mitochondrial respiratory chain activity. For FD, gene therapy is beginning to be applied as a treatment. In view of the loss of mitochondrial function reported in FD, we have considered here the impact of loss of mitochondrial respiratory chain activity on the ability of a GLA lentiviral vector to increase cellular α-gal-A activity and participate in cross correction. Jurkat cells were used in this study and were exposed to increasing viral copies. Intracellular and extracellular enzyme activities were then determined; this in the presence or absence of the mitochondrial complex I inhibitor, rotenone. The ability of cells to take up released enzyme was also evaluated. Increasing transgene copies was associated with increasing intracellular α-gal-A activity but this was associated with an increase in Km. Release of enzyme and cellular uptake was also demonstrated. However, in the presence of rotenone, enzyme release was inhibited by 37%. Excessive enzyme generation may result in a protein with inferior kinetic properties and a background of compromised mitochondrial function may impair the cross correction process.

Eliminating HIV-1 Packaging Sequences from Lentiviral Vector Proviruses Enhances Safety and Expedites Gene Transfer for Gene Therapy.

Lentiviral vector genomic RNA requires sequences that partially overlap wild-type HIV-1 gag and env genes for packaging into vector particles. These HIV-1 packaging sequences constitute 19.6% of the wild-type HIV-1 genome and contain functional cis elements that potentially compromise clinical safety. Here, we describe the development of a novel lentiviral vector (LTR1) with a unique genomic structure designed to prevent transfer of HIV-1 packaging sequences to patient cells, thus reducing the total HIV-1 content to just 4.8% of the wild-type genome. This has been achieved by reconfiguring the vector to mediate reverse-transcription with a single strand transfer, instead of the usual two, and in which HIV-1 packaging sequences are not copied. We show that LTR1 vectors offer improved safety in their resistance to remobilization in HIV-1 particles and reduced frequency of splicing into human genes. Following intravenous luciferase vector administration to neonatal mice, LTR1 sustained a higher level of liver transgene expression than an equivalent dose of a standard lentivirus. LTR1 vectors produce reverse-transcription products earlier and start to express transgenes significantly quicker than standard lentiviruses after transduction. Finally, we show that LTR1 is an effective lentiviral gene therapy vector as demonstrated by correction of a mouse hemophilia B model.

BMI-1 extends proliferative potential of human bronchial epithelial cells while retaining their mucociliary differentiation capacity.

Air-liquid interface (ALI) culture of primary airway epithelial cells enables mucociliary differentiation providing an in vitro model of the human airway, but their proliferative potential is limited. To extend proliferation, these cells were previously transduced with viral oncogenes or mouse Bmi-1 + hTERT, but the resultant cell lines did not undergo mucociliary differentiation. We hypothesized that use of human BMI-1 alone would increase the proliferative potential of bronchial epithelial cells while retaining their mucociliary differentiation potential. Cystic fibrosis (CF) and non-CF bronchial epithelial cells were transduced by lentivirus with BMI-1 and then their morphology, replication kinetics, and karyotype were assessed. When differentiated at ALI, mucin production, ciliary function, and transepithelial electrophysiology were measured. Finally, shRNA knockdown of DNAH5 in BMI-1 cells was used to model primary ciliary dyskinesia (PCD). BMI-1-transduced basal cells showed normal cell morphology, karyotype, and doubling times despite extensive passaging. The cell lines underwent mucociliary differentiation when cultured at ALI with abundant ciliation and production of the gel-forming mucins MUC5AC and MUC5B evident. Cilia displayed a normal beat frequency and 9+2 ultrastructure. Electrophysiological characteristics of BMI-1-transduced cells were similar to those of untransduced cells. shRNA knockdown of DNAH5 in BMI-1 cells produced immotile cilia and absence of DNAH5 in the ciliary axoneme as seen in cells from patients with PCD. BMI-1 delayed senescence in bronchial epithelial cells, increasing their proliferative potential but maintaining mucociliary differentiation at ALI. We have shown these cells are amenable to genetic manipulation and can be used to produce novel disease models for research and dissemination.

Vps33b is crucial for structural and functional hepatocyte polarity.

