The Residual granulocyte colony-stimulating factor (G-CSF) mobilized peripheral blood (mPB) product, remaining after allogeneic stem cell transplantation, was obtained from the Department of Hematology, Christian Medical College, Vellore, following Institutional Review Board (IRB) approval. The Peripheral blood mononuclear cells (PBMCs) were separated by lymphoprep density gradient centrifugation. The CD34⁺ cells from PBMNCs were isolated using the CD34⁺ selection kit (STEMCELL Technologies) and subsequently expanded using AC media or Hibernation medium. AC contains StemSpan™ SFEM II medium supplemented Stem cell factor (SCF, 240 ng/mL), FMS-like tyrosine kinase three ligand (Flt3-L, 240 ng/mL), Thrombopoietin (TPO, 80 ng/mL), and Interleukin-6 (IL-6, 40 ng/mL). For the hibernation medium, the cells are cultured in (SCF, 300 ng/mL), (IL11, 20 ng/mL), (L-Glutamine, 2 mM), and (2-Mercaptoethanol, 100 μM) (Oedekoven et al., 2021). Both the culture media were supplemented with small molecule cocktail of Resveratrol, Stem Reginin-1 and UM729 (RUS) as described in our previous work (Christopher et al., 2022). In both the conditions the cells were cultured at a confluency of 2 × 10⁵/mL. HSPCs were analyzed for cell-surface marker expression both immediately after purification and subsequent days of culture using a BD FACSAria™ III flow cytometer.

For sorting CD90⁺ cells from CD34⁺ HSPCs, the purified CD34⁺ cells were cultured with the above-mentioned cytokines for 16–24 h. Then the cultured cells were stained with the CD90⁺ antibody for 20 min at room temperature (8µg/1 × 10^6 cells). Following incubation, the cells were washed with PBS and CD90⁺ cells were sorted using the flow cytometer BD FACS ARIA III in purity mode.

On reaching 60%–80% confluency, transfect HEK-293T cells with 2 µg of the transgene plasmid (hTERT + HLF or hTERT + MLLT3), 1 µg of the packaging plasmid (psPAX2), and 1 µg of the envelope plasmid (VSV-G) using FuGENE at a 1:3 ratio in OptiMEM medium in 6 cm dish. The transfection mix were incubated for 15 min to allow complex formation. Add the mix dropwise to the cells immediately. For virus collection, harvest the media containing the virus at 48 and 72 h post-transfection, pool the collected media, and perform ultracentrifugation at 20,000rpm for 2 h to pellet the virus and resuspend the viral pellet in plain SFEM-II. Aliquot the virus and store at −80 °C. The plasmid PSJL224 was a kind gift from st jude childrens' research hospital.

Retronectin was coated onto 96-well plates according to the manufacturer’s instructions to facilitate efficient transduction. The transduction mix was prepared in SFEM-II medium by supplementing 6 mM of HEPES buffer and 10 μg/mL of Polybrene. CD90⁺ sorted cells were then resuspended in this transduction mix and the viral suspension was added to the wells. The plates were centrifuged at 800 g for 30 min at 24 °C to enhance viral contact with the cells, followed by incubation in a 5% CO₂ incubator. After 24 h, viral washouts were performed to replace with fresh medium. The cells were cultured and analyzed based on experimental requirements. For testing the transduction enhancers, the following reagents were tested: Retronectin (50 μg/mL), Polybrene (10 μg/mL), Cyclosporin H (8 µM), LentiBoost (0.7 mg/mL), Protamine sulfate (5 μg/mL), and Prostaglandin E2 (PGE2; 10 µM).

Based on experimental requirements, 500 HSPCs were seeded in the 1.5 mL of Methocult Optimum (STEMCELL Technologies). After 14 days of culture, hematopoietic progenitor colonies were scored based on morphology and categorized as Burst forming unit-erythroid (BFU-E), Colony forming unit-erythroid (CFU-E), Colony forming unit-granulocyte-monocyte progenitor (CFU-GM) and Colony forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte (CFU-GEMM).