BACKGROUND & AIMS: In the normal liver, hepatocytes form a uniquely polarised cell layer that enables movement of solutes from sinusoidal blood to canalicular bile. Whilst several cholestatic liver diseases with defects of hepatocyte polarity have been identified, the molecular mechanisms of pathogenesis are not well defined. One example is arthrogryposis, renal dysfunction and cholestasis syndrome, which in most patients is caused by VPS33B mutations. VPS33B is a protein involved in membrane trafficking that interacts with RAB11A at recycling endosomes. To understand the pathways that regulate hepatocyte polarity better, we investigated VPS33B deficiency using a novel mouse model with a liver-specific Vps33b deletion. METHODS: To assess functional polarity, plasma and bile samples were collected from Vps33b liver knockout (Vps33bfl/fl-AlfpCre) and control (Vps33bfl/fl) mice; bile components or injected substrates were quantitated by mass spectrometry or fluorometry. For structural analysis, livers underwent light and transmission electron microscopy. Apical membrane and tight junction protein localisation was assessed by immunostaining. Adeno-associated virus vectors were used for in vivo gene rescue experiments. RESULTS: Like patients, Vps33bfl/fl-AlfpCre mice showed mislocalisation of ATP-binding cassette proteins that are specifically trafficked to the apical membrane via Rab11a-positive recycling endosomes. This was associated with retention of bile components in blood. Loss of functional tight junction integrity and depletion of apical microvilli were seen in knockout animals. Gene transfer partially rescued these defects. CONCLUSIONS: Vps33b has a key role in establishing structural and functional aspects of hepatocyte polarity and may be a target for gene replacement therapy. LAY SUMMARY: Hepatocytes are liver cells with tops and bottoms; that is, they are polarised. At their bottoms they absorb substances from blood. They then, at their tops, secrete these substances and their metabolites into bile. When polarity is lost, this directional flow of substances from blood to bile is disrupted and liver disease follows. In this study, using a new mouse model with a liver-specific mutation of Vps33b, the mouse version of a gene that is mutated in most patients with arthrogryposis, renal dysfunction and cholestasis (ARC) syndrome, we investigated how the Vps33b gene product contributes to establishing hepatocyte polarity. We identified in these mice abnormalities similar to those in children with ARC syndrome. Gene transfer could partly reverse the mouse abnormalities. Our work contributes to the understanding of VPS33B disease and hepatocyte polarity in general, and may point towards gene transfer mediated treatment of ARC liver disease.

Systemic gene delivery following intravenous administration of AAV9 to fetal and neonatal mice and late-gestation nonhuman primates.

Several acute monogenic diseases affect multiple body systems, causing death in childhood. The development of novel therapies for such conditions is challenging. However, improvements in gene delivery technology mean that gene therapy has the potential to treat such disorders. We evaluated the ability of the AAV9 vector to mediate systemic gene delivery after intravenous administration to perinatal mice and late-gestation nonhuman primates (NHPs). Titer-matched single-stranded (ss) and self-complementary (sc) AAV9 carrying the green fluorescent protein (GFP) reporter gene were intravenously administered to fetal and neonatal mice, with noninjected age-matched mice used as the control. Extensive GFP expression was observed in organs throughout the body, with the epithelial and muscle cells being particularly well transduced. ssAAV9 carrying the WPRE sequence mediated significantly more gene expression than its sc counterpart, which lacked the woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) sequence. To examine a realistic scale-up to larger models or potentially patients for such an approach, AAV9 was intravenously administered to late-gestation NHPs by using a clinically relevant protocol. Widespread systemic gene expression was measured throughout the body, with cellular tropisms similar to those observed in the mouse studies and no observable adverse events. This study confirms that AAV9 can safely mediate systemic gene delivery in small and large animal models and supports its potential use in clinical systemic gene therapy protocols.

Impulsivity predicts time to reach euthymia in adults with bipolar disorder.

OBJECTIVES: Specific demographic and illness characteristics have been identified as predictors of overall morbidity and treatment course among individuals with bipolar disorder. However, the role of specific cognitive limitations on disease severity and treatment response is unclear. The present study evaluated whether impulsiveness during acute mania was a significant predictor of achieving euthymia within one year following psychiatric hospitalization. METHODS: Participants were 94 adult inpatients (60 manic) with bipolar I disorder. Baseline symptom severity was assessed using the Young Mania Rating Scale and the Montgomery-Åsberg Depression Rating Scale. Impulsivity was measured with the Stop Signal Task, Degraded Stimulus Continuous Performance Task, Delayed Response Task, and Barratt Impulsiveness Scale-11. RESULTS: Individual predictors of time to reach euthymia included fewer depressive symptoms and better impulse control at baseline, later age at illness onset, shorter illness duration, and the absence of comorbid attention-deficit hyperactivity disorder. Self-reported impulsivity was a significant independent predictor of time to euthymia, even after accounting for relevant clinical variables. CONCLUSIONS: Better trait impulse control may be associated with better treatment responsiveness among adults with bipolar disorder.