The erythroid differentiation protocol for HSPCs was adapted from our previously published protocol (Venkatesan et al., 2023). The three-stage protocol begins with phase I, where CD34⁺ cells are cultured at a density of 5 × 10^4 cells/mL from day 0 to day 8, with a media change on day 4. The phase I medium consists of IMDM GlutaMAX Supplement with 5% AB serum, insulin (20 μg/mL), heparin (2 U/mL), EPO (3 U/mL), holotransferrin (330 μg/mL), SCF (100 ng/mL), IL3 (50 ng/mL), and hydrocortisone (1 μg/mL). In phase II, from day 8 to day 12, cells are seeded at 2 × 10^5 cells/mL in medium identical to phase I but without hydrocortisone and IL3. In phase III, from day 12 to day 20, cells are cultured at 5 × 10^5 cells/mL in medium similar to phase II but excluding SCF, with a media change on day 16. On day 20, cells are harvested for quantifying the percentage of reticulocytes marked by CD235a⁺, Hoechst⁻.

Macrophage differentiation of HSPCs was performed following our previously published protocol (Karuppusamy et al., 2022). In brief, HSPCs were seeded in non-tissue culture-treated polystyrene plates using macrophage differentiation medium (SFEM-II supplemented with 100 ng/mL SCF, 50 ng/mL Flt3-L, 10 ng/mL IL-6, 10 ng/mL IL-3, 10 ng/ml GM-CSF, and 10 ng/ml M-CSF). Every 72 h, non-adherent cells were collected and replated in fresh macrophage differentiation medium. Adherent cells were maintained in RPMI medium with 10% FBS, 10 ng/ml GM-CSF, and 10 ng/ml M-CSF. After 14–16 days, adherent cells were examined under a microscope for morphological characteristics, detached using accutase and stained with antibody CD14. The stained cells were analyzed using a BD FACS Aria III flow cytometer.

For megakaryocyte differentiation of immortalized HSCs, HSCs were seeded in megakaryocyte differentiation medium (SFEM-II supplemented with 100 uL of StemSpan™ Megakaryocyte Expansion Supplement (100X). Once in 3–4 days, cell were replenished with fresh media. After 14 days, cells were examined under a microscope for morphological characteristics, stained with antibody CD61 and CD41 for surface marker expression.

48 h post transduction of hTERT, hTERT + HLF, and hTERT + MLLT3, a total of 1 × 10⁶ cells that were collected and RNA were extracted using RNeasy Mini Kit (Qiagen). 1μg of RNA were used for reverse transcription using Prime-script RT reagent kit (Takara Bio Inc.). Quantitative PCR was performed using SYBR Premix Ex Taq II (Takara Bio), and amplification was carried out on a QuantStudio 6 Flex Real-Time PCR System (Life Technologies). The primer sequences used for qPCR are listed in Supplementary Table S3.

To generate immortalized HSPCs, we engineered three distinct constructs: hTERT, hTERT + HLF, and hTERT + MLLT3 (Figure 1A). In the hTERT construct, ZsGreen and hTERT were co-expressed under the constitutive EF1A promoter, separated by a P2A linker. For the hTERT + HLF and hTERT + MLLT3 constructs, we employed a bi-cistronic doxycycline-inducible system. In this design, hTERT and the Tet-On transactivator are driven by the constitutive MND promoter, while the TRE3G promoter controls the Dox inducible expression of mCherry together with HLF or MLLT3 separated by P2A linker. Upon doxycycline administration, TRE3G activation enables co-expression of mCherry and HLF (or MLLT3), allowing temporal control of stemness factor expression during the expansion phase versus the differentiation phase.