A small molecule modulator of prion protein increases human mesenchymal stem cell lifespan, ex vivo expansion, and engraftment to bone marrow in NOD/SCID mice.

Human mesenchymal stem cells (hMSCs) have been shown to have potential in regenerative approaches in bone and blood. Most protocols rely on their in vitro expansion prior to clinical use. However, several groups including our own have shown that hMSCs lose proliferation and differentiation ability with serial passage in culture, limiting their clinical applications. Cellular prion protein (PrP) has been shown to enhance proliferation and promote self-renewal of hematopoietic, mammary gland, and neural stem cells. Here we show, for the first time, that expression of PrP decreased in hMSC following ex vivo expansion. When PrP expression was knocked down, hMSC showed significant reduction in proliferation and differentiation. In contrast, hMSC expanded in the presence of small molecule 3/689, a modulator of PrP expression, showed retention of PrP expression with ex vivo expansion and extended lifespan up to 10 population doublings. Moreover, cultures produced a 300-fold increase in the number of cells generated. These cells showed a 10-fold increase in engraftment levels in bone marrow 5 weeks post-transplant. hMSC treated with 3/689 showed enhanced protection from DNA damage and enhanced cell cycle progression, in line with data obtained by gene expression profiling. Moreover, upregulation of superoxide dismutase-2 (SOD2) was also observed in hMSC expanded in the presence of 3/689. The increase in SOD2 was dependent on PrP expression and suggests increased scavenging of reactive oxygen species as mechanism of action. These data point to PrP as a good target for chemical intervention in stem cell regenerative medicine.

Effective gene therapy with nonintegrating lentiviral vectors.

Retroviral and lentiviral vector integration into host-cell chromosomes carries with it a finite chance of causing insertional mutagenesis. This risk has been highlighted by the induction of malignancy in mouse models, and development of lymphoproliferative disease in three individuals with severe combined immunodeficiency-X1 (refs. 2,3). Therefore, a key challenge for clinical therapies based on retroviral vectors is to achieve stable transgene expression while minimizing insertional mutagenesis. Recent in vitro studies have shown that integration-deficient lentiviral vectors can mediate stable transduction. With similar vectors, we now show efficient and sustained transgene expression in vivo in rodent ocular and brain tissues. We also show substantial rescue of clinically relevant rodent models of retinal degeneration. Therefore, the high efficiency of gene transfer and expression mediated by lentiviruses can be harnessed in vivo without a requirement for vector integration. For therapeutic application to postmitotic tissues, this system substantially reduces the risk of insertional mutagenesis.

Hematopoietic stem cell and gene therapy corrects primary neuropathology and behavior in mucopolysaccharidosis IIIA mice.

Mucopolysaccharidosis IIIA (MPS IIIA or Sanfilippo disease) is a neurodegenerative disorder caused by a deficiency in the lysosomal enzyme sulfamidase (SGSH), catabolizing heparan sulfate (HS). Affected children present with severe behavioral abnormalities, sleep disturbances, and progressive neurodegeneration, leading to death in their second decade. MPS I, a similar neurodegenerative disease accumulating HS, is treated successfully with hematopoietic stem cell transplantation (HSCT) but this treatment is ineffectual for MPS IIIA. We compared HSCT in MPS IIIA mice using wild-type donor cells transduced ex vivo with lentiviral vector-expressing SGSH (LV-WT-HSCT) versus wild-type donor cell transplant (WT-HSCT) or lentiviral-SGSH transduced MPS IIIA cells (LV-IIIA-HSCT). LV-WT-HSCT results in 10% of normal brain enzyme activity, near normalization of brain HS and GM2 gangliosides, significant improvements in neuroinflammation and behavioral correction. Both WT-HSCT and LV-IIIA-HSCT mediated improvements in GM2 gangliosides and neuroinflammation but were less effective at reducing HS or in ameliorating abnormal HS sulfation and had no significant effect on behavior. This suggests that HS may have a more significant role in neuropathology than neuroinflammation or GM2 gangliosides. These data provide compelling evidence for the efficacy of gene therapy in conjunction with WT-HSCT for neurological correction of MPS IIIA where conventional transplant is ineffectual.