To validate construct designing, the hTERT was transduced into HEK-293T cells, and the proportion of GFP positive (GFP⁺) cells was quantified by flow cytometry 48 h post-transduction. Compared to untransduced controls, approximately 45.7% of cells were GFP⁺(Supplementary Figure S1A). Additionally, relative hTERT mRNA expression was significantly elevated compared to untransduced cells (Supplementary Figure S1B). Similarly, hTERT + HLF and hTERT + MLLT3 were transduced and the mCherry expression (35.2% and 32.1%) (Supplementary Figure S1C) was measured respectively 48 h following post transduction. Furthermore, relative mRNA expression levels of hTERT, HLF, and MLLT3, normalized to β-actin, were significantly increased in both constructs compared with untransduced cells (Supplementary Figure S1D,E). Since the latter two cassettes exploit the Dox inducible system, we wanted to check the impact of Dox on the stemness and the proliferation of HSPCs. The impact was evaluated by culturing HSPCs with doxycycline concentrations ranging from 0 to 1 μg/mL to assess potential toxicity on HSPC subsets marked by CD34⁺CD90⁺ cells. Flow cytometry analysis on day 5 of culture revealed no notable differences in the HSPCs subsets across doxycycline doses (0.25–1 μg/mL) compared to the control (0 μg/mL) (Supplementary Figure S1F). These findings demonstrate that doxycycline-regulated HLF and MLLT3 expression systems are suitable for conditional immortalization of HSPCs.

For immortalization, it is essential to target the HSPC fraction with the stemness and long-term repopulating potential, rather than the bulk population, to ensure that immortalized cells preserve stem cell properties. Among HSPC subsets, CD34⁺CD90⁺ cells are well recognized for their robust and durable engraftment capacity. Equally critical is the establishment of culture conditions that prevent exhaustion and sustain stemness over extended periods. To address this, we initiated our studies by sorting CD90⁺ HSCs 16–24 h after CD34⁺ cell isolation from peripheral blood mononuclear cells (PBMNCs). These cells were then cultured in hibernation medium (hib), which has recently been reported to support the maintenance of long-term Cord blood (CB) derived HSCs in vitro (Oedekoven et al., 2021). We subsequently compared three conditions including our previously established AC + RUS medium [stem cell factor (SCF, 240 ng/mL), FMS-like tyrosine kinase three ligand (Flt3-L, 240 ng/mL), thrombopoietin (TPO, 80 ng/mL), and interleukin-6 (IL-6, 40 ng/mL) and small molecule cocktail of Resveratrol, Stem Reginin-1 and UM729 (RUS)] (Christopher et al., 2022), hibernation medium alone (Hib) [SCF 300 ng/mL, IL11 20 ng/mL, L-Glutamine 2mM, 2-Mercaptoethanol 100 μM], and hibernation medium supplemented with the RUS cocktail (Hib + RUS). Over an 8-day culture period, we observed distinct proliferation dynamics, while AC + RUS promoted robust expansion, whereas both Hib and Hib + RUS led to poor survival and diminished proliferative capacity (Supplementary Figure S1G) with similar frequency of CD34⁺ CD90⁺ cells (Supplementary Figure S1H). Since the hibernation protocol has been shown to preserve stemness in CB derived HSPCs, and given compelling evidence from the literature (Sakurai et al., 2023) that FLT3, and TPO signaling pathways are critical for the survival of mobilized peripheral blood (mPB) HSPCs, we supplemented the hibernation medium with cytokines at 100%, 20%, and 10% of the standard AC concentration. We then cultured CD90⁺ cells for 21 days. By day 21, analysis of HSPC subsets revealed that the optimized culture particularly the 10% cytokine condition in combination with hibernation medium retained the highest proportion of CD34⁺CD90⁺ CD49f⁺ and CD34⁺CD90⁺ CD49f⁻ cells (Figure 1B; Supplementary Figure S2A). This suggests that the medium helps mitigate exhaustion by limiting proliferation (Supplementary Figure S2B). Importantly, the colony-forming potential of cells expanded under these conditions for 21 days remains unaffected compared to standard conditions (Supplementary Figure S2C).

We next evaluated viral envelopes for their ability to efficiently transduce CD90⁺ HSCs. Given prior evidence that the Baboon envelope (BaEV) enhances transduction of primitive HSCs (Bernadin et al., 2019), we compared its performance against the conventional VSV-G envelope. As expected, VSV-G achieved higher transduction efficiency in HEK293T cells (Supplementary Figure S2D). However, in CD90⁺ HSCs, the BaEV envelope outperformed VSV-G (Figure 1C). Despite this improvement, overall transduction efficiency in HSCs remained low, prompting us to test a panel of transduction enhancers (Figure 1D). The combination of retronectin coating, polybrene, and cyclosporine-H proved most effective, yielding a 3.4-fold increase in transduction efficiency relative to polybrene alone.

After optimizing culture and transduction conditions, we transduced CD90⁺sorted HSCs from a healthy donor with either hTERT + HLF or hTERT + MLLT3 for immortalizing HSPCs (Figure 1E). Flow cytometry analysis on day 10 post-transduction revealed 5.72% mCherry⁺ cells in the hTERT + HLF group, compared with only 0.92% in the hTERT + MLLT3 group (Figure 1F). Given the relatively low transduction efficiency, we enriched the mCherry⁺ fraction by sorting at day 11 to better assess the effects. Sorted cells expressing hTERT + HLF expanded rapidly and maintained proliferation for up to 39 days in vitro, followed by a gradual decline that extended to approximately 70 days (Figures 2A,B). In contrast, the mCherry⁺ fraction transduced with hTERT + MLLT3 failed to sustain long-term growth, instead showing progressive differentiation and cell death. As expected, control untransduced HSPCs exhibited limited expansion, highlighting the partial immortalization effect of the hTERT + HLF system. Microscopic inspection on day 19 further confirmed that hTERT + HLF transduced cells retained a small, round, stem cell-like morphology, consistent with their preserved stemness, whereas cells expressing hTERT + MLLT3 did not adopt this morphology and instead exhibited features of apoptosis or differentiation into committed progenitors (Figure 1G).

By day 40 post-expansion, the colony forming capacity of hTERT + HLF transduced cells were reduced compared with wild-type fresh HSPCs (Figure 2C), indicating a loss of functional progenitor activity associated with prolonged proliferation. To evaluate their differentiation competence, day 45 expanded cells were differentiated into erythroid, macrophage, and megakaryocyte lineages. These cells successfully generated terminally differentiated reticulocytes at detectable levels (Figure 2D), confirming retained terminal differentiation potential. However, assessment of erythroid progenitor fold expansion revealed that the hTERT + HLF system failed to generate larger numbers of cells, despite supporting progression to terminal differentiation (Figure 2E). Differentiation toward the myeloid lineage generated comparable percentages of CD14⁺ macrophages (Figure 2F), indicating that myeloid output capacity was largely preserved during extended culture. However, the fold expansion of macrophage progenitors was markedly reduced in the hTERT + HLF group (Figure 2G). Similarly, megakaryocyte differentiation of the hTERT + HLF cells yielded lesser proportion of terminally differentiated cells marked by CD61⁺/CD41⁺ (Figure 2H) and lower progenitor expansion (Figure 2I) compared to the wild-type HSPCs. Together, these results demonstrate that expression of hTERT combined with inducible HLF supports the partial immortalization of healthy donor HSPCs with limited differentiaion potential. Expanded cells maintained proliferative capacity for over 2 months in culture while partially retaining multilineage differentiation potential across erythroid, myeloid, and megakaryocytic pathways. The partial preservation of both progenitor expansion and functional output highlights this strategy as a promising approach, however further optimization are required for generating long-lived, functional HSPCs in vitro.