Gene therapy: is IL2RG oncogenic in T-cell development?

The gene IL2RG encodes the gamma-chain of the interleukin-2 receptor and is mutated in patients with X-linked severe combined immune deficiency (X-SCID). Woods et al. report the development of thymus tumours in a mouse model of X-SCID after correction by lentiviral overexpression of IL2RG and claim that these were caused by IL2RG itself. Here we find that retroviral overexpression of IL2RG in human CD34+ cells has no effect on T-cell development, whereas overexpression of the T-cell acute lymphoblastic leukaemia (T-ALL) oncogene LMO2 leads to severe abnormalities. Retroviral expression of IL2RG may therefore not be directly oncogenic--rather, the restoration of normal signalling by the interleukin-7 receptor to X-SCID precursor cells allows progression of T-cell development to stages that are permissive for the pro-leukaemic effects of ectopic LMO2.

Lentiviral vector integration profiles differ in rodent postmitotic tissues.

Lentiviral vectors with self-inactivating (SIN) long terminal repeats (LTRs) are promising for safe and sustained transgene expression in dividing as well as quiescent cells. As genome organization and transcription substantially differs between actively dividing and postmitotic cells in vivo, we hypothesized that genomic vector integration preferences might be distinct between these biological states. We performed integration site (IS) analyses on mouse dividing cells (fibroblasts and hematopoietic progenitor cells (HPCs)) transduced ex vivo and postmitotic cells (eye and brain) transduced in vivo. As expected, integration in dividing cells occurred preferably into gene coding regions. In contrast, postmitotic cells showed a close to random frequency of integration into genes and gene spare long interspersed nuclear elements (LINE). Our studies on the potential mechanisms responsible for the detected differences of lentiviral integration suggest that the lowered expression level of Psip1 reduce the integration frequency in vivo into gene coding regions in postmitotic cells. The motif TGGAA might represent one of the factors for preferred lentiviral integration into mouse and rat Satellite DNA. These observations are highly relevant for the correct assessment of preclinical biosafety studies, indicating that lentiviral vectors are well suited for safe and effective clinical gene transfer into postmitotic tissues.

Insertion sites in engrafted cells cluster within a limited repertoire of genomic areas after gammaretroviral vector gene therapy.

Vector-associated side effects in clinical gene therapy have provided insights into the molecular mechanisms of hematopoietic regulation in vivo. Surprisingly, many retrovirus insertion sites (RIS) present in engrafted cells have been found to cluster nonrandomly in close association with specific genes. Our data demonstrate that these genes directly influence the in vivo fate of hematopoietic cell clones. Analysis of insertions thus far has been limited to individual clinical studies. Here, we studied >7,000 insertions retrieved from various studies. More than 40% of all insertions found in engrafted gene-modified cells were clustered in the same genomic areas covering only 0.36% of the genome. Gene classification analyses displayed significant overrepresentation of genes associated with hematopoietic functions and relevance for cell growth and survival in vivo. The similarity of insertion distributions indicates that vector insertions in repopulating cells cluster in predictable patterns. Thus, insertion analyses of preclinical in vitro and murine in vivo studies as well as vector insertion repertoires in clinical trials yielded concerted results and mark a small number of interesting genomic loci and genes that warrants further investigation of the biological consequences of vector insertions.

Insertional mutagenesis combined with acquired somatic mutations causes leukemogenesis following gene therapy of SCID-X1 patients.

X-linked SCID (SCID-X1) is amenable to correction by gene therapy using conventional gammaretroviral vectors. Here, we describe the occurrence of clonal T cell acute lymphoblastic leukemia (T-ALL) promoted by insertional mutagenesis in a completed gene therapy trial of 10 SCID-X1 patients. Integration of the vector in an antisense orientation 35 kb upstream of the protooncogene LIM domain only 2 (LMO2) caused overexpression of LMO2 in the leukemic clone. However, leukemogenesis was likely precipitated by the acquisition of other genetic abnormalities unrelated to vector insertion, including a gain-of-function mutation in NOTCH1, deletion of the tumor suppressor gene locus cyclin-dependent kinase 2A (CDKN2A), and translocation of the TCR-beta region to the STIL-TAL1 locus. These findings highlight a general toxicity of endogenous gammaretroviral enhancer elements and also identify a combinatorial process during leukemic evolution that will be important for risk stratification and for future protocol design.

Gene delivery of a mutant TGFß3 reduces markers of scar tissue formation after cutaneous wounding.

The transforming growth factor-ß (TGFß) family plays a critical regulatory role in repair and coordination of remodeling after cutaneous wounding. TGFß1-mediated chemotaxis promotes the recruitment of fibroblasts to the wound site and their resultant myofibroblastic transdifferentiation that is responsible for elastic fiber deposition and wound closure. TGFß3 has been implicated in an antagonistic role regulating overt wound closure and promoting ordered dermal remodeling. We generated a mutant form of TGFß3 (mutTGFß3) by ablating its binding site for the latency-associated TGFß binding protein (LTBP-1) in order to improve bioavailability and activity. The mutated cytokine is secreted as the stable latency-associated peptide (LAP)-associated form and is activated by normal intracellular and extracellular mechanisms including integrin-mediated activation but is not sequestered. We show localized intradermal transduction using a lentiviral vector expressing the mutTGFß3 in a mouse skin wounding model reduced re-epithelialization density and fibroblast/myofibroblast transdifferentiation within the wound area, both indicative of reduced scar tissue formation.

Vector integration is nonrandom and clustered and influences the fate of lymphopoiesis in SCID-X1 gene therapy.

Recent reports have challenged the notion that retroviruses and retroviral vectors integrate randomly into the host genome. These reports pointed to a strong bias toward integration in and near gene coding regions and, for gammaretroviral vectors, around transcription start sites. Here, we report the results obtained from a large-scale mapping of 572 retroviral integration sites (RISs) isolated from cells of 9 patients with X-linked SCID (SCID-X1) treated with a retrovirus-based gene therapy protocol. Our data showed that two-thirds of insertions occurred in or very near to genes, of which more than half were highly expressed in CD34(+) progenitor cells. Strikingly, one-fourth of all integrations were clustered as common integration sites (CISs). The highly significant incidence of CISs in circulating T cells and the nature of their locations indicate that insertion in many gene loci has an influence on cell engraftment, survival, and proliferation. Beyond the observed cases of insertional mutagenesis in 3 patients, these data help to elucidate the relationship between vector insertion and long-term in vivo selection of transduced cells in human patients with SCID-X1.

Gammaretrovirus-mediated correction of SCID-X1 is associated with skewed vector integration site distribution in vivo.

We treated 10 children with X-linked SCID (SCID-X1) using gammaretrovirus-mediated gene transfer. Those with sufficient follow-up were found to have recovered substantial immunity in the absence of any serious adverse events up to 5 years after treatment. To determine the influence of vector integration on lymphoid reconstitution, we compared retroviral integration sites (RISs) from peripheral blood CD3(+) T lymphocytes of 5 patients taken between 9 and 30 months after transplantation with transduced CD34(+) progenitor cells derived from 1 further patient and 1 healthy donor. Integration occurred preferentially in gene regions on either side of transcription start sites, was clustered, and correlated with the expression level in CD34(+) progenitors during transduction. In contrast to those in CD34(+) cells, RISs recovered from engrafted CD3(+) T cells were significantly overrepresented within or near genes encoding proteins with kinase or transferase activity or involved in phosphorus metabolism. Although gross patterns of gene expression were unchanged in transduced cells, the divergence of RIS target frequency between transduced progenitor cells and post-thymic T lymphocytes indicates that vector integration influences cell survival, engraftment, or proliferation.

The differentiation and engraftment potential of mouse hematopoietic stem cells is maintained after bio-electrospray.

The bio-electrospray technique has been recently pioneered to manipulate living, immortalised and primary cells, including a wide range of stem cells. Studies have demonstrated that the creation of viable, fully functional in vitro microenvironments is possible using this technique. By modifying the bio-electrospray procedure (referred to as cell electrospinning), a variety of microenvironment morphologies have been fabricated. Because bio-electrospraying of biological material is a relatively new technique, it is important to determine if there are any unwanted consequences to the manipulated cells as a result of the procedure. Here, we establish the validity of the process using a heterogeneous, living population of hematopoietic stem/progenitor cells, using a functional in vitro assay and in vivo mouse model to investigate for side-effects that previous in vitro assays may not have detected. Our studies demonstrate that these bio-protocols have no obvious negative effects, thus indicating significant promise for utility in biological sciences and for a plethora of healthcare applications.